recurrent selection in rice, using a male-sterile gene

xi

Cover photo: Male-sterile (center) and fertile (left and right)spikelets of rice.

Photo by: Photographic Section, CIAT.

The Centre de coopération internationale en recherche agronomique pour le développement (CIRAD) isa French research organization that specializes in agriculture in the tropics and subtropics. It is astate-owned body and it was established in 1984 following the consolidation of French agricultural,veterinary, forestry, and food technology research organizations for the tropics and subtropics.

CIRAD’s mission is to contribute to the economic development of these regions through research,experiments, training, and dissemination of scientific and technical information.

The Centre employs 1800 persons, including 900 senior staff, who work in about 50 countries. Itsbudget amounts to approximately 1 billion French francs, more than half of which is derived from publicf u n d s .

CIRAD is made up of seven departments: CIRAD-CA (annual crops), CIRAD-CP (tree crops),CIRAD-FLHOR (fruit and horticultural crops), CIRAD-EMVT (livestock production and veterinary medicine),CIRAD-Fôret (forestry), CIRAD-SAR (food technology and rural systems), and CIRAD-GERDAT (management,common services and laboratories, documentation). CIRAD operates through its own research centres,national agricultural research systems, or development projects.

The International Center for Tropical Agriculture (CIAT, its Spanish acronym) is dedicated to the alleviationof hunger and poverty in developing countries of the tropics. CIAT applies science to agriculture toincrease food production while sustaining the natural resource base.

CIAT is one of 16 international agricultural research centers sponsored by the Consultative Group onInternational Agricultural Research (CGIAR).

The Center’s core budget is financed by 25 donor countries, international and regional developmentorganizations, and private foundations. In 1997, the donor countries include Australia, Belgium, Canada,China, Colombia, Denmark, France, Germany, Japan, the Netherlands, Norway, Spain, Sweden, Switzerland,the United Kingdom, and the United States of America. Donor organizations include the European Union(EU), the Ford Foundation, the Inter-American Development Bank (IDB), the International DevelopmentResearch Centre (IDRC), the International Fund for Agricultural Development (IFAD), the Nippon Foundation,the Rockefeller Foundation, the United Nations Development Programme (UNDP), and the World Bank.

Information and conclusions reported in this document do not necessarily reflect the position of anydonor agency.

Recurrent Selection in Rice,Using a Male-Sterile Gene

i i

Recurrent Selection in Rice, Using a Male-Sterile Gene

We are pleased to acknowledge the financial support received from the Centre decoopération internationale en recherche agronomique pour le développement,Département des cultures annuelles (CIRAD-CA), Montpellier, France.

iii

ISBN 958-9439-90-X

Recurrent Selection in Rice,Using a Male-Sterile Gene

Centro Internacional de Agricultura Tropical

International Center for Tropical Agriculture

Marc ChâtelElcio P. Guimarães

Centrede coopérationinternationaleen rechercheagronomiquepour ledéveloppement

DépartementDépartementDépartementDépartementDépartementdes culturesdes culturesdes culturesdes culturesdes culturesannuellesannuellesannuellesannuellesannuellesCIRAD-CACIRAD-CACIRAD-CACIRAD-CACIRAD-CA

iv


About the authors

Marc Châtel is plant breeder at the CIRAD-CA Rice Program in Montpellier, France,and is currently seconded to the CIAT Rice Program.

Elcio P. Guimarães, formerly plant breeder at the CIAT Rice Program, Cali, Colombia,is now plant breeder for the Rice Program at the Centro Nacional de Pesquisa deArroz e Feijão (CNPAF) of the Empresa Brasileira de Pesquisa Agropecuária(EMBRAPA), Brazil.

Centre de coopération internationale en rechercheagronomique pour le développement,

Département des cultures annuelles2477, avenue du Val de MontferrandBP 503534032 Montpellier cedex 1, France

Centro Internacional de Agricultura TropicalInternational Center for Tropical AgricultureApartado aéreo 6713Cali, Colombia

CIAT Publication No. 276ISBN 958-9439-90-XPress run: 300Printed in ColombiaJune, 1997

Châtel, Marc ; Guimarães, Elcio P. Recurrent selection in rice, using amale-sterile gene. -- Cali, Colombia : Centro Internacional de AgriculturaTropical ; Centre de Coopération Internationale en Recherche Agronomique pourle Développement, Département des Cultures Annuelles, 1997. 70 p. -- (CIAT Publication ; no. 276) ISBN 958-9439-90-X

First published in Spanish as “Selección Recurrente con Androesterilidad en Arroz” byMarc Châtel and Elcio P. Guimarães, CIRAD-CA and CIAT, Cali, Colombia, 1995.

1. Rice. 2. Rice -- Selection (plant breeding). 3. Rice -- Male sterility in plants.4. Rice -- Genetic combination. 5. Rice -- Case studies. I. Guimarães, Elcio P.II. Centro Internacional de Agricultura Tropical. III. Centre de CoopérationInternationale en Recherche Agronomique pour le Développement. IV. Title.

v

ContentsContentsContentsContentsContents

Page

Preface vii

Acknowledgments ix

Acronyms and Abbreviations x

Introduction: Improving Rice Populations 1

The Developing Rice Grain 1

The Gene for Male Sterility 2

References 3

Chapter 1: Methodologies for Managing Recurrent Selection 7

Developing Populations of Recombinant Genotypes 7A. Monocytoplasmic population 7B . Polycytoplasmic population 7

Active Maintenance of Populations 1 2A. Maintenance based on male-sterile plants 1 2B . Maintenance based on fertile plants 1 2

Population Improvement 1 5A. Recurrent selection, using male-sterile plants 1 5B . Recurrent selection, using fertile plants 2 4

Selecting Genotypes for Line Development 2 4A. Extraction by the pedigree method 3 2B . Extraction by anther culture 3 6

Introducing New Variability into Populations 3 7

Literature Cited 4 6

Recent Publications 4 6

Conference Posters, March 1994 4 7

Papers Presented at Workshops Held in 1996 4 7

vi


Page

Appendix A 4 8

Appendix B 4 9

Appendix C 5 0

Appendix D 5 2

Chapter 2: Using Recurrent Selection: Five Case Studies 5 3

C a s e 1 : Irrigated Rice in Goiânia, Brazil: Recurrent SelectionConducted by EMBRAPA-CNPAF 5 3

2 : Upland Rice in Villavicencio, Colombia: Recurrent SelectionConducted by CIRAD-CA and CIAT 5 5

3 : Resistance to Blast and Yellow Mottle Virus in Rice,Bouaké, Côte d’Ivoire: Recurrent Selection Conducted byCIRAD-CA, IDESSA, and WARDA 5 7

4 : High-Altitude Rice in Vinaninony and Antsirabe, Madagascar:Recurrent Selection Conducted by CIRAD-CA and FOFIFA 5 9

5 : Lowland Rice Subject to Alternate Periods of Flooding andDrought in Sikasso, Mali: Recurrent Selection Conducted byCIRAD-CA and IER 6 1

Results and Prospects 6 2

Chapter 3: Nomenclature for Rice Improvement by Recurrent Selection 6 3

In t roduct ion 6 3

Existing Nomenclatures 6 3

Proposed Standard Nomenclature 6 5Existing gene pools and populations 6 5New gene pools or populations 6 5Nomenclature system for a gene pool and population catalog 6 5Identifying the status of gene pools and populations 6 6

Selecting Elite Lines 6 7Gene pools or improved populations 6 7Different selection and recombination cycles (working material) 6 8

Seed Exchange and Conservation 6 8

How to Register a Population 6 8

Bibliography 7 0

vii

PrefacePrefacePrefacePrefacePreface

Plant breeding is a time-consuming process by which people manipulate plants toexpress genetic traits of interest. At the same time, those traits that are potentiallydetrimental are eliminated. Historically, the first stage of breeding was todomesticate a wild species, then, through the patient efforts of generations offarmers, select the best-performing descendants of both mutations and naturalcrosses. Breeders of the 20th century, however, have freed farmers of this task,accelerating breeding by applying more appropriate technologies.

Rice breeders, as do breeders of other autogamous species, usually apply thebasic principle of genealogical selection to descendants that almost always come fromsimple crosses. The breeders explicitly take advantage of natural or inducedmutants, whose importance was evidenced, for example, in the gene of semidwarfism.The best-performing lines so obtained tend to be used, in their turn, as progenitorsfor new crosses.

Genealogical selection is effective: countless numbers of improved varieties havebeen produced that have helped solve difficult agricultural situations. This method,however, does not allow the available genetic variability to be fully exploited, forseveral reasons. One is that genealogical selection limits the recombinations and,accordingly, the possibility of selecting favorable genes that code for traits with lowheritability.1 Another reason—related to the narrow genetic base of the progenitorssubmitted to short-term selection—is that this selection method does not use thehundreds of varieties, qualified as potential progenitors, that are stored in germplasmb a n k s .

Recurrent selection initially expands the genetic base and then enriches it withintroductions. The succession of short-selection cycles and crossings-over of selectedgenotypes increase the recombinations and, accordingly, gradually improve thepopulation, without significantly affecting the genetic variability.2 Recurrentselection, therefore, enables planning for the long term, but with responses in theshort term, first, by maintaining and enriching genetic variability; and, second, byselection, during every stage of the process, of plants for the development of fixedlines. Hence, breeding objectives—the production of fixed lines and the increasedcapacity of combination to select hybrid varieties—are reached.

Several autogamous species have been submitted to recurrent selection, forexample, oat, wheat, barley, sorghum, soybean, bean, tobacco, and cotton. Bestresults were obtained when the techniques of that selection method were adequatelyused. Rice is not listed because recurrent selection was applied for the first time tothis crop only 10 years ago, when a joint project between CIRAD andEMBRAPA-CNPAF was established in Brazil. Of the literature on the work carried outby this project, that of Veillet3 should be highlighted.

1 . Gallais, A. 1977. Amélioration des populations, méthodes de sélection et création de varietés;1: Synthèse sur les problèmes généraux et sur les bases théoriques pour la sélectionrécurrente intra-population. Ann. Amélior. Plant. (Paris) 277:281-330.

2 . Kervella, J.; Goldringer, I.; and Brabant, P. 1991. Sélection récurrente chez les autogames pourl’amélioration de varietés lignées pures: Une revue bibliographique. Agronomie 11:335-352.

viii


This manual is addressed to research teams who, to guarantee the futuredevelopment of recurrent selection, are committed to establishing recurrent selectionprograms, streamlining the methodology, developing varieties, and publishingscientific reports. Indeed, this manual is written by one such team, which works forthe CIRAD/CIAT collaboration project on recurrent selection. The exchange ofgermplasm and scientific information is a basic activity. Other teams of researchershave preceded them and other new teams will continue their work because,apparently, successful groups of breeders are also “recurrent”. I wish them all thebest in their careers.

Michel Jacquot

Head (formerly, rice breeder)Plant Breeding OfficeCIRAD

3 . Veillet, S. 1993. Organisation of the genetic variability and recurrent selection in rice (Oryza sativa L.).Thesis. Institut national d’agronomie, Paris-Grignon (INA-PG), France.

ix

AcknowledgmentsAcknowledgmentsAcknowledgmentsAcknowledgmentsAcknowledgments

We trust that this manual, “Recurrent Selection in Rice, Using the Male-Sterile Gene”,will be useful to the community of rice breeders. The manual was made possible bythe constant support that rice recurrent-selection programs received from the variousresearch institutions and colleagues who breed rice in Latin America and Africa.

For the role they played in the development of rice gene pools and the use of therecurrent selection method in their rice breeding programs, we especially want toacknowledge the Centro Nacional de Pesquisa de Arroz e Feijão (CNPAF) of theEmpresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Goiânia, Brazil; theDépartement des cultures annuelles (CA) of the Centre de coopération internationaleen recherche agronomique pour le développement (CIRAD), Montepellier, France; andthe Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia.

We are most grateful to those colleagues who reviewed the drafts and advised uson its improvement: Dr. J. Taillebois from CIRAD-CA—he developed the first basicgene pools in Brazil—and Drs. P. C. Neves, O. P. Morais, and P. H. N. Rangel fromEMBRAPA-CNPAF.

We also appreciate the readiness with which the following colleagues providedus with detailed information and documents on their ongoing rice breeding programs,using recurrent selection: Drs. N. Ahmadi, J. Enjalbert, R. Dechanet, M. Vales, andS. Veillet from CIRAD-CA in Mali, Madagascar, Côte d’Ivoire, and France, respectively;Drs. O. P. Morais, P. C. Neves, and P. H. N. Rangel from EMBRAPA-CNPAF, Brazil;Dr. R. Alvarado from the Instituto de Investigaciones Agropecuarias (INIA), Chile; andDr. C. P. Martínez from CIAT, Colombia.

Marc Châtel Elcio P. GuimarãesCIRAD-CA CIAT

x


Acronyms and AbbreviationsAcronyms and AbbreviationsAcronyms and AbbreviationsAcronyms and AbbreviationsAcronyms and Abbreviations

CNA Abbreviation of CNPAF (in germplasm nomenclature)

CNPAF Centro Nacional de Pesquisa de Arroz e Feijão of EMBRAPA

EMBRAPA Empresa Brasileira de Pesquisa Agropecuária, Brazil

FOFIFA Centre national de la recherche appliquée au développement rural,Madagasca r

IDESSA Institut des savannes, Côte d’Ivoire

IDSA Abbreviation of IDESSA (in germplasm nomenclature)

IER Institut d’économie rurale, Mali

IRAT Institut de recherches agronomiques tropicales et des culturesvivrières, France (now incorporated into CIRAD-CA). The old acronymis still used as an institutional code in germplasm nomenclature.

Ms, ms Coding of genes for male sterility

RYMV Rice yellow mottle virus

URB Upland Rice Breeders, Indonesia

URRC Upland Rice Research Consortium, Indonesia

WARDA West African Rice Development Association, Côte d’Ivoire

1

Introduction: Improving Rice Populations

IntroductionIntroductionIntroductionIntroductionIntroduction

Improving Rice PopulationsPopulation improvement is based on the evaluation of individual plants of apopulation, the selection of the best representatives of that population, and theirrecombination. This manual describes a suitable methodology for developing andmanaging rice populations—especially those that segregate for a recessive gene formale sterility.

Population improvement aims to (a) develop germplasm that gathers thegenetic variability found in several individual genotypes; (b) progressively increasethe genetic value of one or several agronomic characteristics of a given material;(c) create a genetic base to obtain fixed lines that suitably express the selected traitor traits; and (d) develop germplasm as a source of potential parents for breedingprograms.

Sarkarung (1991) described a simple technique to conduct manual crossing inrice, which, compared with traditional methods, increased the number of possiblecrosses over a given period. The technique is currently applied at severalinstitutions: at CIAT, it is used to develop improved populations through recurrentselection (Guimarães et al., 1995); and, in the breeding programs of several LatinAmerican countries, it is used to increase the number of crossings. The geneticmechanisms of male sterility in rice are similar to those found in sorghum andsoybean, thus facilitating population improvement.

This manual provides guidelines for developing, maintaining, and improvingpopulations, and selecting rice lines. It also shows how to introduce variability intorice populations that segregate for a recessive gene for male sterility.

The Developing Rice GrainThe spikelet—or flower—is the reproductive structure of rice. The male part is thestamen, which is formed by the anther and filament. The anther contains thepollen grains, which fertilize the ovary. Each spikelet has six stamens. The pistilrepresents the female part, and consists of an ovary, two styles, and two featherystigmas (Figure 1).

Anthesis is the period during which the spikelet opens, in preparation forreproductive activity. In the stage before anthesis, the filament develops within thespikelet and, just before the anthers touch the upper part of the spikelet andanthesis begins, they open and let the pollen fall onto the feathery stigmas. Rice isthus self-pollinated. The rate of allogamy (cross-pollination) is less than 1% undernormal climatic conditions.

Seed formation begins with pollination, that is, when the anther’s pollengrains fall on the stigmas. Fertilization begins when the pollen grains germinateand form pollen tubes, down which the male nuclei descend to enter the ovary andfuse with the female nuclei. This stage lasts 18 to 24 hours. Complete grainformation, that is, from fertilization to full grain, takes 25 to 30 days.

2


The panicle may contain from 50 to 250 spikelets. Flowering begins when thepanicle emerges from its sheath; the first spikelets to flower are located in theupper part of the panicle. After 5 to 7 days, the entire panicle will have flowered.Each spikelet remains open for 45 minutes or longer, and its stigmas are receptiveto pollen for 4 or 5 days. In contrast, pollen grains survive outside the anther foronly a few minutes.

The Gene for Male SterilityRice is an autogamous plant, with its female and male reproductive organs locatedin the same flower. Cross-pollination (or allogamy) occurs naturally in certainenvironments, but is never important. Consequently, to obtain a rice populationcomposed of individuals of different genotypes (i.e., recombinant), the floral biologyof rice has to be modified so that cross fertilization can occur and recombinantsobtained.

Modification can be done in two ways: first, by manual crossing in which thestamens are removed from the flowers (castration), converting them into “females”that can be fertilized with the pollen of other rice plants. The second way is tointroduce recessive genes for male sterility to make rice plants perform as ifallogamous.

Figure 1. Rice spikelet with its male and female reproductive organs.

Rachilla

Pedicel

Ovary

Sterilelemma

Awn FilamentAnther

Stamen

Palea

Sterile glumes

Rudimentary glumes

Nerves

Apicula

Stigma

StylePistil

3


The most well-known gene for male sterility in rice was discovered by Singh andIkehashi (1981) in a mutant of the irrigated rice variety IR 36, and can be found inother, already existing, male-sterile populations. The mutant IR 36 has a recessivenuclear gene, called “ms”, that, when homozygous (msms), causes pollen grain to besterile. Male-sterile plants have normal panicles, but their pollen is sterile. Pollengrains can be identified in the laboratory by their reaction to potassium iodide(Iodine®): fertile grains turn purple, whereas sterile grains do not stain (Color plate1). In the field, normal plants have full, yellow anthers; male-sterile plants, incontrast, have smaller, shriveled, whitish anthers (Color plate 2).

Recessive plants (msms) for sterile pollen produce seeds only when fertilized bypollen, containing Ms or ms haploid cells, produced by fertile plants (MsMs or Msms).As a result, a population with these recessive plants behaves as if allogamous. Tomaintain male sterility, the male-sterile mutant of the IR 36 female plant (msms) iscrossed with male-fertile (MsMs) IR 36. The hybrid seed is genetically heterozygous(Msms), and will produce fertile plants (Msms). These fertile plants are selfed—theyhave normal flowers—and produce both homozygous (MsMs and msms) andheterozygous (Msms) seed. If these seeds are sown, those of genotypes MsMs andMsms will produce fertile plants and those of genotype msms, male-sterile plants.

Plant breeders use the gene for male sterility to facilitate the development andimprovement of populations by the recurrent selection method.

ReferencesGuimarães, E.P.; Correa-Victoria, F.; and Tulande, E. 1995. GC-91, a broad-based rice

synthetic population for blast (Pyricularia grisea Sacc.) resistance. Rev. Bras. Genet.18(4):553-561.

Sarkarung, S. 1991. A simplified crossing method for rice breeding: a manual. CIAT, Cali,Colombia. 32 p.

Singh, R.J. and Ikehashi, H. 1981. Monogenic male-sterility in rice: induction,identification and inheritance. Crop Sci. 21:286-289.

4


Fertile pollen

Male-sterile pollen

Color plate 1. Fertile (purple) and sterile (unstained) pollen grains after treatment with potassiumiodide (Iodine®).

5


Fertile anther

Male-sterile anther

Color plate 2. Fertile (above) andmale-sterile (below)plants.

Male-sterile spikelet

Fertile plant

Male-sterile plant

Fertile spikelet

Planta androestértil

7

Methodologies for Managing Recurrent Selection

Chapter 1Chapter 1Chapter 1Chapter 1Chapter 1


Developing Populations of Recombinant GenotypesTo develop a recombinant population, several factors should be considered. One is thepercentage of plants with the gene for male sterility (ms) that will be maintained in thepopulation. Another is the number of recombinations that should be made beforestarting selection.

Alternatives for developing, maintaining, and selecting a population are discussedbelow. To calculate the frequency of the gene for male sterility (ms), segregation isassumed to occur independently (without linkages) and gamete segregation to beunbiased—in other words, segregation is not preferential.

A. Monocytoplasmic populationThe first step is to select the lines and makeup of the future population

(original or base population) on the basis of the objectives of each breeding program.Activity 1 describes the stages involved in developing original populations with thesingle source of cytoplasm from the IR 36 mutant.

B . Polycytoplasmic populationUnlike the previous technique, this one aims to develop polycytoplasmic

populations, the individuals of which have cytoplasm from diverse origins(IR 36 and constitutive lines used for the development of the population). Activity 2describes the stages required to develop these populations.

8


Activity 1. Developing a monocytoplasmic population.

Plant ing Field methodology

1 s t CrossingOnce the lines that will form the population are chosen, fertile plants(MsMs) are crossed individually with male-sterile plants (msms) ofvariety IR 36 or other existing populations. Each crossing will resultin heterozygous hybrid seed (Msms) that will receive, from itsmale-sterile parent, its cytoplasm and 50% of its genes. The hybridseeds (Msms) of all crosses are then mixed in similar or differentproportions, according to the expected contribution of each malepa ren t .

In some cases, and because of the genetic distance between parents(for example, Indica x Japonica crosses), the sterility obtained inseveral F1 plants does not reflect the action of the male-sterile gene,but the incompatibility between these genotypes. F1 plants shouldtherefore be observed, F2 seeds harvested separately, and then mixedin the proportions required by the breeder.

2 n d SelfingThe F1 seed mixture is planted in an isolated site, producing onlyfertile plants (Msms). These undergo selfing, and their seeds—F2—willhave the genetic constitution, MsMs, Msms, and msms, in a ratioof 1:2:1.

3 r d RecombinationThe original lines—in other words, those conforming the population—are recombined when the mixture of F2 seeds is sown. Plants willsegregate for the three genotypes mentioned. In this stage, themale-sterile plants (msms) will be fertilized at random by both Ms(75%) and ms (25%) pollen; the seeds obtained will have genotypesMsms (75%) and msms (25%).

Recombinants of the original parents exist in these seeds, but theircytoplasm comes only from the source of male sterility.

Population: This third stage can be replicated several times, until the plantbreeder considers that the population is ready for selection. Thismeans that there has been a sufficient recombination between thegenes of the original parents. The mixture of these seeds is the basicrecombined population.

Advantage: A population is obtained quickly and easily.

Disadvantages: (a) The cytoplasm of the population originates only from themale-sterile variety.

(b) Fifty percent of the genetic variability of the populationcomes from the male-sterile variety.

9


Crossing

• Selection of lines to form the base material for recombination (V1, V2, V3, ..., Vn).

• Individual crosses of these fertile plants with male-sterile plants:

• Harvest and mixture of the hybrid seed of each crossing:

Selfing

• Sowing of the mixture (fertile F1 plants):

• Harvest of F2 seed.

Recombination

• Sowing of F2 seed.

• The resulting population segregates for the gene for male sterility:

• Harvest and mixture of seeds produced by male-sterile plants (original population).

Population

• Sowing of the previous mixture:

• Population of recombinant plants obtained.

x V3 xmsms Vn...xms

ms V1 xmsms V2

MsMs

MsMs

msms

MsMs

MsMs

↓↓↓↓↓

...Ms V2ms

Ms V3ms

Ms Vnms

Ms V1ms

Ms V2ms

Ms VnmsMs V3

ms

Ms V1ms

↓↓↓↓↓

↓↓↓↓↓

msms V1

MsMs V3

msmsVn

MsMs V3

Msms

V1Msms

V2Msms V1

Msms

Vn VnMsMs

msms V2

V1Msms

Vn

Msms

Msms

msms Vn

V3Msms

V3MsMs

V2

Msms

msms

MsmsMsms

Msms

Msms

Msms

msms

msmsMsmsMsms

msms

↓↓↓↓↓

10


Activity 2. Developing a polycytoplasmic population.


1 s t CrossingAfter selecting the lines with which the population will be formed, theplants (MsMs) of these lines are crossed individually with male-sterileplants (msms) of variety IR 36 or of other already existing populations.Each cross will produce heterozygous hybrid seed (Msms), whose cellshave genes of the male-sterile parent (50%) and cytoplasm of thevariety source of male sterility. This technique differs from theprevious one in that, once the hybrid seeds are planted and fertileplants (Msms) obtained from each crossing, these are backcrossedwith the male parent, thus diversifying the cytoplasm.

2 n d BackcrossingSeeds of the F1 generation (Msms) and of recurrent parents (MsMs) areplanted. These will be used as the mother parent in each backcross.Once this round of crosses is completed, the genetic contribution ofthe source of male sterility to population variability is reduced to 25%.As a result, seeds of genotypes MsMs and Msms are produced at aratio of 1:1 in each backcrossing (F1BC).

3 r d SelfingThe F1BC seed of each cross are planted during this third stage,yielding fertile plants (MsMs and Msms) that are left to self. Thisselfing produces F2BC seed of genotypes MsMs (62.5%), Msms (25%),and msms (12.5%) (see Appendix A).

4 t h RecombinationAfter planting the F2BC seeds of each cross, a population withmale-sterile plants (msms) is obtained. These plants are pollinated atrandom, and produce seeds that represent the first occurrence ofrecombination between the original parents.

Population: The plant breeder may repeat this stage several times—providingsufficient opportunities for recombination of parental genes—until thebreeder judges that the population is ready for selection. Fujimaki(1978) recommends at least three recombinations. The seed mixtureproduced in this stage constitutes the basic recombined population.

Advantages: (a) Backcrossing allows the cytoplasm of the original parents to bemixed.

(b) The male-sterile variety usually contributes 25% of thepopulation’s genetic variability.

(c) By increasing the number of backcrosses, the geneticcontribution of the male-sterile variety to the base populationdecreases to less than 25%.

Disadvantages: (a) Four plantings are required to obtain the base population.

(b) This population will have only 14.3% of male-sterile plants(see Appendix A). Accordingly, a larger population will beneeded to increase the number of male-sterile plants.

11


Crossing and backcrossing

• Selection of lines to form the base material for recombination (V1, V2, V3, ..., Vn).

• Individual crosses of these fertile plants with male-sterile plants:

• Harvest of the hybrid seed of each cross separately:

• Backcross ing:

• Harvest and mixture of the seed from backcrossing.

Selfing

• Sowing of the seed mixture:

• A population of fertile homozygous andheterozygous plants is obtained.

• Harvest and mixture of the seedsfrom that population.

Recombination

• Sowing of the seed mixture:

• A population of plants segregating for thegene for male sterility is obtained.

• Harvest and mixture of seeds produced bymale-sterile plants (original population).

Population

• Sowing of the previous mixture:

• Population of recombinant plantsob ta ined .

x V3 xmsms Vn...xms

ms V1 xmsms V2Ms

MsMsMs

msms

MsMs

MsMs

↓↓↓↓↓

↓↓↓↓↓

↓↓↓↓↓

...Ms V2ms

Ms V3ms

Ms Vnms

Ms V1ms

↓↓↓↓↓

↓↓↓↓↓

↓↓↓↓↓

Ms V3ms

MsMs V3 x Ms

Ms Vn x Ms Vnms

...V2 xMsMs

Ms V2ms

MsMsV1 x Ms V1

ms

Ms V1ms

MsMs Vn

Ms V3ms

MsMs V2

Ms Vnms

Ms V2ms

MsMs Vn

MsMs V3

MsMs

msms

msms

MsMs

Msms

Msms

Msms

Msms

Msms

msms

Msms

MsMs

msms

Msms

MsMs

Msms

↓↓↓↓↓

↓↓↓↓↓

Msms

msms

msms

Msms

Msms

msms

msms

12


Active Maintenance of PopulationsThe populations already developed are stored in a germplasm bank until required forbreeding. If the amount of original seed stored decreases, then the seed should bemultiplied, but care must be taken not to alter the genetic constitution of the originalpopulat ion.

In this manual, the multiplication of seed from populations is referred to asactive maintenance, and is carried out with either the male-sterile or fertile plantspresent in these populations. Samples of the seeds produced in each generation ofmultiplication should be kept. After several years, the genetic variability contained inthe different seed lots should be analyzed.

Active maintenance should not be based only on fertile plants because thisquickly reduces the frequency of the ms gene. Best results are obtained by alternatingthe two methods described below:

A. Maintenance based on male-sterile plantsThe active maintenance of a population, using male-sterile plants, represents an

additional recombination cycle. Male-sterile plants (msms) are pollinated by fertileplants (Msms) to reconstitute the population (Activity 3).

Genetic drift—the loss of genes caused by seed multiplication—must be avoided.This problem is commonly caused by the difference in flowering time of the plantswithin a population. This means that only the plants of the same cycle can becross-pollinated and, when that characteristic is linked in some way to other genes,recombination will be limited. To reduce that risk, planting should be conducted ontwo or three different dates, separated by intervals of about 10 days. All themale-sterile plants will then have the same probability of being fertilized by the fertileplants of the population. For example, if a population of 3,000 plants is sown, then1,000 seeds should be planted in the same physical area on the first date, 1,000 moreon the second, and the remaining 1,000 on the third.

Care should be taken when harvesting the fertilized male-sterile plants. All seed-producing, male-sterile plants are harvested individually, and seeds from each plantare mixed in equal amounts. This condition tends to limit the total number of seedsobtained in a multiplication.

B . Maintenance based on fertile plantsAs in the previous case, and to avoid genetic drift, all fertile plants of the

population are harvested and their seed mixed (similar amount per plant). There is noneed to plant on different dates because the plants are self-pollinated (Activity 4).

Seed production from these plants is high and therefore population seedabundant. However, the seed collected from fertile plants will have a reducedfrequency of the ms gene: only 25% of the resulting population’s plants will bemale-sterile. If this process is replicated, the frequency of the msms genotypes willdecrease even more because it will be impossible to phenotypically differentiate afertile MsMs plant from a Msms plant, either during plant growth or at harvest.

13


• Original population:

• Harvest and mixture of seeds from all male-sterile plants.

• Storage of the mixture.

• Sowing of part of the stored seed (the other part is kept for further use):

• Fertile and male-sterile plants obtained.

Activity 3. Active maintenance (multiplication) of a population, usingmale-sterile plants.


O n e Multiplication with recombination only A sample of stored seed is planted. In the field, the plants will be of

two types: male-sterile (msms) and fertile (Msms). Fertile plantspollinate male-sterile plants. Seeds of these plants (Msms and msms)are mixed, and this mixture is the multiplied original population.

Advantages: (a) The frequency of the male-sterile gene (50%) is maintained.

(b) Population maintenance and recombination become a singleopera t ion .

Disadvantage: A relatively small amount of seed is produced.

↓↓↓↓↓

Msms

msms

msms

Msms

msms

Msms

Msms

msms

msms

Msms

Msms

msms

Msms

msms

Msms

msms

Msms

msms

14



• Harvest and mixture of seeds from fertile plants.

• Storage of the mixture.

• Sowing of part of the stored seed (the other part is kept for further use):

• Fertile (homozygous and heterozygous) and male-sterile plants obtained.

Activity 4. Active maintenance (multiplication) of a population, using fertilep lant s .


O n e Multiplication only Seed from the original population is planted. The plants developed

from that sample segregate as fertile heterozygous (Msms) and sterile(msms) plants. Pollen (Ms and ms) of fertile plants not only helps inthe fertilizing of male sterile plants, but also in the selfing of fertileones. Seeds harvested from self-fertilized plants are fertile (MsMs andMsms) or male sterile (msms), and represent the multiplication of theoriginal population.

Advantage: A large amount of seed is obtained from the population.

Disadvantage: The frequency of the ms gene in the population is reduced.

↓↓↓↓↓

Msms

msms

msms

Msms

msms

Msms

Msms

msms

msms

Msms

Msms

msms

Msms

msms

Msms

MsMs

Msms

MsMs

MsMs

15


Population ImprovementPopulation improvement is a medium- to long-term activity. In each cycle, thepopulation is assessed, individual plants are selected (selection units), and thebest-performing individuals are recombined (recombination units).

This selection method, known as recurrent selection, aims to increase graduallyand continuously the frequency of those genes in the population that are of interest tobreeding program objectives. Different strategies of selection can be adopted,according to trait heritability and available time and resources.

The efficiency of this method—measured, for example, in terms of genetic gainper unit of time or per selection cycle—will depend on the strategy used. In someareas of Latin America and the Caribbean, two rice crops can be grown per year.Selection should therefore be conducted during the main cropping season, andrecombination during the off season.

A. Recurrent selection, using male-sterile plantsMale-sterile plants, selected according to program objectives, constitute the selectionunits. Of these, those that perform best will later be used as recombination units.Several techniques can be used to select and recombine materials, using male-sterile(msms) plants.

1. Mass selection. The mass selection technique is based on the observation ofplant phenotypes. Therefore, all male-sterile plants of a population that express thedesired trait (or traits) will be selected, and their seed harvested separately. This seed(Msms and msms) is then mixed so that the selected individual plants are representedin equal proportions (Activity 5).

2. Plant selection with progeny evaluation. This and other techniques withwhich the progenies of male-sterile plants are evaluated present several variants of themethodology. In this manual, the following will be discussed: nonisolation of theprogenies and use of stored seed for recombination (Activity 6); nonisolation of theprogenies, without using the stored seed for recombination (Activity 7); and isolation ofthe progenies, without using the stored seed (Activity 8).

(Text continues on p. 24)

16


Activity 5. Mass selection of male-sterile plants.


1 s t SelectionA population that will produce both male-sterile (msms) and fertile(Msms) plants is planted. Of these, the best-performing male-sterile(msms) plants are selected and fertilized by fertile plants, yieldingMsms and msms seed, which form the basis of the recombinations tage .

2 n d RecombinationMsms and msms seed, harvested in the previous generation, are thensown. The male-sterile plants (msms) obtained will be fertilized by thefertile plants (Msms). Seeds of genotypes Msms (50%) and msms(50%)—the basis of the improved population—are produced, thuscompleting a selection cycle.

Advantages: (a) This methodology is simple and time-saving.

(b) It is efficient for highly heritable characteristics.

(c) The improved population will include 50% male-sterile plantsand 50% fertile plants.

Disadvantage: The methodology is rather ineffective for characteristics with lowheritabili ty.

Comments: All the selection units are recombination units, and the breeding cycleis completed in two cropping seasons.

17


msms

Msms

msms

Msms

Msms

Msms

msms

msms

• Harvest of seeds from male-sterile plants.

↓↓↓↓↓

↓↓↓↓↓

msms

Msms

msms

Msms

Msms

Msms

msms

msms

msms

Msms

msms

Msms

Msms

msms

msms

Msms

• Selection of male-sterile plants.

• Harvest and mixture of seeds from selected male-sterile plants.

↓↓↓↓↓

Selection


Recombination

• Sowing of the mixture:


• Improved recombinant population obtained.

• Improved population obtained.

18


Activity 6. Selection of male-sterile plants, evaluation of nonisolatedprogeny, and recombination from stored seed.


1 s t Selection with recombination at randomThe base population is planted, and all fertilized, male-sterile plants(msms) that express the desired trait (or traits) are selected. Eachplant is harvested separately. One part of these seeds is stored in thegermplasm bank, and the other is used to evaluate progenyperformance. These seeds will yield fertile (Msms) and male-sterile(msms) plants.

2 n d EvaluationSeed of each selected plant of the previous generation will be plantedas a line. The best-performing progenies are then selected andrecombined. Stored seed is used for this recombination. The seeds ofmale-sterile plants present in the selected (evaluated) progeniesshould not be used because these plants may have been fertilized bypollen from fertile plants of neighboring progenies discarded by theevaluator.

3 r d Recombination of selected plantsA recombination cycle is carried out with stored seed, in whichmale-sterile plants—from msms seed—will be fertilized by Ms and mspollen of fertile plants. The recombinant seed harvested forms thebasis of the improved population. A selection cycle is thus completed.

Advantages: (a) The technique is efficient for characteristics of low heritability.

(b) Progenies are not isolated.

(c) The recombinant plant population will contain 50% male-sterileplants and 50% fertile plants.

Disadvantages: (a) Evaluating each progeny is expensive.

(b) Seed storage installations are needed.

(c) The relationship between the mother plant and the progenyshould be carefully monitored.

(d) The amount of seed produced by male-sterile plants can limitthe type of tests conducted with the progeny.

Comments: In this technique, only the selection units chosen for progenyperformance are recombination units. A selection cycle involves threecropping seasons.

•

•

•

19


Recombination



• Harvest and mixture of seeds frommale-sterile plants.


• Improved recombinant populationobta ined .

msms

Msms

msms

Msms

Msms

msms

msms

Msms

• Storage of part of the seed from eachselected plant:

Seed of plant 1 ... seed of plant n

Msms

msmsMsmsmsms

msmsMsmsmsmsMsms

Msms

msmsmsms

Msms

Evaluation

• Individual sowing:

↓↓↓↓↓

Msms

Msms...Ms

msmsms

↓↓↓↓ ↓

• Mixture of stored seedfrom male-sterile plantsthat produced thebest-performing progeny(according to the previousevaluation).

Selection


msms

Msms

msms

Msms

msms

Msms

↓↓↓↓↓

↓↓↓↓↓

msms

Msms

msms

Msms

Msms

msms

Msms

msms

msms

Msms

msms

Msms

Msms

msms

Msms

msms

20


Activity 7. Selection of male-sterile plants, evaluation of their nonisolatedprogeny, and recombination from fertile plants (no seed storage).


1 s t Selection with recombination at randomThe base population is planted. All fertilized male-sterile plants(msms) expressing the desired trait (or traits) are selected, and eachplant is harvested separately. The potential of the progeny of theseplants will be evaluated. These seeds will produce 50% fertile plants(Msms) and 50% male-sterile plants (msms).

2 n d EvaluationThe lines that best fulfill breeding objectives are selected, and, fromthese, the best-performing fertile plants (Msms). Selfing in theseplants produces seed of genotypes MsMs, Msms, and msms. Thisseed, mixed in equal proportions, is the starting point of therecombination cycle.

3 r d Recombination of selected fertile plantsRecombination units are planted. Their progenies will have genotypesMsMs (25%) and Msms (50%), which are fertile, and genotype msms(25%), which is male-sterile. The seeds harvested from male-sterileplants form the basis of the improved population, thus completing theselection cycle.

Advantages: (a) This alternative is efficient for characteristics of low heritability.

(b) Two types of selection are combined: between and withinprogenies .

(c) Isolation of progenies is unnecessary.

Disadvantage: The improved population contains 33% male-sterile plants(see Appendix B).

Comments: In this alternative, recombination units consist of selected, fertileplants from the progenies chosen from selection units. A selectioncycle involves three cropping seasons.

21


Selection



• Individual harvest of seed from eachselected plant.

Evaluation


• Evaluation ofprogeny andselection of bestperformers ofprogeny and offertile plants.

• Harvest and mixture of seeds from selected fertile plants.

Recombination






msms

Msms

msms

Msms

msms

Msms

msms

Msms

Msms

msms

Msms

msms

msms

Msms

msms

Msms

Msms

msms

msms

Msms

msms

Msms

Msms

Msms

↓↓↓↓↓

↓↓↓↓↓

msms

msms

msms

Msms

msms

Msms

Msms

Msms

Msms

msms

Msms

Msms

msms

Msms

MsMs

MsMs

msms

Msms

msms

Msms

Msms

msms

msms

Msms

↓↓↓↓↓

↓↓↓↓↓

22


Activity 8. Selection of male-sterile plants, evaluation of their isolatedprogeny, and recombination from male-sterile plants (no storedseed) .


1 s t Selection and recombination at randomThe base population is planted, and all fertilized, male-sterile plants(msms) that express the desired trait (or traits) are selected. Theseplants are harvested separately, and their progenies will havegenotypes Msms and msms.

2 n d EvaluationThe seeds harvested from each plant in the previous stage are plantedin separate rows so that the individual plants of each row representthe mother plant. Each progeny is isolated from its neighbors by rowsof either maize (upland conditions) or tall, late-maturing rice varieties(irrigated conditions). Those male-sterile plants (msms) that show thebest agronomic characteristics (selection between and withinprogenies) are selected among the assessed progenies. Thesemale-sterile plants will be fertilized only by pollen of fertile plants(Ms and ms) of the same progeny. The seed thus obtained (Msms andmsms) is mixed in similar amounts to begin the recombination cycle(see Appendix C).

3 r d RecombinationHarvested seed from male-sterile plants (Msms and msms) are planted.The resulting population will have 50% fertile (Msms) and 50%male-sterile (msms) plants. Only seed from msms plants are harvestedto recombine the genotypes selected during the evaluation process.The improved population is obtained from this seed mixed in similaramounts (proportional mixture). The selection cycle is thuscompleted.

Advantages: (a) Recombination occurs in each cropping season.

(b) The technique is efficient for characteristics of low heritability.

(c) Two types of selection—between and among progeny—arecombined .

(d) The improved population contains 50% male-sterile plants.

Disadvantages: (a) Progeny must be isolated.

(b) The amount of seed produced by the male-sterile plant to yieldmsms plants is not always sufficient for recombination.

(c) At flowering, male-sterile plants should be identified within theprogeny, a process that can be highly time-consuming withnumerous progenies.

Comments: The recombination units in this technique are the male-sterile plantsselected among the progeny of the selection units. A selection cyclecovers three cropping seasons.

23


Selection



• Individual harvest of seeds from eachselected plant.

↓↓↓↓↓

msms

Msms

msms

Msms

msms

Msms

msms

Msms

msmsMsms

msmsMsms

Msmsmsms

msmsMsms

msmsMsms

MsmsMsms

Msmsmsms

Msmsmsms

↓↓↓↓↓

Recombination






• Evaluation ofprogeny, selectionof the bestperformers, andselection ofmale-sterile plants.

↓↓↓↓↓

msms

msms

Msms

Msms

Msms

msms

msms

Msms

msms

Msms

msms

Msms

Msms

msms

Msms

msms

↓↓↓↓↓

Evaluation

• Individual sowingwith isolation(maize or rice lines):

msms

Msms

msms

Msms

Msms

msms

msms

Msms

• Harvest and mixture of seeds from male-sterile plants.

24


Population Improvement (Continued from p. 15)

B . Recurrent selection, using fertile plantsFertile plants are selected according to breeding program objectives. These plants

are the selection units that will later be recombined.

1. Mass selection. Mass selection is based on observations of plant phenotype(Activity 9).

2. Plant selection with progeny evaluation. The selection method is commonto Activities 10, 11, and 12. For recombination, using male-sterile plants from theprogenies, all progeny should be isolated to prevent the pollination of male-sterileplants by pollen of fertile plants from other progenies.

Selecting Genotypes for Line DevelopmentPopulation improvement aims at developing sources of germplasm from whichbreeders can obtain, by selection, lines that express, in the best way possible, thedesired trait or traits. These genotypes, once extracted from the population, areselected by different breeding methods to obtain fixed lines, which, in turn, will beused as progenitors or will be released as varieties by national programs.

Another reason to extract lines from populations is the convenience of evaluating,at the end of each cycle of recurrency, the progress made by applying a recurrentselection strategy. Population improvement itself is not the final stage of the process,but an intermediate stage that increases the overall breeding efficiency. The finalstage is the development of fixed lines by national institutions. This objective may bemore quickly reached by using previously improved populations.

Regardless of population origin, genotype extraction will be conducted bytraditional breeding methods that are based on the use of fertile plants from theselected population or populations.


25


Activity 9. Mass selection of fertile plants.


1 s t SelectionIn this case, all fertile plants (Msms) expressing the desired trait (ortraits) are selected within the population and harvested separately.Similar amounts of seed are taken from each plant and mixedproportionately.

2nd RecombinationThe seeds in the mixture have genotypes MsMs, Msms, and msms.A recombination cycle is made from the male-sterile plants, which arefertilized by Ms or ms pollen. Then similar amounts of the seedsharvested from male-sterile plants are mixed proportionately. Thismixture will be the basis of the improved population.

Advantages: (a) This methodology is simple and time-saving.

(b) It is efficient for highly heritable characteristics.

Disadvantage: The improved population contains 33% male-sterile plants.

Comments: The recombination units are the selection units. The selection cyclecovers two cropping seasons.

Recombination






Selection


• Selection of fertile plants.

• Harvest and mixture of seeds fromselected fertile plants.

↓↓↓↓↓

↓↓↓↓↓

msms

Msms

msms

Msms

Msms

msms

Msms

msms

msms

Msms

msms

Msms

Msms

msms

Msms

msms

msms

Msms

msms

Msms

Msms

MsMs

Msms

MsMs

↓↓↓↓↓

26


Activity 10. Selection of fertile plants, evaluation of their nonisolatedprogeny, and recombination from stored seed.


1 s t SelectionSeed of the base population is planted. All fertile plants (Msms)expressing the desired trait (or traits) are selected, and their seed isharvested separately. The progenies of this seed will be evaluated.Part of the seed from each selected plant is stored.

2 n d EvaluationThe progenies (genotypes Msms, MsMs, and msms) of plants selectedfrom the base population are planted and evaluated. Those showingsuperior agronomic potential are selected. Similar amounts of seedsfrom the mother plants of each progeny—previously stored seed—are then taken and mixed proportionately.

3 r d RecombinationThe planting of this mixture initiates the recombination cycle. Theseeds harvested from male-sterile plants (msms) of this mixture formthe basis of the improved population.

Advantages: (a) This technique is simple.

(b) It is efficient for characteristics of low heritability.

Disadvantages: (a) The improved population contains 33% male-sterile plants.

(b) It requires seed storage facilities.

(c) The relationship between the mother plant and its progenymust be closely monitored.

Comments: The recombination units correspond to the selection units chosen onthe basis of the performance of their progeny. A selection cycle coversthree cropping seasons.

27


Selection



msms

Msms

msms

Msms

Msms

msms

msms

Msms

Evaluation


MsmsmsmsMsms

msms

msmsMsmsmsms

Msms

Msmsmsms

msmsMsms

Recombination



• Harvest and mixture of seedsfrom male-sterile plants.



msms

Msms

msms

Msms

Msms

MsMs

Msms

MsMs

msms

Msms

Msms

msms

• Mixture ofstored seedfrom fertileplants that hadproduced thebest -performingprogeny(according tothe evaluation).

↓↓↓↓ ↓

Seed of plant 1 ... seed of plant n

...Msms

MsMs

msms

Msms

MsMs

msms

• Evalua t iona n dselection ofthe best-performingprogeny.

MsMs

msms

Msms

MsMs

msms

Msms

MsMs

msms

Msms

• Individual harvest of seeds fromeach selected plant.

• Part of the seed from each selectedplant is stored.

msms

Msms

Msms

msms

↓↓↓↓↓

28


Activity 11. Selection of fertile plants, evaluation of their nonisolatedprogeny, and recombination from fertile plants (no seed storage).


1 s t Selection (see Activity 10).

2 n d EvaluationThe progeny (genotypes Msms, MsMs, and msms) of fertile plantsselected from the base population are planted. Those showingsuperior agronomic potential are selected, and the seeds from eachfertile plant are then harvested. A mixture is made with similaramounts of seed from each.

3 r d RecombinationThis seed mixture constitutes the basis of the recombination cycle.The seeds produced by the male-sterile (msms) plants are harvested,and a mixture is made with similar amounts of seed per plant. Thisnew mixture is the basis of the improved recombined population.

Advantages: (a) This methodology is efficient for characteristics of lowheritability.

(b) Two types of selection—between and within progeny—arecombined .

(c) No isolation of progeny is required.

Disadvantage: The improved population includes 33% male-sterile plants.

Comments: The recombination units include superior-performing fertile plants,selected among the progeny of the selection units. A selection cyclecovers three cropping seasons.

29


Selection



• Individual harvest of seeds from eachselected plant.

Evaluation


Recombination


msms

Msms

msms

Msms

Msms

MsMs

Msms

MsMs




msms

Msms

msms

Msms

Msms

msms

Msms

msms


↓↓↓↓↓

↓↓↓↓↓• Mixture of seeds from selected fertile plants.

• Evaluation ofprogeny andselection offertile plants(homozygousand heterozygous).

Msms

msms

MsmsMsMs

Msms

msms

MsMs

Msms

Msms

MsMs

msms

Msms

msms

Msms

MsMs

msms

MsMs

MsMs

Msms

msmsMsms

msms

Msms

msms

Msms

Msms

msms

Msms

msms

30


Activity 12. Selection of fertile plants, evaluation of their isolated progeny,and recombination from male-sterile plants (no seed storage).


1 s t Selection (see Activity 10).

2 n d EvaluationThe progenies of fertile plants (Msms) selected from the basepopulation are planted. In this planting, each progeny should beisolated from its neighbors by rows of either maize or tall,late-maturing rice varieties. Those with superior agronomic potentialare then selected, and the seed produced by male-sterile plantsharvested. A similar amount of seed per plant is mixedproportionately, forming the improved population.

3 r d RecombinationSeeds of the previous mixture are planted. Recombination occurswhen the male-sterile plants of this population are pollinated. Theseeds harvested from these plants form the basis of the improvedrecombined population.

Advantages: (a) Recombination occurs during each cropping season.

(b) The methodology is efficient for characteristics of lowheritabili ty.

(c) Two types of selection occur: between and within progenies.

(d) The improved population has 50% male-sterile plants.

Disadvantages: (a) All progeny must be isolated.

(b) The amount of seed produced by the male-sterile plants mustbe sufficient to yield several msms plants in the progeny forrecombinat ion .

(c) At flowering, male-sterile plants must be identified in theprogeny—a most time-consuming task when dealing withnumerous progenies.

Comments: Recombination units are the seed harvested from male-sterile plantsselected from superior-performing progenies. A selection cycle coversthree cropping seasons.

31


Selection


msms

Msms

msms

Msms

Msms

msms

msms

Msms



• Individual harvest of seeds fromeach selected plant.

↓↓↓↓↓

Evaluation

• Individual sowingwith isolation (maize orrice lines):

• Evaluation ofprogeny, selectionof the bestperformers andof male-sterile plants.

• Mixture of seeds from male-sterile plants selected from the selected progeny.

• Harvest and mixture of seedsfrom male-sterile plants.

Recombination


msms

Msms

msms

Msms

Msms

msms

Msms

msms


↓↓↓↓↓


msms

msms

Msms

msms

Msms

Msms

Msms

msms

Msmsmsms

MsmsMsMs

Msms

msms

MsMs

Msms

msms

MsMs

Msms

MsMs

MsmsMsMs

msmsMsms

msms

MsmsMsMs

Msms

msms

32


Selecting Genotypes for Line Development (Continued from p. 24)

A. Extraction by the pedigree method1. From populations maintained by harvesting male-sterile plants

(Activity 13a). This population is composed of fertile (Msms) and sterile (msms) plants.

2. From populations maintained by harvesting fertile plants (Activity 13b).These populations contain 25% male-sterile (msms) plants and 75% fertile plants.Of these, one-third are homozygous (MsMs) and two-thirds heterozygous (Msms).If fertile plants of genotype Msms are selected for extraction, the same type ofsegregation that occurs in Activity 13a will appear. If MsMs plants are selected, theirprogeny will not segregate. The main difference between Activities 13a and 13b is thepresence or absence of male-sterile plants in the progenies of the fertile plants inActivity 13a.


Activity 13a. Extraction of lines, using the pedigree method, from apopulation maintained with male-sterile plants.


1 s t Selection 1The population will have 50% male-sterile plants (msms) and 50%fertile plants (Msms). Fertile plants expressing the desired trait ortraits are then selected, and seeds from each plant are harvestedseparately. Of these seeds, some will have fertile genotypes, bothhomozygous (MsMs) and heterozygous (Msms), and others will bemale-sterile genotypes (msms).

2 n d Selection 2Seed from each selected plant is planted. Progenies of this materialwill segregate for male sterility because they derive from selfed,heterozygous (Msms), fertile plants. The best-performing progeniesare selected and, from these, those fertile plants showing superioragronomic potential. Each of these selected plants is then harvestedseparately.

Comments: Selection should continue on the basis of fertile plants (MsMs andMsms), which will either segregate or not, depending on theirgenotype. If the process is to be continued in segregating (fertile andmale-sterile plants) lines, the same type of segregation will occur insubsequent generations. Male-sterile plants will not reappear intotally fertile lines.

33


msms

Msms

msms

Msms

Msms

msms

msms

Msms

↓↓↓↓↓

MsMs

Msms

MsMs

MsMs

MsMs

msms


MsMs

msms

Msms

MsMs

MsMs

MsMs

• Evaluation of progeny:

Progeny from heterozygous fertile plants produces fertileand male-sterile plants.

msms

Msms

msms

Msms

msms

Msms

msms

Msms

MsMs

MsMs

↓↓↓↓↓ ↓↓↓↓↓

Progeny fromhomozygous fertileplants producesonly fertile plants.

↓↓↓↓↓

↓↓↓↓↓

Selection continues until completely fertile,fixed lines are obtained.

... ...

... ...

• Original population, maintained with male-sterile plants:

• Selection of fertile plants, all heterozygous.


↓↓↓↓↓

↓↓↓↓↓↓↓↓↓↓


Selection of the best-performing progeny and selection offertile plants (heterozygous or homozygous).

...

Msms

MsMs

msms

msms

Msms

msms

Msms

MsMs

msms

Msms

...

MsMs

Msms

msms

MsMs

msms

Msms

↓↓↓↓↓

34


Activity 13b. Extraction of lines, using the pedigree method, from apopulation maintained with fertile plants.


1 s t Selection 1The population will have 75% fertile plants (MsMs and Msms) and 25%male-sterile plants (msms). Fertile plants expressing the desired traitor traits are then selected, and seeds from each plant are harvestedseparately. Of these seeds, some will have fertile genotypes, bothhomozygous (MsMs) and heterozygous (Msms), and others will bemale-sterile (msms).

2 n d Selection 2Seed from each selected plant is planted. Progenies of this materialwill segregate for male sterility because they derive from selfed,heterozygous (Msms), fertile plants. The best-performing progeniesare selected and, from these, those fertile plants showing superioragronomic potential. Each of these selected plants is then harvestedseparately.

Comments: Selection should continue on the basis of fertile plants (MsMs andMsms), which will either segregate or not, depending on theirgenotype. If the process is to be continued in segregating (fertile andmale-sterile plants) lines, the same type of segregation will occur insubsequent generations. Male-sterile plants will not reappear intotally fertile lines.

35


• Original population, maintained with fertile plants:

msms

Msms

msms

Msms

Msms

MsMs

MsMs

Msms

MsMs

Msms

MsMs

MsMs

MsMs

msms


MsMs

msms

Msms

↓↓↓↓↓

MsMs

MsMs

MsMs


Progeny from heterozygous fertile plants produces fertileand male-sterile plants.

msms

Msms

msms

Msms

msms

Msms

msms

Msms

MsMs

MsMs

↓↓↓↓↓ ↓↓↓↓↓

↓↓↓↓↓

↓↓↓↓↓

... ...

... ...

• Selection of fertile plants, both homozygous and heterozygous.


↓↓↓↓↓↓↓↓↓↓


Msms

MsMs

msms

msms

Msms

msms

Msms

MsMs

msms

Msms

MsMs

MsMs

MsMs

Selection of the best-performing progeny and selectionof fertile plants (heterozygous or homozygous).

↓↓↓↓↓

↓↓↓↓↓

Selection continues until completely fertile,fixed lines are obtained.

... ...

Progeny fromhomozygous fertileplants producesonly fertile plants.

MsMs

Msms

MsMs

MsMs

MsMs

msms

MsMs

msms

Msms

... ...

↓↓↓↓↓

36


Selecting Genotypes for Line Development (Continued from p. 32)

B . Extraction by anther cultureThis method reduces the time spent in developing fixed lines. When used in

populations that segregate for the gene for male sterility, certain problems arise thatwould not occur in germplasm that does not involve such a gene.

The populations consist of male-sterile (msms) and fertile plants. The latter areeither both heterozygous (Msms) and homozygous (MsMs) or only heterozygous (Msms),if the population was maintained by harvesting male-sterile plants. Because the male-sterile plants cannot be identified before flowering, the laboratory will therefore behandling anthers that will not form calli because of their genetic origin.

To avoid anther processing male-sterile plants, these plants should be identifiedwithin the population at flowering of the first panicles and then discarded. Only plantsidentified as fertile should be used.

To minimize the drawbacks caused by the presence of male-sterile plants,populations maintained by harvesting fertile plants should be preferred because thefrequency of male-sterile plants within these populations is only 25%.

The fertile lines obtained by anther culture are then submitted to selection,following the normal procedure of the breeding program, whether for yield evaluation,crossing, or other program objectives.

37


Introducing New Variability into PopulationsPopulation improvement is a dynamic process that combines the selection of individualplants and the recombination of the superior-performing ones. This dynamism can befurther intensified by introducing new genotypes, which will either broaden the geneticbase of the target population or improve it by introducing one, or several, traits that itpreviously did not have. In some cases, both effects are achieved.

New variability is introduced into the population by crossing male-sterile (msms)from the population and fertile (MsMs) plants of those lines that the breeder wishes tointroduce as a source of variability into the population. These crossings can bedirected or done at random. The contribution of each progenitor is controlled in thefirst case, and estimated in the second.

When developing a new population, with either directed (Activities 14 and 15) orrandomized crossings (Activities 16 and 17), crossings could be evaluated before theplanting in which recombination will occur.

38


Activity 14. Introducing new variability by directed crossing, withoutevaluating the F1 generation, but using a mixture of hybrid seed.


1 s t CrossingEach line (MsMs) to be introduced as a source of variability is crossedwith several male-sterile plants (msms) of the population. Thesecrosses will produce hybrid seed (Msms).

2 n d SelfingHybrid seed (Msms) is mixed, either in similar amounts or inproportions equivalent to the desired contribution of every newprogenitor. Thus begins the recombination cycle of the sources ofvariability with the original population. Seed of all plants in the F1generation will be harvested in bulk to form the F2 population. FertileF1 plants (Msms) are selfed and produce F2 seeds that are bothmale-sterile (25%, msms) and fertile (25% MsMs and 50% Msms).

3 r d RecombinationIn the F2 population, male-sterile plants (msms) fertilized by pollen offertile plants will be harvested. The mixture of these seeds willconstitute the new population, which will have both male-sterile(msms) and fertile (Msms) plants.

Comments: Of the variability present in the new population, 50% derives from thegermplasm with the gene for male sterility, and the other 50% fromintroduced lines. Three cropping seasons are required to introducenew variability into a population by this method.

39


Crossing


msms

Msms

msms

Msms

Msms

msms

msms

Msms

↓↓↓↓↓

x V3msms

MsMs

• Harvest and mixture of hybrid seeds from all crosses:

xmsms V2

MsMs xms

ms V4MsMsxms

ms V1MsMs

• Individual crosses between male-sterile plants of the population and four sourcesof variability (V1 to V4):

↓↓↓↓↓

Ms V2ms

Ms V3ms

Ms V1ms

Ms V4ms


• Population of fertile F1 plants obtained.Msms

Msms

• Harvest and mixture of F2 seeds in bulk.

V1 V2

↓↓↓↓↓Msms Ms

ms

V3V4

Recombination


Msms

V3MsMsV1

MsMs

V4MsMs ms

ms V3

V2MsMs

msms V2

V2Msms

msms V1

V1MsmsV3

Msms

msms V4

• Population of F2 plants obtained.


• New population obtained:

- 50% of original population.

- 50% of new sources of variability(V1, V2, V3, and V4).

New population


msms

Msms

msms

Msms

msms

msms

Msms

V4

Msms

↓↓↓↓↓


40


Activity 15. Introducing new variability by directed crossing, using individualcrossing and selection in each F1 generation.


1 s t CrossingEach line (MsMs) to be introduced as a source of variability will becrossed with several male-sterile plants (msms) of the population.These crosses will produce hybrid seed (Msms).

2 n d Selection and selfingThe hybrid seed (Msms) of each cross is planted separately. Theprogeny of each combination (or cross) is assessed and F1 plants ofsuperior performance are selected. Selected fertile plants (Msms) ofthe F1 generation are left to self. The resulting seed constitutes the F2generation and, of these, 25% are male-sterile (msms) and 75% fertile(25% MsMs and 50% Msms).

3 r d RecombinationThe seed of the F2 generation is planted in such a way that (a) allcrosses are coupled, (b) the crosses are grouped according to thebreeder’s own objectives, or (c) the seed of each cross is mixed in equalproportions. Of the F2 generation plants, only the seeds produced bymale-sterile plants will be harvested. The mixture of this seed willform the new population.

Comments: In the new population, 50% of the variability comes from thegermplasm containing the gene for male sterility and the remaining50% from introduced lines after their evaluation in the F1. Threecropping seasons are required to introduce new variability into apopulation by this method.

41


msms

Msms

msms

Msms

Msms

msms

msms

Msms

↓↓↓↓↓

• Individual harvest of hybrid seeds from each cross.

x V2MsMs

msms

• Individual crosses between male-sterile plants of the population and four sourcesof variability (V1 to V4):

x V3msms

MsMs xms

ms V4MsMsx V1

MsMs

msms

New population


msms

Msms

msms

Msms

Msms

Msms

msms

msms

Msms V4

MsMs

MsMs

Msms

msms V2

V4

MsMs

Msms

V2

msms V1 ms

ms V4

Recombination


• Population of F2 plants obtained.

• Harvest of seeds from male-sterile plants.V2

V1V1

Crossing


Evaluation

• Sowing and evaluation of each F1:

↓↓↓↓↓

• Harvest and mixture of F2 seeds from each selected F1.

Ms V2ms

Ms V3ms

Ms V4ms

Ms V1ms


• New population obtained:

- 50% of original population.

- 50% of new sources of variability(V1, V2, V3, and V4).

42


Activity 16. Introducing new variability by completely randomized crossing,using seed mixtures.


O n e Crossing and recombination only Seeds from lines that are sources of variability (MsMs) are mixed

physically—either in equal or different proportions—with the seedsfrom the male-sterile population. The mixture is then planted, andthe seeds produced by male-sterile plants harvested. These plants(msms) are fertilized by Ms pollen of fertile plants of introducedvarieties and also by pollen (Ms and ms) of the population in which thenew variability is to be introduced. The seeds harvested frommale-sterile plants will have, accordingly, genotypes Msms and msms.

Comments: The method aims to develop a population with new lines in which50% of the genes are from the population that provided the gene formale sterility and the other 50% from the mixture of the varietiesused as sources of variability. But this goal is not achieved becausethe contribution of the sources of variability is less than 50%(see Appendix D).

43


msms

Msms

msms

Msms

Msms

msms

msms

Msms

Crossing


↓↓↓↓↓• Physical mixture of seeds from the population and from sources of variability

(V1 to V4):

Recombination


MsMs V3 Ms

ms

Msms

msms

MsMs V1 Ms

Ms V2

MsMs V4

msms

msms

msms

Msms

Msms

• Harvest and mixture of seeds produced by male-sterile plants.

New population


• New population obtained.

msms

Msms

msms

Msms

Msms

Msms

msms

msms

44


Activity 17. Introducing new variability by directed individual mixtures ofseed, using a crossing block technique.


1 s t CrossingSeeds from each source of variability (V1 to Vn) are mixed with seedsfrom the population containing the gene for male sterility (individualmixtures). Each mixture is planted in plots or “crossing blocks”,separated from each other by barriers of either maize or tall,late-maturing rice varieties.

Randomized crosses occur within each block, in which male-sterileplants (msms) of the population are pollinated by Ms pollen of fertileplants (MsMs) of the sources of variability, and Ms and ms pollen offertile plants (Msms) of the population. The seeds produced bymale-sterile plants are harvested in each plot. This methodologyimproves the control of the genetic contribution of each source ofvariability.

2 n d RecombinationSeeds from the male-sterile plants of each directed cross are mixed inequal or different amounts and then planted.

Male-sterile plants (msms) of this population will be fertilized by fertileplants (Msms) from the different crosses. The seeds produced in themsms plants are harvested and mixed; this mixture will be the basis ofthe new population.

Comments: The contribution of each variety used as a source of variability shouldbe estimated (see Appendix D).

The technique was developed by J. Taillebois, CIRAD-CA, FrenchGuiana, to produce F1 hybrid rice seed.

45


msms

V1Msms

Msms

msms

V2Msms

Msms

msms

V3Msms

Msms

msms

V4Msms

Msms

New population


msms

• New population obtained.

Recombination


Msms

msms

Msms

Msms

msms

Msms

msms

Msms

Msms

Msms

msms

Msms


V2 V3

V1 V4

• Harvest and mixture of seeds from male-sterile plants of each cross:

Crossing

• Seeds from each source of variability are mixed separately with seeds from theoriginal population:

msms

MsMs V4

Msms

msms

MsMs V3

Msms

msms

MsMs V2

Msms

msms

MsMs V1

Msms

Msms

Msms

msms

Msms

Msms

msms

Msms

Msms

msms

Msms

Msms

46


Literature CitedFujimaki, H. 1978. Recurrent selection by using genetic male sterility for rice improvement.

Jpn. Agric. Res. Q. 13:153-156.

Recent PublicationsAhmadi, N. and Diaby, M. 1993. Amélioration variétale pour la riziculture innondée: Rapport

1993. CIRAD-CA and Institut d’économie rurale (IER, Mali), Montpellier, France. 25 p.

Bueno M., J.M. 1994. Evaluación del progreso genético por resistencia a Pyricularia grisea Sacc.en un ciclo de selección recurrente en la población de arroz Oryza sativa GC-91. Thesis(Ing. Agrón.). Facultad de Ciencias Agropecuarias, Universidad Nacional, Palmira,Colombia. 80 p.

Châtel, M. and Guimarães, E.P. 1994. Review of the present status and proposals of ricegermplasm enhancement for Latin America and the Caribbean, using recurrent selection.In: Proceedings of the CIRAD-CA First International Upland Rice Breeders Workshop,September 1994. CIRAD-CA, Montpellier, France. 10 p.

__________ and __________. 1996. Catalogue registration for rice gene pools and populations,1996 issue. CIRAD-CA and CIAT, Cali, Colombia.

__________; __________; Ospino, Y.; and Borrero, J. 1994. Upland rice improvement in recessivemale-sterile gene pool and populations. CIRAD-CA/CIAT rice special project for1993-1994. CIAT, Cali, Colombia. 30 p.

CIRAD-CA and CIAT. 1996. Upland rice improvement: using gene pools and populations withrecessive male-sterile gene. The 1994-1995 CIRAD-CA/CIAT Rice Project report. Cali,Colombia. 31 p.

Enjalbert, J. 1993. La sélection récurrante sur le riz d’altitude a Madagascar; rapport decampagne 1992-1993. CIRAD-CA and Centre national de la recherche appliquée audéveloppement rural (FOFIFA, Madagascar), Montpellier, France. 21 p.

__________. 1994. Sélection récurrante; rapport de campagne 1993-1994. CIRAD-CA and Centrenational de la recherche appliquée au développement (FOFIFA, Madagascar), Montpellier,France. 23 p.

Marín G., J.M. 1994. Efecto del número de ciclos de recombinación en la variabilidad genética depoblaciones de arroz (Oryza sativa L.). Thesis (Ing. Agrón.). Facultad de CienciasAgropecuarias, Universidad Nacional, Palmira, Colombia. 50 p.

Rangel, P.H.N. 1992. La selección recurrente mejora el arroz brasileño. Arroz en las Américas13(1):4-5.

__________; Neves, P.C.; and Morais, O.P. 1992. La selección recurrente recombina genes en elarroz de riego. Arroz en las Américas 13(3):2-4.

__________; Zimmermann, F.; and Neves, P.C. n.d. Aumento do potencial produtivo do arrozirrigado através de seleção recorrente. Rev. Bras. Genet. (In press.)

Vales, M. 1992. Recurrent selection for partial rice blast resistance in Côte d’Ivoire. In: Biologiede la reproduction et amélioration des plantes; rapport du 13ème Congrés Eucarpia,juillet 1992, Angers, France.

__________. 1994. Pathologie du riz; rapport de synthèse 1986-1994. CIRAD-CA and Institut dessavannes (IDESSA, Côte d’Ivoire), Montpellier, France.

Veillet, S. 1993. Organization of the genetic variability and recurrent selection in rice Oryzasativa L. Ph.D. dissertation. Institut national agronomique, Paris-Grignon, France.

47


Conference Posters, March 1994The following posters were presented at the IX International Rice Conference for LatinAmerica and the Caribbean, held in Goiânia, Brazil, in March 1994:

Châtel, M.; Taillebois, J.; Ahmadi, N.; and Dechanet, R. Rice recurrent selection at CIRAD-CA.

__________; Guimarães, E.P.; and Huertas, C. Rice germplasm enhancement for Latin Americaand the Caribbean (LAC).

Cordeiro, A.C. and Rangel, P.H.N. Ensaios de avaliação de familias S2 de arroz irrigado emRoraima (Brasil).

Filippi, M.C.; Neves, P.C.; Notteghem, J.L.; and Prabhu, A.S. Recurrent selection for partialresistance to leaf blast in upland rice.

Guimarães, E.P.; Correa-Victoria, F.; and Tulande, E. Selección recurrente, metodología paradesarrollar resistencia al añublo del arroz.

Morais, O.P.; Silva, J.C.; Cruz, G.D.; Regazzi, A.J.; and Neves, P.C. Parâmetros genéticos dapopulação de arroz irrigado, CNA-IRAT 4/0/3.

__________; __________; __________; __________; and __________. Análise multivariada dadivergência genética dos progenitores da população de arroz irrigado, CNA-IRAT 4/0/3.

Rodrigues, R.E.; Rangel, P.H.N.; and Zimmermann, F. Ganhos de seleção numa população dearroz irrigado oriunda de intercruzamientos usando macho-esterilidad genética.

Veillet, S.; Châtel, M.; Filippi, M.C.; and Neves, P.C. Index selection for yield and partial blastresistance in upland rice recurrent selection.

Papers Presented at Workshops Held in 1996Châtel, M. Recurrent selection populations. At: The Upland Rice Research Consortium (URRC)

Workshop, Padang, West Sumatra, Indonesia, 4-13 January 1996.

__________ and Guimarães, E.P. Nomenclature system for managing gene pools and populations.At: The second International Workshop of Upland Rice Breeders (URB), Padang, WestSumatra, Indonesia, 4-13 January 1996.

__________ and __________. Catalog registration for gene pools and populations. At: Thesecond International Workshop of Upland Rice Breeders (URB), Padang, WestSumatra, Indonesia, 4-13 January 1996.

__________ and __________. Rice recurrent selection: review of the present status and progress.At: The second International Workshop of Upland Rice Breeders (URB), Padang, WestSumatra, Indonesia, 4-13 January 1996.

__________; __________; Correa, F.; Ospina, Y.; Borrero, J.; and Tulande, E. Mejoramientopoblacional para piricularia y suelos ácidos. At: The Taller Regional de Agrociencia yTecnología Siglo XXI, Orinoquia Colombiana, Villavicencio, Meta, 13-15 November 1996.

48


Appendix A. Calculating the frequency of male-sterile plants in apolycytoplasmic population segregating for the msgene for male sterility

Cross:Fertile line X IR 36 (male sterile)

MsMs m s m sF1 ---> M s m s

Backcross:Fertile line X F 1

MsMs M s m sF1BC ----> MsMs Msms

( 1 / 2 ) ( 1 / 2 )5 0 % 5 0 %

Selfing:MsMs (1/2) ----------> MsMs (1/1)Msms (1/2) ----------> MsMs (1/4)

----------> Msms (1/2)----------> msms (1/4)

F2BC ----> MsMs (1/2) x (1/1) + (1/2) x (1/4) = (1/2) + (1/8) = 5/8 = 62.5%

Msms (1/2) x (1/2) = 1/4 = 2/8 = 25.0%

msms (1/2) x (1/4) = 1/8 = 12.5%

Percentages in “Backcross” and “Selfing” indicate the proportion of genotypes in thepopula t ion .

Recombination:The total number of fertile plants (MsMs and Msms) produced by selfing represents87.5% of that population (62.5% + 25.0%). These plants will fertilize the male-sterileplants, which account for 12.5% of the same population.

Of the self-fertilized fertile plants, 87.5% become 100% fertile plants, supplying pollenfor male-sterile plants. Plants of genotype MsMs (that were 62.5% of the self-fertilizedpopulation) therefore represent 71.4% of the new population (62.5% ÷ 87.5%); plantsof genotype Msms now represent 28.6% of the new population (25.0% ÷ 87.5%).

(a) Male-sterile plants X Fertile plantsm s m s MsMs

(71.4%)

Pollen: ms = 100.0% Pollen: Ms = 35.7%Ms = 35.7%

(b) Male-sterile plants X Fertile plantsm s m s M s m s

(28.6%)

Pollen: ms = 100.0% Pollen: Ms = 14.3%ms = 14.3%

Only ms pollen of the fertile Msms plants will produce (in male-sterile plants) 14.3%of seeds of the msms genotype. Ms pollen of fertile plants, both homozygous (MsMs, 71.4%)and heterozygous (Msms, 14.3%) will produce in male-sterile plants 83.7% of seeds of theMsms genotype.

49


Appendix B. Calculating the frequency of male-sterile plants in apopulation segregating for the ms gene for malesterility: selection of male-sterile plants, evaluationof their nonisolated progeny, and recombinationfrom fertile plants

Population:Male-sterile plants Fertile plants

m s m s M s m s

Selection of sterile plants and harvest of their seed:M s m s m s m s( 1 / 2 ) ( 1 / 2 )

Evaluation:The progeny of each sterile plant selected is evaluated.

Selfing:Selection of fertile plants of the best-performing lines; plants are self-fertilized:

M s m s X M s m s

F1 --------> M s M s M s m s m s m s( 1 / 4 ) ( 1 / 2 ) ( 1 / 4 )

The seed of fertile plants is mixed and planted.

Recombination:MsMs (1/4) ---------> Pollen: Ms (1/4) = 25.0%

Msms (1/2) ---------> Pollen: Ms (1/4) = 25.0%---------> ms (1/4) = 25.0%

The male-sterile (msms) plants do not produce fertile pollen. Accordingly, only75.0% of the self-fertilized plants (50% Msms and 25% MsMs) will produce pollencapable of fertilizing male-sterile plants. In this new population (100%) of fertileplants, the proportion of Ms pollen produced by plants of MsMs genotype changes to33.3% (25% ÷ 75%); the proportion of Ms pollen and that of ms pollen, both producedby plants of the Msms genotype, have the same value (25% ÷ 75%):

MsMs (1/4) ---------> Pollen: Ms = 33.3%

Msms (1/2) ---------> Pollen: Ms = 33.3%---------> ms = 33.3%

Only this ms pollen, produced by the fertile plants (Msms), will produce—in themale-sterile plants it fertilizes—seeds of the msms genotype that will yield male-sterileplants. These therefore represent 33.3% of the population of recombined plants.

Ms pollen from the fertile MsMs (33.3%) and Msms (33.3%) plants willproduce—in the male-sterile plants it fertilizes—fertile seeds that will yield fertileplants. These represent, accordingly, 66.6% of the population of recombined plants.

50


Appendix C. Ideal method of mixing seeds of selected progeniessegregating for the ms gene for male sterility

(a) The progenies (or descendants) A, B, C, and D are planted, isolated one fromanother by furrows of maize or tall rice varieties:

Isolation

A1---msms *- -Msms---msms---Msms---Msms---Msms---

Progeny A---Msms---Msms---msms*- -Msms---msms---Msms---

A2

Isolation

B1---msms *- -Msms---msms---Msms---Msms---Msms---

Progeny B---Msms---Msms---Msms---Msms---msms*--Msms---

B2

Isolation

---msms---Msms---msms---Msms---Msms---Msms---Progeny C(discarded)

---Msms---Msms---Msms---Msms---msms---Msms---

Isolation

---Msms---Msms---msms---Msms---msms---Msms---

Progeny D---Msms---msms *--Msms---Msms---msms *--Msms---

D1 D2

Isolation

* A1, A2, B1, ... are plants selected within every progeny.

51


(b) Number of seeds produced by each selected plant:

A1 = 55 B1 = 80 D1 = 25A2 = 50 B2 = 30 D2 = 35

(c) Mixture of seed of selected plants in each progeny:

(i) Balanced mixture. In each progeny, the same number of seeds aretaken from each selected plant. In the following example, we use theentire production of the plants yielding the smallest number of seeds(A2 50, B2 30, and D2 25) to make the mixture:

progeny A = 1 0 0 seeds (50 A1 and 50 A2)progeny B = 6 0 seeds (30 B1 and 30 B2)progeny D = 5 0 seeds (25 D1 and 25 D2)

(ii) Unbalanced mixture. In each progeny, a different number of seeds istaken from each selected plant, according to the breeder’s criterion:

progeny A = 5 0 seeds (20 A1 and 30 A2)progeny B = 9 0 seeds (60 B1 and 30 B2)progeny D = 6 0 seeds (25 D1 and 35 D2)

(d) Seed mixture of all progenies:

(i) Balanced final mixture. The same number of seeds are taken, perprogeny, of the previous balanced mixture (c[i]). The followingexample is based on progeny yielding the lowest number of seeds(progeny D, 50):

progeny A: 50 s e e d sprogeny B: 50 s e e d sprogeny D: 5 0 s e e d s

Total : 1 5 0 s e e d s

(ii) Unbalanced final mixture. A different number of seeds are taken, perprogeny, of the mixture already made in each progeny (c[i] or c[ii]),according to the breeder’s criterion:

progeny A: 45 s e e d sprogeny B: 85 s e e d sprogeny D: 45 s e e d s

Total : 1 7 5 s e e d s

52


Appendix D. Calculating the contribution of each source ofvariability in a completely randomized crossing,using seed mixtures with a male-sterile population

If we assume that four varieties are sources of variability and that the initial mixturecontains 4,000 plants, then half of the population of new lines will correspond to theoriginal population and half to the four sources.

The original population contains 1,000 male-sterile msms plants (referred to asS) and 1,000 fertile Msms plants (referred to as F). There are also 500 plants ofsource variety number 1 (abbreviated as V1), 500 of variety 2 (V2), 500 of variety 3(V3), and 500 of variety 4 (V4), thus reaching a total of 4,000 plants.

The 1,000 S plants of this population will receive pollen from the 1,000 F andthe 2,000 plants of source varieties. All the pollen (100%) comes from fertile plants.The F plants therefore contribute 33.3% (1,000 male plants of a total of 3,000) andthe source varieties (V1 to V4) with 66.6% (2,000 male plants of a total of 3,000).

The breeder should not forget that, at pollination, fertile (male) plants contribute50% of the genetic base of the crossings and male-sterile (female) plants theremaining 50%.

Consequently, 50% of the genes of the resulting population will belong tomale-sterile plants; 16.7% to fertile plants of the original population (i.e., 33.3% of thetotal pollen multiplied by 50% of masculine gene contribution); and 33.2% to the fourvarieties that are sources of variability, each variety contributing 8.3%.

53

Using Recurrent Selection: Five Case Studies



Several national programs and international institutes are using, in theirrice-breeding programs, the recurrent selection method to develop and improve basicpopulations and to develop lines. How recurrent selection is used to improve irrigatedand upland rice is described below for Brazil, Colombia, Côte d’Ivoire, Madagascar, andMali.

Case 1: Irrigated Rice in Goiânia, Brazil: RecurrentSelection Conducted by EMBRAPA-CNPAF

Paulo H. N. Rangel

Work was based on the CNA-IRAT 4/0/5 gene pool, which had been developed by a5-year collaborative project between EMBRAPA-CNPAF and CIRAD-CA (formerly IRAT)that began in 1984. By introgressing improved lines or selecting from the existinggene pool, the following populations were developed:

CNA 1: by introgressing three lines (CT 7363-5-3-10-M, CT 6910-2-3-3-M,and Bluebelle) into the CNA-IRAT 4/0/5 gene pool.

CNA 5: by introgressing nine lines (Metica 1, IRGA-409, CICA 8, De Abril,Paga Divida, Quebra Cacho, Brejeiro, IR 1342, and Basmati 370) intothe CNA 1 population.

CNA-IRAT 4PR: by selecting and recombining early maturing, fertile plantsselected from S2 lines of the CNA-IRAT 4/1/1 population.

CNA-IRAT 4ME: by selecting and recombining intermediate maturing, fertileplants selected from S2 lines of the CNA-IRAT 4/1/1 population.

CNA 2M and CNA 2R: by introgressing maintainer or restorer lines for the WAmale-sterile cytoplasm in the CNA-IRAT 4 gene pool.

The Brazilian National Rice Program aims to improve the grain yield and diseaseresistance of rice germplasm. Research is based on effective collaboration among stateresearch institutions participating in the project. The materials developed from thedifferent populations are assessed as S2 families; those considered superior byparticipating institutions are recombined.

Each participating institution has a defined responsibility: EMBRAPA-CNPAF isresponsible for developing and recombining selected S2 families, whereas stateinstitutions are responsible for the multilocational evaluations that indicate whichmaterials are the most appropriate for a given state. These materials will not only formthe base population for improvement, but will also be used as a source of improvedlines for direct release.

54


Developing the CNA-IRAT 4PR and CNA-IRAT 4ME populationsThe CNA-IRAT 4PR and CNA-IRAT 4ME populations were developed through

selection of S1 (first stage) and S2 (second stage) families. In the first stage, 100families were derived from fertile plants selected from the CNA-IRAT 4/0/3 gene pool.Once these were evaluated, the 14 best-performing families were selected and, withineach family, 10 plants. These 140 plants were then recombined to produce theCNA-IRAT 4/1/1 population.

In the second stage, the new population was planted and evaluated in Goiânia,where 121 early and 102 intermediate cycle, fertile plants were selected. Two groupsof materials were formed. The S2 families were evaluated and the best families of eachgroup were recombined to develop the CNA-IRAT 4PR (early) and the CNA-IRAT 4ME(intermediate) populations.

Improving the CNA-IRAT 4PR and CNA-IRAT 4ME populationsPopulation improvement is based on the selection of S2 families. In 1991,

breeders selected 164 plants of each population to evaluate advances after oneselection cycle. The S2 families derived from those populations were evaluated inGoiânia, State of Goiás, and at Formoso do Araguaia, State of Tocantins.

All the characteristics analyzed presented statistical differences between eachgroup of families and their controls. Average yields were 4,649 and 4,513 kg/ha forthe early and intermediate groups, respectively. The six best families of the first groupyielded more than 6,000 kg/ha, while the two best families of the second group yieldedmore than 7,000 kg/ha. The coefficients (%) of genetic variation for the early andintermediate cycle groups were:

Genetic variation Populat ion

CNA-IRAT 4PR CNA-IRAT 4ME(early) (intermediate)

Grain yield 1 0 . 4 0 1 0 . 9 9Time to heading 2.50 2 . 9 2Resistance to Helminthosporium oryzae 6 . 2 8 9 . 5 4Resistance to neck blast 1 2 . 0 1 9 . 0 7

Maize breeders consider a value of 7.0% or more as a good indicator of thegenetic potential of a population.

Results indicated that recurrent selection was efficient in improving populations.Furthermore, considering the current levels of genetic gain, the grain yield of the CNA-IRAT 4PR and CNA-IRAT 4ME populations will double, if eight selection cycles areapplied.

Line developmentUsually, a breeder has three objectives when selecting genotypes from a

population: (a) to evaluate the population’s present level of genetic variability;(b) to improve the population by selecting and recombining the best genotypes; and(c) to develop fixed lines and, subsequently, commercial varieties.

(%) for:

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This project embraces these three objectives. State institutions are responsiblefor developing improved lines. During the evaluation of rice lines for subsequentrecombination, each state chooses those lines best adapted to local conditions toincorporate them into breeding programs.

This process has already undergone two cycles, and the selected material is stillbeing evaluated. The positive benefits of this process will be evident only within 2 or 3years—that is, when the lines selected by the programs are incorporated into theactivities of Brazil’s rice improvement network.

Case 2: Upland Rice in Villavicencio, Colombia: RecurrentSelection Conducted by CIRAD-CA and CIAT

M. Châtel and E. P. Guimarães

This project aims to develop and improve upland rice gene pools and populations forLatin American savannas with good rainfall distribution. Breeding objectives are acid-soil tolerance, earliness, grain quality, and disease resistance.

In Brazil, CIRAD-CA and EMBRAPA-CNPAF developed the tropical Japonica genepool CNA-IRAT 5/0/3, and by incorporating new variability into this gene pool, thefollowing populations were developed:

CNA-IRAT A/0/1: by introgessing early upland Japonica lines.

CNA-IRAT P/1/0F: by introgressing modern lowland Indica lines.

IRAT Lulu/0/1: by introgressing modern lowland Indica lines and aromaticirrigated lines.

These materials were introduced to Colombia in 1992 by CIRAD-CA and CIAT,and evaluations began in Villavicencio in 1993. Each group of germplasm wasrepresented by 2,000 seeds planted on individual hill plots. Each material wascharacterized by sampling 400 plants for the following traits: acid-soil tolerance, initialvigor, time to heading, plant height, number of tillers, tolerance of neck blast, numberof fertile and male-sterile plants at flowering, and male-sterile seed set.

These populations performed well in acid soils. In the CNA-IRAT 5,CNA-IRAT A, and CNA-IRAT P germplasm groups, more than 82% of the plants weretolerant of acid soils, while in the IRAT Lulu population, only 66% of the plants weretolerant .

The Indica-Japonica populations CNA-IRAT P and IRAT Lulu showed,respectively, the highest percentages of vigorous (50.8% and 38.4%), late flowering(42.6% and 67.5%), and short plants (40.9% and 50.3%). About 80% of tillerproduction varied between 17 and 36 tillers per plant.

The groups of pure Japonica germplasm were more tolerant of neck blast(6.8% of CNA-IRAT 5 and 3.7% of CNA-IRAT A had susceptible plants) than were theIndica-Japonica populations, CNA-IRAT P and IRAT Lulu. About 52% of the IRAT Lulupopulation was susceptible.

The genetic constitution of the different germplasm, which has been influencedby materials introduced into the CNA-IRAT 5 gene pool, explains these results.

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After one evaluation cycle, the CNA-IRAT A/0/1 population was assessed as beingthe best adapted to program objectives and therefore to be used as a source of malesterility in the development of a new population.

Developing the PCT-4\0\0\0 populationBased on information available in CIAT’s traditional breeding program, eight lines

were selected as sources of variability for developing a new population. These lineswere CT 11231-2-2-1-4-M, CT 11231-2-2-3-1-M, CT 11231-2-2-2-1-2-M,CT 11608-8-6-M-2-M, CT 11608-9-2-1-2-M, CT 6196-33-11-1-3-M, A 8-394, andIR 53167-3-M. Each line was manually crossed with male-sterile plants of theCNA-IRAT A/0/1 population.

The resulting F1 generations of these crosses were planted at the CIAT ExperimentStation at Palmira. The performance of each combination was monitored, and the onewith line CT 11608-9-2-1-2-M was discarded because it showed a very high number ofsterile spikelets. The F2 seeds of the remaining seven crosses were mixed in differentproportions according to the genetic origin of the introduced lines. These seeds are thebasis of the PCT-4\0\0\0 population. Evaluations of the population will begin inVillavicencio after the first recombination cycle.

Improving the CNA-IRAT 5/0/3, CNA-IRAT A/0/1,CNA-IRAT P/1/0F, and IRAT Lulu populationsThe improvement of the CNA-IRAT 5 gene pool, and the CNA-IRAT A and

CNA-IRAT P populations began in 1993. Selection focused on plant type, acid-soiltolerance, earliness, grain quality, vigor, and disease resistance. Mass selection wasused in male-sterile plants, thus selecting the best plants within the evaluatedgermplasm. The seed produced by these plants was planted in the following generation,and male-sterile plants were again selected.

One cycle of recurrent selection was equivalent to one generation of planting. Theprocess was repeated three times. In 1994, these materials were recombined at Palmirawithout selection. The seed harvested from male-sterile plants was the basis of threenew improved populations: PCT-5\0\0\0, PCT-A\0\0\0, and PCT-P\0\0\0.

Line developmentSelection criteria, similar to those mentioned previously, were used to develop

fixed lines: acid-soil tolerance, flowering at 86 days after sowing or before, plant heightbelow 95 cm, more than six tillers per plant, and resistance to neck blast.

In 1993 A (first semester), 213 fertile S0 plants were selected from three of the fouroriginal groups of germplasm evaluated: CNA-IRAT 5/0/3 (76 plants), CNA-IRAT A/0/1(111 plants), and CNA-IRAT P/1/0F (16 plants). The IRAT Lulu population wasdiscarded because of its high susceptibility to neck blast. In 1993 B (second semester),148 fertile plants were selected from the groups of germplasm in the first cycle ofrecurrent selection for acid soils; these groups were CNA-IRAT 5\SA\0\3(59 plants), CNA-IRAT A\SA\0\1 (82 plants), and CNA-IRAT P\SA\0\0F (7 plants).

In 1994 A, 229 fertile plants were selected from the groups of germplasm in thesecond cycle of recurrent selection for acid soils; these groups were CNA-IRAT5\SA\1\3,5\SA\0 (31 plants), CNA-IRAT A\SA\1\1,A\SA\0 (167 plants), andCNA-IRAT P\SA\1\0F,P\SA\0 (31 plants). In 1994 B, all lines selected from the fertileplants of the original groups of germplasm and those with one or two cycles of recurrentselection were planted at CIAT, Palmira, for seed increase.

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The lines derived from selected plants in both the original CNA-IRAT P/1/0Fpopulation and the first cycle of recurrent selection were discarded because theypresented undesirable agronomic characteristics.

Each selection step in the recurrent populations will be used as a source ofgermplasm for developing lines for the acid-soil savanna system. The traditionalbreeding program will continue to screen those plants selected before 1994 to developfixed lines.

Case 3: Resistance to Blast and Yellow Mottle Virus inRice, Bouaké, Côte d’Ivoire: Recurrent SelectionConducted by CIRAD-CA, IDESSA, and WARDA

M. Vales, N. Yoboue, and A. Sy

This project aims to develop upland rice populations with durable resistance to blast,and create new rice populations targeting resistance to rice yellow mottle virus(RYMV) under irrigated conditions in Africa.

Developing the IDSA-IRAT 1, 8, 10, and 12 populationsThe IDSA-IRAT 1 Japonica population is the base population for blast resistance

in the recurrent selection project. It was developed by selecting resistant plants inthe CNA-IRAT 5 gene pool, inoculated with blast race CI 69. This race was chosenbecause it can overcome the resistance of major genes present in CNA-IRAT 5.IDSA-IRAT 8 was derived from the IDSA-IRAT 1 population after two selection cyclesfor grain type.

The IDSA-IRAT 10 Indica-Japonica buffer population was developed fromIDSA-IRAT 8 by introducing the following 20 Indica and Japonica lines: MANA 1,55-55, Alicombo, BG 90-2, Bouake 189, Ceysvoni, TOX 1011-4-1, Abongoua 88,Chokoto, Fossa Man 2, IDSA 11, IRAT 216, IDSA 85, IRAT 247, LAC 23, Khao DawkMali 105, Tangara, IRAT 112, IRAT 115, and Moroberekan. The purpose was tocombine, in this population, resistance to both blast and RYMV.

The IDSA-IRAT 12 Indica-Japonica population is being developed for resistanceto RYMV, using as base material the IDSA-IRAT 1 population into which the followingsources of resistance to RYMV—of both Indica and Japonica types—are beingintroduced: Koto Ouro S5, TOX 3100-37-3-3-3-2, Super IRAT 216, Dioukeme,Progresso, TOX 3052-46-E2-2-2-4-3, TOX 3058-28-1-1, TOX 3211-14-1-2-1-2,TOX 3226-5-2-2-2, TOX 3233-31-6-2-1-2, TOX 3440-16-3-3-2-2-3,TOX 3440-171-1-1-1-1, TOX 3440-176-1-2-1, and TOX 3553-36-2-2-2.

Improving the IDSA-IRAT 8 population for blast resistanceThree strategies have been used to improve this population: the first consists of

inoculating rice plants in plastic trays. Resistant plants are then transplanted to thefield and the seed produced by male-sterile plants harvested. Two cycles of recurrentselection are conducted per year.

The second strategy is based on the selection of fertile plants, resistant toboth leaf and neck blast, under field conditions. Disease-spreader lines are

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inoculated with a specific blast race. Resistant, fertile plants are selected, and theirseed mixed and planted for recombination. The seeds harvested from male-sterileplants are the basis of the next cycle of recurrent selection. Each cycle requires twosowings.

The third strategy involves selecting, harvesting, and mixing, then sowing theseed mixtures of selected, resistant, fertile plants. Seed from the resulting male-sterileplants is then harvested, mixed, and planted. The fertile plants of this sowing areharvested and their seed will form the basis for the next cycle of recurrent selection.Each cycle therefore requires three sowings.

The IDESSA/CIRAD-CA project applied the following selection process:

(a) In 1993, 3,000 seeds harvested from male-sterile plants of the IDSA-IRAT 8population were planted in plastic trays. The resulting plants wereinoculated with the CI 69 blast race. The 1,000 most resistant plants weretransplanted to the field. Of these, 450 fertile plants were harvestedindividually and male-sterile plants were bulked.

(b) 150 S1 plants were selected for grain type. Their seed was planted in the fieldat three sites (Bouaké, Ferké, and Man) and the resulting lines assessed.50 S1 lines were selected for their reaction to blast and for their agronomiccharacter is t ics .

(c) Further population improvement was achieved with off-season planting of the150 S1 plants assessed in 1993, a year in which disease pressure was low.The resulting 150 S2 lines were assessed again in 1994 for their resistance toblast at Bouaké and Man, Côte d’Ivoire, and at Farako-Bâ, Burkina Faso. InCôte d’Ivoire, plants were inoculated with the CI 69 blast race. The 50 mostresistant S2 families were selected and recombined.

(d) To take advantage of both tray and field selection, the 50 S2 families selectedin 1993 were then recombined with male-sterile plants resulting from the1993 tray selection process. Each S2 family was combined with threemale-sterile plants of the population, yielding 150 combinations. Thesematerials were planted in trays and inoculated with race CI 69. The mostresistant plants of the most resistant combinations were selected andtransplanted to the field. The best 150 plants regarding agronomiccharacteristics and blast resistance were selected to give continuity, alongwith their seed, to the selection process.

Line developmentFertile plants were selected in the 1993 and 1994 plantings and incorporated

into the line development process of the joint IDESSA/CIRAD-CA project in Côted’Ivoire. No significant advances can be reported so early in the improvement process.

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Case 4: High-Altitude Rice in Vinaninony and Antsirabe,Madagascar: Recurrent Selection Conducted byCIRAD-CA and FOFIFA

R. Dechanet and J. Enjalbert

This project aims to develop and improve two types of germplasm: (a) Japonicagermplasm for irrigated conditions at high altitudes (1,800 m), with tolerance of lowtemperatures and resistance to Pseudomonas fuscovaginae (a bacterial sheath rot);and (b) Japonica germplasm for upland conditions (1,500 m), with tolerance of lowtemperatures and rice blast.

Developing the MD 1 gene poolThe CNA-IRAT 1/0/2 gene pool was the source of male sterility used to develop

MD 1. Thirteen introduced lines were chosen after intense germplasm assessmentand selection of those best adapted to local irrigated conditions. These lines wereLatsidahy, Latsibavy, Tokambana, Mitsangana, Rojofotsy Vinaninony, IBPGR 115,IBPGR 118, IBPGR 138/2, IBPGR 141/4, AS 37, AS 40, AS 39, and AS 92. Each linewas crossed with male-sterile plants of the gene pool to produce, after one cycle ofbackcrossing for cytoplasm mixing, the MD 1 gene pool.

Improving the MD 1 gene poolTolerance of low temperatures and resistance to P. fuscovaginae are two basic

selection criteria, but progress has been difficult because of the large variationsobserved from year to year and among sites.

Vinaninony was chosen as the main test site, and mass selection was applied.In 1993, 121 fertile S0 plants were selected from the MD 1/0/3 gene pool to begin theprocess. In the following off-season, these plants were recombined at the MahitsyExperiment Station. Seed produced by the 773 male-sterile plants in therecombination cycle of the population selected in MD 1/1/0 was mixed in equalproportions to originate the MD 1/1/1 population, after one cycle of selection andrecombinat ion.

Line developmentIn 1992, 1993, and 1994, plants with good spikelet fertility and resistance to

P. fuscovaginae were selected from the MD 1/0/3 gene pool. In 1992, because ofexternal factors, selection was conducted at Soanindrariny, where 179 S0 plants werechosen. These materials and the gene pool were planted at Vinaninony in 1993;38 S1 lines were selected from the 179 tested and 121 S0 plants selected from thegene pool, which was again submitted to selection.

The gene pool was planted for the third time in 1994, and again 280 S0 plantswere selected. From the 38 S1 lines, 9 S2 lines were selected, and from the 121 S1lines, 30 were selected. The project will continue in 1995 with the planting of theselines, which will yield 280 S1, 13 S2, and 9 S3 lines, all derived from the MD 1/0/3gene pool.

Developing the MD 2 gene poolDeveloping the MD 2 gene pool followed a similar process to that of the MD 1

gene pool. Eleven lines adapted to upland conditions were crossed with male-sterile

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plants of the gene pool. These lines were IAC 25, Latsidahy, Latsibavy, FOFIFA 62,FOFIFA 116, Daniela, Shin Ei, Rikuto Norin 15, Guarani, F4 C 58-L10, and PratãoPrecoce. The CNA-IRAT 1/0/2 population was used as a source of male sterility.After backcrossing to mix the cytoplasm, the MD 2 population was obtained.

Improving the MD 2 gene poolBefore starting selection, we conducted two recombination cycles, identified as

MD 2/0/2 and MD 2/O/3. In 1994, improvement began by selecting fertile S0 plantsfrom the MD 2 gene pool, which had already been submitted to two recombinationcycles (MD 2/0/2).

That same year, part of the seed was stored for future recombination and theother part planted to assess the S1 progenies. But because only 44 S1 lines wereselected, the evaluation was repeated and the progeny reselected in larger plots, usingthe stored seed. The genotypes that will be used to develop the improved populationwill come from this new selection.

Observations indicated that the phenotypes of this gene pool are close to thoseof irrigated rice, and that their susceptibility to blast increased. This drift probablyoccurred because the population was recombined under irrigated conditions, greatlyenhancing the contribution of those plants best adapted to that system.

To counteract the drift, the genetic base of the gene pool is being adjusted byintrogressing 11 Japonica upland lines that were developed by traditional breedingfor high altitudes.

Line developmentIn 1993 and 1994, plants showing good agronomic characteristics, good spikelet

fertility, and resistance to blast were selected from the MD 2/0/2 and MD 2/0/3 genepools.

In 1993, 393 fertile S0 plants were selected from the MD 2/0/2 gene pool, theirseed planted in 1994, and 44 S1 lines were selected. In the same year, concurrentlywith the previous work, 30 S0 plants were selected, according to an index for differentyield components, and 70 plants were harvested at random.

The program is using these materials to develop lines that will be cold tolerantand blast resistant. Genetic correlation studies can also be conducted with thesel ines.

In 1994, 127 S0 plants were selected from the MD 2/0/3 gene pool. Theseplants are still being evaluated and are expected to produce lines adapted to theupland conditions of Madagascar’s highlands.

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Case 5: Lowland Rice Subject to Alternate Periods ofFlooding and Drought in Sikasso, Mali: RecurrentSelection Conducted by CIRAD-CA and IER

N. Ahmadi and M. Diaby

This project aims to develop rice lines that tolerate flooding and drought, and resistthe rice yellow mottle virus (RYMV) under the rainfed conditions of Africa’s lowlandswamps. This ecosystem is characterized by alternate periods of flooding anddrough t .

Developing gene pools and populationsAfrican gene pool. A gene pool of African germplasm adapted to the African

swamp ecosystem was first developed, beginning in 1991, and completed in 1994.

The process consisted of crossing and backcrossing nine African lines withmale-sterile plants of the CP 122L and CP 126 gene pools. The African lines wereBentoubala B Mali, Dissi 14, Ebendioula, Fossa Man 1, Gambiaka, Gambiaka Terra,Samba Badi, Siantane Diofor, and Sikasso H.

Indica-Japonica gene pools. These gene pools allow the complementaritybetween the Japonica and Indica groups to be exploited. The former can contributeits resistance to several diseases and drought, and the latter, its ability to adapt toirrigated conditions and its high grain-yield potential.

Initial crosses were made in 1990, and the populations SIK.P1.0 and SIK.P2.0were obtained in 1992. The origin of these two populations is basically the same,differing only in the material used as source of male sterility.

The procedure followed to obtain these populations was, first, to pollinatemale-sterile plants of the CP 122L and CP 126 gene pools (both Indica) with fertileplants of CNA-IRAT 5/0/2F (Japonica). Second, the resulting seeds were mixed andthe SIK.P1.0 population formed. Third, the same process was repeated, but withmale-sterile plants of CNA-IRAT 5/0/2F, which were pollinated by fertile plants ofCP 122L and CP 126. The final result was the SIK.P2.0 population.

Improvement for resistance to RYMVFertile S0 plants, tolerant of flooding and resistant to RYMV, were selected from

the CNA-IRAT 4/0/2F, CP 122L, and CP 126 gene pools. S1 progenies were selectedfrom CNA-IRAT 4/0/2F (24%), CP 122L (32%), and CP 126 (38%). These selections,once recombined, gave three improved populations, identified as SIK.P3.3, SIK.P4.3,and SIK.P5.3.

Line developmentThe aim is to produce fixed lines from the gene pools and improved populations.

Overall, 296 lines are under selection. Their origin is as follows: 13 lines,CNA-IRAT 4; 3 lines, CNA-IRAT 5; 14 lines, CP 122; 25 lines, CP 126; 81 lines,SIK.P3; 102 lines, SIK.P4; and 58 lines, SIK.P5. These materials will continue beingevaluated in 1995 to develop varieties adapted to rainfed lowland ecosystems,characterized by alternate periods of flooding and drought.

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Results and ProspectsSince CIRAD-CA and EMBRAPA-CNPAF developed the first rice gene pools in Brazil,in 1984, significant advances have been made and valuable experience has beenacquired in germplasm management and recurrent selection of this autogamouscereal .

Several national and international programs of Latin America and Africa,particularly that of Madagascar, are using this methodology in their breedingstrategies to complement traditional methods, such as pedigree and mass breeding,and backcrossing. Preliminary results indicate that improving gene pools andpopulations may help solve problems so far not solved by traditional breedingmethods, such as the grain-yield plateau in irrigated rice germplasm or the poorresistance to blast in upland rice.

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Nomenclature for Rice Improvement by Recurrent Selection


Nomenclature for Rice Improvementby Recurrent Selection

Introduct ionIn 1984 a recurrent selection program for rice was initiated as part of a collaborativeresearch project between CIRAD-CA (previously IRAT, France) and EMBRAPA-CNPAF,Brazil. A few years later, CIAT and IRRI also developed rice gene pools for populationimprovement. The international agricultural research centers (IARCs) and thenational agricultural research systems (NARS) have used this new germplasm in theirbreeding programs, either directly or as a source of male sterility to develop newgermplasm adapted to diverse local conditions.

Although several gene pools and populations are currently used in rice breeding,there is no clear, standardized nomenclature system. Accordingly, not only thetracking of background information and selection criteria applied to this germplasmbecomes increasingly difficult, but also the comparison of results of breeding workand strategies. A standardized nomenclature system is necessary as traditionalbreeding programs increasingly use the recurrent selection method and as theinterinstitutional germplasm exchange increases.

In this chapter we propose a standard nomenclature system to be adopted bybreeders. This system can be used to publish a catalog in which all availableinformation on rice gene pools and populations is recorded. Each entry would befully described, with special emphasis on development, genetic constitution, status ofgermplasm, targeted objectives and ecosystems, and major agronomic characteristics.A rice research institution would be responsible for coordinating and updating thecatalog.

Existing NomenclaturesFirst, we must define what is a gene pool and what is a population. This proposalconsiders a gene pool as the specific and unique genetic background that results fromdifferent combinations of a group of genotypes. These genotypes may be pooledtogether in similar or different proportions through crossing, with or without a genefor male sterility.

A population is the result of applying a breeding procedure to that geneticbackground, and is characterized by enhanced traits and genetic modifications,compared with the parental gene pools to which as much as 50% new variability isin t roduced.

EMBRAPA-CNPAF, CIRAD-CA, IRRI, and CIAT have developed gene pools namedas follows:

CNA-IRAT: Developed in Brazil by EMBRAPA-CNPAF and ClRAD-CA(previously IRAT).

CP: Prepared at IRRI and currently used by CIRAD-CA in Mali, Africa.

GC: Developed by CIAT.

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Each gene pool received a code number for identification (CP 122, GC 91,CNA-IRAT 4). The plant breeders who developed these gene pools relate those codenumbers to their breeding activities, for example:

(a) In CNA-IRAT germplasm, CNA-IRAT represents the standard institutionalcode and a number identifies a gene pool (e.g., CNA-IRAT 4). This is followedby two additional numbers, separated by a slash. The first numbercorresponds to the number of selection cycles conducted in the gene pool(CNA-IRAT 4/0). The second number refers to the number of recombinationsapplied to that gene pool (CNA-IRAT 4/0/3). Therefore, CNA-IRAT 4/0/3refers to the gene pool CNA-IRAT 4, where there was no selection, althoughthree cycles of recombination were conducted.

A few populations were developed from this gene pool, following the samedenomination scheme. An example is CNA-IRAT 4/1/3, received by CIAT in1993; this population came from the CNA-IRAT 4 gene pool after oneselection cycle conducted in the third recombination cycle.

(b) The CNA-IRAT gene pools were formed, using the gene for male sterility fromIR 36 and resulting in a mixture of sterile and fertile plants. The lastnumber of several gene pools is therefore followed by “F,” for example,CNA-IRAT 4/0/1F. This code number indicates that the gene pool wassubmitted to one recombination cycle and that the seed increase wasconducted by harvesting fertile heterozygous plants (/1F).

The same sequence of code numbers applies to the CP gene pools in Mali.An example is the CP 126/1/1F population, which means that the CP 126gene pool was submitted to one selection cycle and one recombination cyclein which seed increase originated from fertile heterozygous plants.

(c) At CIAT, the letters are the initials of the scientists who participated in genepool development and the numbers refer to the year in which S0 seed wasobtained. For example, in 1991, Drs. Guimarães and Correa-Victoriasynthesized GC-91.

(d) When gene pools or populations are used as sources of male sterility tointroduce new variability, the resulting germplasm can be identified in twoways. In practice, new lines are crossed with male-sterile plants of the genepool or population so their genetic contribution represents at least half of thenew germplasm.

The two name variations are, first, the name of the original germplasm(source of male sterility) is maintained, but a specific identification is added.For example, CNA-IRAT 1 MD 1 is the first germplasm developed for high-altitudeareas in Madagascar (MD). It was derived by introducing 13 locally adaptedlines into the CNA-IRAT 1 gene pool.

In the second name variation, the name of the original germplasm (sourceof male sterility) is removed and the new population receives a differentname. For example, CNA 1 comes from introducing three lines into theCNA-IRAT 4/0/5F gene pool by crossing with male-sterile plants.

(e) The nomenclature used to identify the CNA-IRAT gene pools and populationsgives only the number of selection and recombination cycles used in thebreeding process. Further information may be needed for better germplasmcharacterizat ion.

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Proposed Standard NomenclatureExisting or new gene pools and populations should be considered independently.

Existing gene pools and populationsBecause rice breeders already use the germplasm under a given name and code

number, changes are inconvenient. Those names and code numbers should be keptand recorded in a central catalog. Registered germplasm (see p. 68-69) could bepublished in the International Rice Research Newsletter or in Crop Science.

New gene pools or populationsNew germplasm may be a truly new gene pool developed by an institution, or it

may be a population obtained by introducing improved lines into an already existinggene pool or population. There are several ways this can be done:

(a) For new gene pools:

(i) By combining a group of genotypes through manual crossing or by usingmale-sterile genes available in their original genetic background.

(ii) By introducing more than 50% of new germplasm, crossing andbackcrossing rice with male-sterile plants of an already developed genepool or population.

(iii) By crossing two or more gene pools or populations.

(iv) By combining the previous options or any other possible.

(b) For new populations:

(i) By improving germplasm in one or several specific traits throughrecurrent selection in gene pools or populations—both existing and new.

(ii) By introducing at least 50% variability in a gene pool.

In each case, the institution that creates, modifies, or selects gene pools orpopulations will name them according to its own criteria and will later register themin the catalog with detailed information on the genetic constitution and methodologyused. For exchange and publication purposes, the catalog nomenclature is the onethat would be officially recognized and used by breeders.

Nomenclature system for a gene pool and population catalogPrefix. The use of the subprefixes “GP” for gene pools and “P” for populations

(in capital letters) is proposed. Letters should be used by institutions to identify theircrosses (e.g., CT for CIAT, IR for IRRI, CNA for EMBRAPA-CNPAF, WAB for WARDA, orIRAT for CIRAD-CA). No space is left between the letters that form the subprefix andthe institutional code.

Numbering. The entire prefix is immediately followed by a dash and a number,with no spaces. This numbering is consecutive according to the registration catalog.

For example, if the CIAT gene pool GC-91 is the first registered in the catalog,followed by gene pools or populations of EMBRAPA-CNPAF and WARDA, the officialname of the first gene pool would be GPCT-1. The CNA 1 gene pool would be

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registered then as GPCNA-2 and the population proposed by WARDA would appear asPWAB-3. If the next gene pool to be registered is another from CIAT, it would benamed GPCT-4, and so forth.

Identifying the status of gene pools and populationsThe improvement of gene pools and populations is a continuous process of

evaluation, selection, and recombination. It is therefore important to know thegermplasm’s present status. Each institution is free to use its own criteria to namethe germplasm under study. However, plant breeders are recommended to identifytheir work material as proposed in this manual so they can efficiently handle thepopulations and gene pools during the breeding process. This identification is usefulwhen communicating and exchanging information before registering the germplasmas a new population. The following definitions and examples will clarify this proposal:

(a) Identification

(i) A full identification gives the number of recombination cycles of the genepool or population, both before and after selection. The traits used toselect the germplasm are also identified by letters.

(ii) The acronyms of the Standard Evaluation System for Rice, developed by IRRI(1988), should preferably be used to identify these traits. Selected traits andrecombination cycles (after and before selection) are separated by a backslash (“\”), not the slash normally used to identify crosses (“/”).

(b) Examples

(i) Selection was conducted once in the GC-91 gene pool—catalog nameGPCT-1—for leaf blast. CIAT would handle these populations as GPCT-1\Bl.

(ii) Continuing the previous example, the numbers that follow in the sequencewould indicate how many recombination cycles were used after selection,and in which recombination cycle was selection conducted in the originalgene pool. During this semester ’s planting, GPCT-1\Bl, the CIAT populationimproved for resistance to leaf blast, would be called GPCT-1\Bl\1\2because the GPCT-1 gene pool was selected for leaf blast (Bl) in the secondrecombination cycle of the gene pool (\2) and then recombined once (\1).

(iii) In a hypothetical example, GPCT-4\0\0\0 would indicate a gene poolproduced by ClAT—the fourth registered in the catalog—in which norecombination cycles or selection (basic gene pool) have occurred. Afterthree recombinations of this basic gene pool, a breeder decides to work withit and converts it into GPCT-4\NBl\0\3, which means that a selection forneck blast (\NBl), with no recombination cycle after selection (\0), wasmade in the third recombination cycle (\3) of the original gene pool.GPCT-4\NBl\3\3 indicates, then, that there were three recombination cyclesafter selection for neck blast, which was made in the third recombinationcycle of the original gene pool.

(c) Selection cycles. Selection is usually made for several characters. In thiscase, the acronyms of these traits should appear in the code identifying thegermplasm.

(i) For several characters at the same time. For example, if a population isenhanced for leaf blast and brown spot at the same time, its identificationwould be GPCT-4\Bl,BS\2\4.

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(ii) Consecutive selection of several characters. If germplasm has been enhancedfor a certain trait in one season, and for another in the next, itsidentification would be GPCT-4\Bl\3\4,BS\0. The acronyms of eachcharacter are separate, indicating that breeding was done at different times.

(iii) Repeated selection for the same trait. If germplasm has been selected severaltimes for the same trait, then the resulting germplasm can be identified asfollows:

For example, a CNA gene pool underwent four recombinations(GPCNA-4\0\0\4), was submitted once to selection for neck blast andrecombined twice (GPCNA-4\NBl\2\4). It was selected in the followingcropping season for the same trait, and then recombined five times. Thefinal identification of that collection would be GPCNA-4\NBl\2\4,NBl\5.

(d) Registration in catalog. When the germplasm has been sufficiently enhanced forthe trait, or traits, under selection, the breeder can register it, following thesame rules for including new germplasm in the catalog. For example,GPCT-4\NBl\3\4—an improved population derived from the GPCT-4 gene poolby selection for leaf blast—becomes PCT-10, according to the simplestnomenclature of the catalog; 10 refers to the number of registration.

Once the breeder has learned to use this identification system, he or she willhave access to all the information available on the improvement of a givengermplasm.

Selecting Elite LinesElite lines can be extracted two ways: the first is by using gene pools or improvedpopulations, and the second, through selection and recombination cycles duringgermplasm improvement. The nomenclature used here is based on WARDA’sproposal for traditional line development that was accepted by the participants of theFirst International Upland Rice Breeders Workshop (CIRAD-CA, 1993).

Gene pools or improved populationsThe nomenclature of elite lines selected from gene pools or populations should

indicate the germplasm used to develop the line and the institution or institutionsthat participated in the process. The symbol “>” is used to separate the name of thepopulation or gene pool from the assigned pedigree. Three examples follow:

(i) GPWAB-3>78-4-3-2-1. This elite line was extracted by WARDA (afterselection during five generations) from a gene pool they have registered.

(ii) PCT-5>WAB-4-3-2-1. This elite line was extracted by WARDA from apopulation developed and registered by CIAT.

(iii) PIR-7>23-1-WAB-3-4-CNA-4-B. The PIR-7 population was produced andregistered by IRRI. An elite line from that population underwent selection byIRRI during two generations. WARDA received the line PIR-7>23-1 andsubmitted it to selection for two more generations. Finally, the linePIR-7>23-1-WAB-3-4 was sent to EMBRAPA-CNPAF, where it was submittedto selection during one more generation. In the following generation seedswere harvested in bulk (symbolized as “B”).

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Different selection and recombination cycles (workingmaterial)The example presented below illustrates the application of the proposed

nomenclature to this method of extracting elite lines:

PWAB-7\GQ\2\3>79-7-4-2. A WARDA population, PWAB-7, is undergoingselection to improve grain quality (GQ). The institution selects an elite lineduring four generations in the third recombination cyle (\3) of the originalPWAB-7 population, which had been selected once for grain quality (GQ) andthen recombined twice (\2).

Seed Exchange and Conservation(a) Each institution will hold seeds of the gene pools or populations it has

registered (breeders’ seed). This seed will be available for exchange.

(b) Replies to requests for seed should include the information contained in thecatalog.

(c) A minimum of 2,000 seeds should be sent with the request for registration tothe institution responsible for the catalog.

(d) Every institution is responsible for the maintenance and medium-termconservation of its gene pools and populations. A sample of these should besent to IRRI for long-term conservation.

(e) Institutions should forewarn potential users and the institution responsiblefor the catalog of their intention to discard germplasm that is no longer used.The remaining seed should be sent to IRRI for conservation.

(f) Each year the institutions involved with breeding by recurrent selection mustreport their activities to a central coordinator, who is responsible forpreparing a global activity report on recurrent selection and on the use givento the resulting rice gene pools and populations. This report is then sent toall those participating in the institutional information and germplasmexchange system.

How to Register a PopulationTwo researchers registered a rice population in the June 1989 issue of theInternational Rice Research Newsletter [14(3):8-9], a scientific bulletin published byIRRI in the Philippines. The original text in English read as follows:

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CNA-IRAT 5 upland rice populationJ. Taillebois and E. P. Guimarães, Coordenador Arroz, EMBRAPA/Centro Nacionalde Pesquisa-Arroz, Feijão (CNPAF), C.P. 179, 74000 Goiânia, Brazil

“To develop a segregating population for a recurrent selection program for drought andfield blast resistance, CNPAF and Institut de Recherches Agronomiques et des CulturesVivrières (IRAT) used 27 upland rice varieties and a monogenic recessive male sterile geneobtained by mutation on IR36 by R. J. Singh and H. Ikehashi.

“We crossed and backcrossed 26 varieties to male-sterile F2 plants of Palawan/IR36 (MS +)1. The crosses were made to obtain a polycytoplasmic population. F2 seeds ofeach backcross were bulked in different proportions to ensure a different rate of eachcomponent (see table). The bulked seeds were considered the initial populationCNA-IRAT 5/0/0.

“Outcrossed seeds collected on male sterile plants of the initial population were bulked toform the next population, CNA-IRAT 5/0/1. The procedure was repeated three times toobtain the CNA-IRAT 5/0/3 population.

“Samples of bulk seeds harvested on self-pollinated plants of the CNA-IRAT 5/0/2population are available to rice breeders.”

CNA-IRAT 5 initial population.

Variety Paren tage Outcrossing rate(%)

IR36 (ms +)1 Mutant of IR36 1 2 . 5 0Pa lawan a Asian germplasm 1 2 . 5 0C u i a b a n a a IAC47/SR2041-50-1 8 . 1 0IRAT237a IAC25/RS25 6 . 7 3Beira Campo Brazilian germplasm 5 . 3 9CNA4097 63-83 / IAC25 5 . 3 9CNA4145 IAC47/Kinandong Patong 5 . 3 9Cabaçu (IRAT177) Mutant of 63-83 5 . 3 9IREM41-1-1-4 Mutant of Makouta 5 . 3 9Palha Murcha Brazilian germplasm 5 . 3 9TOx 1011-4-2 IRAT13/DP689//TOx 490-1 5 . 3 9CNA5171 IAC47/IRAT13 2 . 6 9IAC165a Dourado Precoce/IAC1246 2 . 6 9IREM247 a Mutant of IAC25 2 . 5 0IAPAR9a Bata ta i s / IACF.3-7 1 . 5 7IRAT112a Dourado Precoce/IRAT13 1 . 4 7CNA4135 a IAC47 /63 -83 1 . 3 6IREM238 a P J 1 1 0 / I A C 2 5 1 . 3 5Arroz de Campoa Brazilian germplasm 1 . 2 5CA 435a African germplasm 0 . 8 4Casca Branca Brazilian germplasm 0 . 8 4CNA5179 IAC47/IRAT13 0 . 8 4CNA770187 Brazilian germplasm 0 . 8 4Comum Crioulo Brazilian germplasm 0 . 8 4J a g u a r i Brazilian germplasm 0 . 8 4L-13 0 . 8 4L-81-24 IAC2091/Jaguar i / IRAT10 0 . 8 4Santa America Brazilian germplasm 0 . 8 4

1. Authors’ note: For this manual, we used Msms instead of MS + and msms instead of ms +.a. Cytoplasms used to form the population.

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BibliographyCIRAD-CA. 1993. Recommendations of the First International Upland Rice Breeders Workshop,

6-10 September 1993, Montpellier, France. 66 p.

IRRI (International Rice Research Institute). 1988. Standard evaluation system of rice. 3rd ed.Los Baños, Philippines. 54 p.

Taillebois, J. and Guimarães, E.P. 1989. CNA-IRAT 5 upland rice population. Int. Rice Res.Newsl. 14(3):8-9.

WARDA (West Africa Rice Development Association). n.d. WARDA’s varietal nomenclature system.4 p. (Typescript.)

CIAT Publication No. 276

CIRAD-CA and theRice Program and Communications Unit at CIAT

Edition: Elizabeth Mc Adam de Páez (editor)Gladys Rodríguez (editorial assistant)

Translat ion: Lynn Menéndez

Production: Graphic Arts Unit, CIATOscar Idárraga (layout)Julio César Martínez (cover design)

Printing: Impresora Feriva S.A., Cali

recurrent selection in rice, using a male-sterile gene

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