Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes Aug. 31st- Sept. 4th, 2014

From genotype to phenotype

Linkage

• Loci that are close enough together on the same chromosome to deviate from independent assortment are said to display genetic linkage

Crossover

Co-dominant Marker

P1 P2 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Dominant Marker P1 P2

1 2 3 4 5 6 7 8 9 10 11 12 13 14

P1 P2 1 2 3 4 5 6 7 8 9 10 11 12 13 14

QTL mapping

• genotype and phenotype individuals • look for statistical correlation between

genotype and phenotype

Genetic Linkage maps

Genetic Linkage maps (also called meiotic maps) rely

on the naturally occurring process of recombination

for determination of the relative order, and distances

between polymorphic markers.

The statistical analysis of the segregating data is then

used to convert the recombination fraction into an

additive unit of distance measured in centiMorgan

(cM), with 1 cM representing a 1% probability that

a recombination has occurred between two markers

on a single chromosome.

Several linkage maps based on intraspecific crosses between upland

cottons have been reported (Shappley et al. 1998, Ulloa

and Meredith 2000, Zuo et al. 2000, Ulloa et al. 2002), but all the maps

were characterized with low marker coverage of the

genome. As molecular polymorphism is limited within ( G. hirsutum L).

Therefore, in this study we are interested to using interspecific hybrids

between G. hirsutum L. and G. barbadense L., as an efficient source for

polymorphism.

4 . Selection of parental varieties

The cotton map will be developed from an interspecific cross between

G. barbadense and G. hirsutum. Polymorphic parental varieties will be

selected among previous studied genotypes.

These parental varieties will be crossed to obtain the F1 generation.

5 . Generation of segregating population

An F1 plant will be selfed to generate the F2 segregating population.

The DNA of the different individual plants representing the F2 population

will be analyzed using the different marker types ( SSR and AFLP ).

The pattern of inheritance of these genetic markers among the F2

Individuals will be examined.

1 . Selection of cotton accessions among the collection available at the

Cotton Research Institute (CRI)

2 . Isolation and purification of genomic DNA from the different accessions.

3 . Fingerprinting of cotton accessions using different molecular markers

(AFLP , SSR and ISSR).

7 . Bulked Segregant Analysis

To rapidly find markers closely linked to the trait of interest

(earliness ), two bulked samples will be prepared for the trait.

Screening for differences between the pooled DNA samples for

the trait will be performed using the different molecular markers.

6 . Screening of Morphological Traits

Morphological trait of interest particularly date of flowering will be

scored for the individual plants of the segregating population.

8 . Analysis of Segregation and Map construction

The segregation of all the studied markers ( molecular and

Morphological ) will be analyzed among the F2 individuals.

The goodness of fit to the expected 3:1 Mendelian ratio for

each segregating locus will be tested by chi-square test and

the linkage analysis between loci determined according to

the maximum likelihood method.

The linkage map will be constructed using the MAPMAKER

version 2.0 software.

Marker Distance

Lin

e 1

Lin

e 2

Lin

e 3

Lin

e 4

Lin

e 5

Lin

e 6

Lin

e 7

Lin

e 8

Lin

e 9

Lin

e 1

0

Lin

e 1

1

Lin

e 1

2

Lin

e 1

3

Lin

e 1

4

Lin

e 1

5

Lin

e 1

6

_3_0363_ 0 A B B A A A B A B B A B B B B B

_1_1061_ 0.8 A B B A A A B A B B A A A B B A

_3_0703_ 1.5 B A A B B B A B A A B B B B B B

_1_1505_ 1.5 B A A B B B A B A B B B B B B B

_1_0498_ 1.5 B B B B B B B B B B B B B B B A

_2_1005_ 3.8 A B B A A A B A B A A B B B B B

_1_1054_ 3.8 A A A A A A A A A B A A A A A A

_2_0674_ 6 A B B A A A B A B A A A A A A B

_1_0297_ 8.8 A A B B B B B A A A A A A A A B

_1_0638_ 10.7 A A B B B B B A A B A A A A A A

_1_1302_ 11.4 B A A A B B A A A B A B B B B A

_1_0422_ 11.4 B A A A B B A A A B A B B B B A

_2_0929_ 15.3 A B B B A A B B B A B A A A A B

_3_1474_ 15.4 A B B B A A B B B A B A A A A A

_1_1522_ 17.3 A B B B A A B B B A B A A A A A

_2_1388_ 17.3 A A A A A A A A A A A A A A A A

_3_0259_ 18.1 B B B B B B B B B B B A A A A A

_1_0325_ 18.1 B B B B B B B B B B B A A A A A

_2_0602_ 20.8 A A B A A A A B A B A A A A A A

_1_0733_ 23.9 B B B B B B B B B B B A A A A A

_2_0729 23.9 B B B B B B B B B B B A A A A A

_1_1272_ 23.9 A B B B A A B B B B B B B B B B

_2_0891_ 26.1 A A A A A A A A A B A A A A A A

_2_0748_ 26.6 B B B B B B B B B A B B B B B B

_3_0251_ 27.4 A B A A A B A A A B A A A B A A

_1_0997_ 35.5 B B A A A B B B B B B B B B B B

_1_1133_ 41.8 B B A A A B B B B A B A A A A A

_2_0500_ 42.5 A A A A A A A A A B A B B B B B

_3_0634_ 43.3 B B B B B B B B B A B A A A A A

0

10

5 Disease severity

Application of QTL Mapping in Crop Improvement

Introduction

Drought is one of the most common

abiotic stressor limiting crops productivity

throughout the world.

Therefore, breeding and selection for high-

yielding crops under drought stress is a

major objective of crop breeders working

under unfavorable environments.

The construction of a molecular linkage map represents the

first step in the genetic dissection of a target trait of interest.

Both, genetic linkage maps and QTL maps are useful in durum

wheat improvement because they provide useful tools for

studying genome structure, evolution, identifying or

manipulating chromosome segments QTL (quantitative trait

loci) controlling important agronomic traits.

Objectives

1. To develop a QTL map of Egyptian durum wheat through the

application of different DNA markers (SSR, RAPD, AFLP,

EST and SCoT) and an F2 segregating population obtained

from an intraspecific cross between two durum varieties

(Baniswif-1 and Souhag-2).

2. To tag QTLs controlling yield and drought tolerance-related

traits: root length, plant height, spike length, number of

branches/plant, number of spike/plant, number of

spikelets/spike, number of kernel/spike, thousand kernel

weight, fresh weight, dry weight and total amino acids.

Mapping Population used (F2, RIL, DH, BC, ….)

Type of markers employed (AFLP, SSR, EST, SNP, DArT, ……)

Percentage of Genome Coverage

Traits of Interest (QTL Results)

Mapping population

Methodology

Two polymorphic varieties (Baniswif -1 and Sohag-2) were selected

among the germplasm available at Wheat Research Dept., Crop

Research Institute, ARC, Egypt.

These two varieties were used to develop an F2 mapping population

comprising 76 plants from the intraspecific cross.

The parents and F2 plants were grown in the year 2009 at one of the Agricultural

Genetic Engineering Research Institute experimental fields.

The F2 plants were grown in two replicates in a randomized complete block design.

Trait measurements

Data for:

- Root length, plant height as described by De Vita et al., 2007.

- Number of spikelets/spike, number of kernel/spike and thousand

kernel weight as described by Nacite et al., 1992.

- Spike length, number of branches/plant, number of spike/plant as

described by Diab et al., 2007.

- Fresh weight, dry weight and total amino acids as described by

Abebe et al., 2003.

DNA isolation

DNA was isolated from the two parents and the 76 F2 plants using DNAeasy

Plant Mini Kit (Qiagen, Santa Clarita, CA).

DNA markers

A preliminary screen of polymorphism between the two parental

genotypes was performed using 42 RAPD, 56 SSR, 32 AFLP, 20 EST and 26

SCoT primers and/or primer combinations.

Only 1 RAPD, 15 SSR, 11 AFLP and 10 SCoT primers revealed discernible

polymorphic patterns.

Therefore, analysis of segregation among the 76 F2 individuals was

performed using these polymorphic primers and/or primer combinations.

RAPD amplification was performed as described by Williams et al. (1990)

with minor modifications.

RAPD analysis

RAPD profile of the two parental genotypes as revealed by different primers

M P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 M P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2

SSR analysis

SSR analyses was performed as described by Hussein et al. (2003).

Table : SSR Primer name, primer sequence and Chromosome.

SSR profile of the two parental genotypes as revealed by different

primers

M P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2

EST analysis EST analyses were performed as described by Adawy (2007).

Table : EST primer code, gene name, primer sequences and expected PCR product.

EST profiles of the two parental genotypes as revealed by different primers

AFLP analysis

AFLP analyses was performed

using 11 AFLP primer

combinations according to the

protocol of Vos et al. (1995) with

minor modifications.

AFLP® Analysis System II

(Invitrogen, USA) was employed .

AFLP profile of the two parental genotypes as revealed by different primer combinations

Table : AFLP primer combination sequences

Start Codon Targeted (SCoT) Polymorphism analysis

SCoT is a novel method for generating plant DNA markers.

This method was developed based on the short conserved

region flanking the ATG start codon in plant genes.

SCoT uses single 18-mer primers in polymerase chain

reaction (PCR) and an annealing temperature of 50°C.

PCR amplicons are resolved using standard agarose gel

electrophoresis.

SCoT Analysis

SCoT analyses were

performed as described

by Collard and Mackil

(2009).

Table : SCoT primers and their sequences

SCoT profiles of the two parental genotypes as revealed by different

primers

M P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2 P1 P1 P2 P2

Genetic linkage map construction and QTL detection

The markers that showed polymorphism between the parental lines were

used to construct the genetic linkage map.

Linkage analysis and map construction were performed by using Map

Manager QTX V1.4 (Manly and Cudmore, 1997) using the Kosambi

function with a minimum LOD score of 3.0 followed by ripple command

for each linkage group to check the final order of markers.

The association between phenotype and genotype was investigated using

single point analysis (SPA), using QTL cartographer (Wang, et al. 2004).

Significance levels of 5%, 1%, 0.1% and 0.01% were used to declare a QTL.

Trait measurements

Results

Phenotypic and physiological data

of root length, plant height, spike

length, number of branches/plant,

number of spike/plant, number of

spikelets/spike, number of

kernel/spike, thousand kernel

weight, fresh weight, dry weight

and total amino acids traits for the

76 F2 plants derived from the

intercross between Baniswif-1 and

Sohag-2.

Trait measurements

Phenotypic and physiological data

of root length, plant height, spike

length, number of branches/plant,

number of spike/plant, number of

spikelets/spike, number of

kernel/spike, thousand kernel

weight, fresh weight, dry weight

and total amino acids traits for the

76 F2 plants derived from the

intercross between Baniswif-1 and

Sohag-2.

Statistics and normality test of traits

The Mean, Variance, Standard

Deviation, Coefficient of Variation,

Skewness and Kurtosis values for

root length, plant height, spike length,

number of branches/plant, number of

spike/plant, number of spikelets/spike,

number of kernel/spike, thousand

kernel weight, fresh weight, dry weight

and total amino acids are presented

in Figure.

All traits showed normal

distribution and large

amount of variation.

High kurtosis value were

observed for No. of Kernel/

spike (NKS) and Total Amino

Acids (TAA) and a large

skewness value was

obtained for No. of Kernel/

spike (NKS)

Primer, sequence, number of total bands and polymorphic bands as revealed by RAPD analysis.

RAPD patterns of the

two parents and F2

individuals derived

from the cross

BaniSwif-1 and

Sohag-2 as revealed

by primer OP-C4. M

is the standard DNA

marker 100 bp

ladder, P1 (cv. Bani

Swif -1) and P2 (cv.

Sohag-2).

RAPD analysis

M P1 P1 P2 P2 F2 individulas

Primer code, Primer

name, primer sequence,

chromosome and

marker size as detected

by SSR

SSR analysis

SSR patterns of the two parents and F2 individuals derived from the cross BaniSwif

-1 and Sohag-2 as revealed by primer S8. M is the standard DNA marker 100 bp

ladder, P1 (cv. BaniSwif -1) and P2 (cv. Sohag-2).

Primer combinations, selective nucleotides, number of total bands, polymorphic bands

and percentage of polymorphism as detected by AFLP primer combinations.

AFLP analysis

AFLP patterns of the two parents and F2 individuals derived from the cross Baniswif -1 and Sohag-2 as revealed by primercomb. 3/6. M is the standard DNA marker 100bp ladder, P1 (cv. Baniswif -1) and P2 (cv. Sohag-2).

SCoT analysis

Primer name, primer sequence, number of total bands, polymorphic bands and

percentage of polymorphism as detected by SCoT

SCoT patterns of the two parents and F2 individuals derived from the cross Baniswif -1 and Sohag-2 as revealed by primer S4. M is the standard DNA marker 100bp ladder, P1 (cv. Baniswif -1) and P2 (cv. Sohag-2).

Distribution of molecular

markers, assignment and

centiMorgan (cM)

coverage across the 14

linkage groups of the

genetic map used in QTL

mapping.

Molecular linkage groups of durum wheat (intercross between Baniswif-1 and Sohag-2) showing positions of QTL influencing root length, plant height, spike length, number of branches/plant, number of spike/plant, number of spikelets/spike, number of kernel/spike, thousand kernel weight, fresh weight, dry weight and total amino acids. Map distances between adjacent markers are in cM

` `

All 56 SSR primer pairs preliminary

screened on the two parents, were

previously mapped on the durum

wheat chromosomes.

However, only 15 SSRs primers

revealed polymorphic patterns

between the two parents. These

primer pairs were applied to the F2

individuals.

Assignment of linkage groups

to the chromosomes

Based on the presence of these SSR

markers, eight linkage groups (LG) were

assigned to chromosomes, i.e., LG1, LG3,

LG5, LG6, LG7, LG9, LG13 and LG14 were

assigned to chromosomes 1B, 3B, 5B,

6A, 6B, 7A, 3A and 2B, respectively.

The nine SSR markers used to assign

eight chromosomes are highlighted in

the linkage groups shown in the figure.

Assignment of linkage groups

to the chromosomes

QTL analysis

A total of 74 QTL at significance

level of 5%, 1%, 0.1% and 0.01%

have been identified for the 11 traits

on twelve linkage groups (1, 2, 3, 4,

5, 6, 7, 8, 9, 12, 13 and 14).

Among these QTLs, 3 QTL for RL, 11

QTL for PH, 7 QTL for SL, 3 QTL for

NBP, 3 QTL for NSP, 8 QTL for NSS,

15 QTL for NKS, 10 QTL for TKW,

4 QTL for FW, 5 QTL for DW and 5

QTL for TAA were identified.

Some genomic regions were found

where QTL for different traits

overlapped on linkage groups 2, 4, 6,

7, 8, 9, 13 and 14.

The linkage groups 6, 7 and 8 showed

the most overlapped traits.

Correlation between Traits

For example, QTL for spike length,

number of branches/plant, number

of spike/plant, number of

spikelets/spike and thousand kernel

weight were mapped to the same

chromosomal location.

Similarly, QTL for spike length, number

of kernel/spike, thousand kernel

weight and total amino acids were

mapped to identical genomic region.

Correlation between traits Correlation coefficient among Root Length, Plant height, Spike length, Number of branches/plant, Number of

spike/plant, Number of spikelets/spike, Number of Kernel/spike, Thousand kernel weight, Fresh Weight, Dry

Weight and Total Amino Acids traits in F2 segregating population.

Positive correlation

Negative correlation

No correlation

QTL Detection Softwares

Conclusion A genetic map comprising 114 molecular markers located on 14

linkage groups and spanning a total of 2040.9cM, was constructed.

This map was useful in detecting 74 significant QTLs related to high

productivity and drought tolerance (including : root length, plant

height, spike length, number of branches/plant, number of

spike/plant, number of spikelets/spike, number of kernel/spike,

thousand-kernel weight, fresh weight, dry weight and total amino

acids), and promises to provide a better understanding of the durum

wheat crop and enhancing breeding programs through MAS.

On the other hand, the constructed linkage map contains RAPD, SSR,

SCoT and AFLP markers that were not mapped collectively in any

other durum wheat maps.