polymorphism of the storage proteins in portuguese rye (secale cereale l.) populations

Polymorphism of the storage proteins in Portuguese rye (Secale cereale L.) populations MIGUEL RIBEIRO 1,2, LU Í S SEABRA 1,2, ANT Ó NIO RAMOS 1,2, SOFIA SANTOS 1,2, OLINDA PINTO-CARNIDE 1,2 , CARLOS CARVALHO1, 2 and GILBERTO IGREJAS 1,2

1 Department of Genetics and Biotechnology, University of Tr á s-os-Montes and Alto Douro, Vila Real, Portugal 2 Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Tr á s-os-Montes and Alto Douro, Vila Real, Portuga l

Ribeiro, M., Seabra, L., Ramos, A., Santos, S., Pinto-Carnide, O., Carvalho, C. and Igrejas, G. 2011. Polymorphism of the storage proteins in Portuguese rye ( Secale cereale L.) populations. – Hereditas 149 : 72-84. Lund, Sweden. eISSN 1601-5223. Received 17 August 2011. Accepted 25 November 2011.

Currently, due to the abandonment of traditional agricultural practices and the decline of rye production in Portugal, there is a need to assess the genetic diversity of rye in order to preserve its biodiversity. Furthermore, a greater knowledge of rye secalins is important for rye bread-making quality and other crop breeding purposes.

The genetic variation and diversity of storage proteins were estimated for fourteen populations of rye ( Secale cereale L.) sampled in northern Portugal. The work showed the high genetic diversity within the Portuguese rye gene-pool as an important source for plant breeding and emphasized the necessity of an integrated resources genetic program to allow a more effi cient management and conservation of these resources. The rye populations were compared with ‘ Picasso ’ and ‘ Marder ’ varieties. Several alleles were identifi ed by the single electrophoretic mobility patterns. We studied a set of 1600 rye seeds, including regional populations and varieties, having observed a total of 24, 5, 21 and 47 alleles for HMW, 75k γ -, 40k γ - and ω -secalins, respectively. The coeffi cient of similarity within populations is presented using cluster representation. The mean value of genetic variation indices ( H ) for rye storage proteins was very high in regional populations, ranging from 0.67 to 0.78, while in the varieties ranged from 0.57 to 0.58. Knowledge of the diversity of secalins will increase our understanding of the quality differences between rye varieties, especially considering the relative small number of rye cultivars grown around the world.

Gilberto Igrejas, Department of Genetics and Biotechnology, University of Tr á s-os-Montes and Alto Douro, PT-5001-801 Vila Real, Portugal . E-mail: [email protected]

Rye ( Secale cereale L.) is an open pollinated, diploid (2n � 14), out breeding species, well adapted to cold winters and acidic soils of low productivity, being culti-vated mostly in the colder northern areas of Europe ( HELBAEK 1970; ZOHARY and HOPF 1993; BRITES et al. 2007; AKHAVAN et al. 2010).

Since the 1960s the area for cultivation and the produc-tion levels of rye ( Secale cereale L.) have generally been declining throughout the world. In Portugal, it declined from about 119 thousand tons in 1961 to 18 thousand tons currently, even importing annually a similar amount (FAOStat 2011). This phenomenon may be related to the lower economic interest in rye compared to other crops such as wheat and barley in western Europe and in the USA ( SHEWRY et al. 1983a, 1983b). In complement while production of rye has declined somewhat during the 1990s, production of the three major cereals, wheat, rice, and maize, has increased ( BUSHUK 1981, 2001). Likewise, and despite being very close taxonomically, the major storage proteins of wheat and barley, namely gliadins and glutenins, and

hordeins, respectively, have been more extensively stud-ied in relation to the proteins of rye (secalins) ( SHEWRY et al. 1984a; BUSHUK 2001; GELLRICH et al. 2002).

Analysis of storage protein alleles is well known to be a powerful tool for genotyping genetic resources, namely in cereals like wheat ( PAYNE and LAWRENCE 1983 ; BRANLARD and LE BLANC 1985; HAMER et al . 1992; IGREJAS et al . 1999; BRANLARD et al . 2003; RIBEIRO et al. 2011).

The storage proteins of rye can be separated into two fractions, the prolamins and glutelins, which correspond to wheat gliadins and glutenins, respectively. The prola-min fraction is extracted by 60 or 70% aqueous ethanol solution ( CHARBONNIER et al. 1981; KASARDA et al. 1983; KUBICZEK et al. 1993) and comprises mainly monomeric proteins. The second fraction, glutelins, is extracted sequentially by 50 or 55% 1-propanol or 2-propanol in the presence of a reducing agent ( SHEWRY et al. 1983a). This fraction consists of small subunits of complex polymers stabilized by inter-chain disulfi de bonds (equivalent to the glutenin of wheat) ( SHEWRY and BECHTEL 2001).

Hereditas 149: 72–84 (2012)

© 2012 The Authors. This is an Open Access article. DOI: 10.1111/j.1601-5223.2012.02239.x

Hereditas 149 (2012) Polymorphism of storage proteins in Portuguese rye 73

Sodium dodecyl sulphate – polyacrylamide gel elec-trophoresis (SDS-PAGE) revealed that the prolamin frac tions of rye contained all four storage protein types ( ω -secalins, 40 000-dalton (40k) γ -secalins, 75 000-dalton (75k) γ -secalins and high molecular weight (HMW) seca-lins), whereas the glutelin fractions contained only HMW and γ -75k secalins ( PRESTON and WOODBURY 1975; FIELD et al. 1982; SHEWRY et al. 1982, 1983a, 1983b; KASARDA et al. 1983).

HMW secalins have been assigned to loci on the long arms of chromosome 1R ( Glu-R1 or Sec-3 ) ( LAWRENCE and SHEPHERD 1981). The 40k γ -secalins are coded at loci Gli-R1 (or Sec-1 ) ( SHEPHERD 1968), ω -secalins at Gli-R3 (or Sec-4 ) ( CARRILLO et al. 1992) on chromosome 1RS and 75k γ -secalins at Gli-R2 (or Sec-2 ) on chromosome 2RS ( SHEWRY et al. 1984b). The 75k group of γ -secalins is clearly related to the 40k group ( SHEWRY et al. 1982). The ω -secalins are regarded as the major cause of poor grain quality in wheat 1BL/1RS translocation lines ( DHALIWAL et al. 1990).

However, rye is important for breeding purposes and for gene introgression in other cereal species like wheat, as a source of favourable agronomic traits. Features such as nutrient effi ciency, tolerance of diseases, allowing a reduced usage of pesticides and fertilizers during production ( BOLIBOK-BR A GOSZEWSKA et al. 2009), and more recently, the recognized dietary value ( ISAKSSON et al. 2009) makes rye an important genetic resource, which contributes to increase the variability and a more effective gene pool.

Rye proteins are important for rye bread-making quality, namely in the dough-mixing step. Different rye varieties show highly different bread-making quality and this can be attributed to signifi cant differences in the content and structure of starch and proteins, which are infl uenced by harvest year and genotype ( HANSEN et al. 2003, 2004).

The aim of the present work was to describe the allelic diversity of the storage proteins encoded at Sec-1 , Sec-2 , Sec-3 and Sec-4 loci in a collection of a Portuguese rye populations and infer the inter and intrapopulation variability using SDS-PAGE, as well to determine the geographical in fl uence on genetic diversity of these rye populations. Thus, the present work will allow a more effi cient management and conservation of the resources, and its incorporation in plant breeding programs.

MATERIAL AND METHODS

Plant material

Materials used in the present work were composed of fourteen regional populations and two varieties of rye ( Secale cereale L.) from the rye germplasm bank of University of Tr á s-os-Montes and Alto Douro. The fi rst

group includes fourteen populations of Secale cereale L. from northern Portugal namely ‘ Montalegre ’ , ‘ Vila Pouca ’ , ‘ Alv ã o ’ , ‘ Padrela ’ , ‘ Centenico ’ , ‘ Gimonde ’ , ‘ Lamego ’ , ‘ Lamas de Ô lo ’ , ‘ Malhadas ’ , ‘ Morgade ’ , ‘ Cepeda ’ , ‘ Vilar Douro ’ , ‘ Edral ’ and ‘ Atilho ’ . For each population 100 seeds were randomly selected for investi-gation. The second set of seeds includes two rye varieties namely ‘ Marder ’ and ‘ Picasso ’ and 100 seeds of each vari-ety were randomly selected for investigation. A total of 1600 seeds (belongs to 14 populations plus two varieties) were analysed and described in the present work. The geo-graphical origin of rye populations under study is shown in Fig. 1 and more specifi c information, including the year of multiplication, is listed in Table 1.

Electrophoresis

Storage proteins were extracted from half of a single seed of each population and separated by SDS-PAGE using the sequential procedure of SINGH et al. (1991). The prolamin fraction of rye storage proteins, essentially ω -secalins, was extracted from crushed endosperm with 50% propa-nol solution. The supernatant was dried at 65 ° C and the residue was dissolved in sample buffer. The glutelin fraction of rye storage proteins present in the pellet were reduced and alkylated in a 50% propanol solution with dithiothreitol (1%) and 4-vinylpyridine (0.74%), respectively. The electrophoresis of secalins was per-formed on vertical gel (180 � 160 � 1 mm) according to the SDS-PAGE protocol described by SINGH et al. (1991) with some modifi cations: constant gel concentration was preferred to gradient gel. The ω -secalins, and HMW, 40k and 75k γ -secalins were separated in a resolving

Fig. 1. Geographic origin of the Portuguese rye populations. 1 – ‘ Montalegre ’ , 2 – ‘ Vila Pouca ’ , 3 – ‘ Alv ã o ’ , 4 – ‘ Padrela ’ , 5 – ‘ Centenico ’ , 6 – ‘ Gimonde ’ , 7 – ‘ Lamego ’ , 8 – ‘ Lamas de Ô lo ’ , 9 – ‘ Malhadas ’ , 10 – ‘ Morgade ’ , 11 – ‘ Cepeda ’ , 12 – ‘ Vilar Douro ’ , 13 – ‘ Edral ’ , 14 – ‘ Atilho ’ .

74 M. Ribeiro et al. Hereditas 149 (2012)

Table 1. Geographical information, years of multiplications and ‘ population cultivars ’ sizes of rye analysed .

Population Location Latitude Longitude Elevation Year of multiplication Size of population

Montalegre Montalegre 41 ° 49′N 7 ° 47′ O 949 1999 100Vila Pouca Vila Pouca 41 ° 31′N 7 ° 37 ′ O 624 2000 100Alv ã o Alv ã o 41 ° 29′N 7 ° 36′O 1024 1999 100Padrela Padrela 41 ° 33′N 7 ° 30′O 994 2000 100Centenico Ribeira de Pena 41 ° 30′N 7 ° 47′O 485 1999 100Gimonde Gimonde 41 ° 48′N 6 ° 41′O 525 1996 100Lamego Lamego 41 ° 05′N 7 ° 47′O 441 2000 100Lamas de Ô lo Lamas de Ô lo 41 ° 22′N 7 ° 47′O 1021 1996 100Malhadas Malhadas 41 ° 32′N 7 ° 19′O 774 2000 100Morgade Montalegre 41 ° 45′N 7 ° 44′O 874 1995 100Cepeda Montalegre 41 ° 48′N 7 ° 40′O 965 1996 100Vilar Douro Mirandela 41 ° 42′N 7 ° 09′O 499 1999 100Edral Vinhais 41 ° 50′N 7 ° 09′O 824 1997 100Atilho Atilh ó – Boticas 41 ° 42′N 7 ° 47′O 1012 1995 100

gel using 10.3% and 12.52% T; 0.97 and 1.3% C, respec-tively. The gels were stained with Coomassie blue R-250 and visually analysed for allelic identifi cation.

For two-dimensional electrophoresis (2-DE) of rye secalins, the total secalin proteins of the rye seed were obtained by adding the extraction solution containing 4% Chaps, urea 7 M, Thiourea 2 M, 1% IPG buffer, 20 mM DTT and Milli-Q water. The fl our plus extraction solution were vortexed, sonicated and centrifuged. The rehydra-tion of the strips to 3-10 pH was carried out using a rehy-dration solution (extraction solution with bromophenol blue). Isoelectric focusing (IEF) conducted at 60 kVh was followed by SDS-PAGE and gels (T: 12.52%; C: 0.97%) were Coomassie stained.

Nomenclature

Due to the absence of a consensus nomenclature for the alleles of different rye storage proteins in the study (HMW, 75k γ -, 40k γ - and ω -secalins), different bands were num-bered according to their electrophoretic mobility as fol-lows: band 1 for the lower mobility band and higher molecular weight, the next number 2 and so on. In the spe-cifi c case of ω -secalins, which were analyzed in different gels, the description continued starting at the band 51.

The most representative electrophoretic patterns of HMW, 75k γ - and 40k γ -secalins, and of ω -secalins, revealed by SDS-PAGE for the rye populations and varie-ties, established to standardize and facilitate the analyses, are shown in Fig. 2 and 3, respectively.

≈ 120 kDa

4HMWsecalins

75k γ-secalins

40k γ-secalins

≈ 97 kDa

111520

252625

28

≈ 66 kDa

30

3435

404142

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

≈ 30 kDa

Fig. 2. Most representative SDS-PAGE pattern of HMW, 75k γ - and 40k γ -secalins.


in Table 2. A total of 97 different alleles for the rye storage proteins were detected.

For HMW secalins a total of 24 alleles were found. In terms of allelic frequencies, bands 4, 5 and 11 were the most frequent in the populations studied. The variety ‘ Marder ’ showed a frequency of 100% for the band 4, followed by the populations ‘ Montalegre ’ , ‘ Vila Pouca ’ , ‘ Alv ã o ’ , ‘ Padrela ’ , ‘ Malhadas ’ and ‘ Morgade ’ with values of 86.67, 84, 80.67, 85.33, 75 and 78%, respectively. Fol-lowing a similar pattern, the band 5 of HMW secalins was present in all seeds studied of the variety ‘ Picasso ’ and population ‘ Atilho ’ and with frequencies of 89 and 92% in the populations ‘ Gimonde ’ and ‘ Vilar Douro ’ , respectively. For the band 11, it presented a frequency of 100% for varieties studied, with values ranging from 19 to about 83% for the populations, except ‘ Morgade ’ , ‘ Cepeda ’ and ‘ Gimonde ’ populations, with residual values between 2 and 6%. Additionally, the presence of bands 10, 15 and 16 of HMW secalins in virtually all popula-tions studied should be noted; only in the population

Statistical analysis

Nei ’ s genetic variation index, H , was calculated for each locus profi le as follows: H � 1 � �p2 i , where p i is the frequency of a particular allele ( NEI 1973). Genetic dissimilarity between rye populations and varieties was calculated according to the model presented by NEI (1972, 1978) and WRIGHT (1978). The presence or absence of each allele was scored in a binary data matrix. Genotypes were grouped by cluster analysis according to their rela-tionship using the average linkage between groups fusion method (UPGMA, unweighted pair group method with arithmetic average) in the NTSYS-pc program, ver. 2-10a ( ROLPH 2000).

RESULTS

Allelic diversity of HMW, 75k g -, 40k g - and w -secalins

The frequencies of the alleles identifi ed at the four loci encoding HMW, 75k γ -, 40k γ - and ω -secalins are shown

565452

≈ 120 kDa

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≈ 97 kDa

ω-s

ecal

ins

69

67

73

84

81

77

80

73

≈ 66 kDa

89

84

≈ 30 kDa

1 2 3 4 5 7 86

Fig. 3. Most representative SDS-PAGE pattern of ω -secalins.

76 M. Ribeiro et al. Hereditas 149 (2012) Ta

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0.00

0.00

0.00

0.00

0.00

0.00

0.00

470.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

670.

000.

0048

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.67

0.00

0.00

490.

000.

670.

000.

000.

000.

000.

000.

000.

000.

000.

004.

002.

000.

000.

000.

0050

0.00

0.67

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

8.00

2.00

0.00

0.00

0.00

ω -s

ecal

ins

Sec-

2 51

0.00

0.00

0.00

81.3

33.

000.

000.

003.

000.

000.

0046

.00

0.00

0.00

10.6

70.

000.

0052

99.3

399

.33

100.

006.

0094

.00

81.0

010

0.00

97.0

089

.00

100.

0098

.00

100.

0010

0.00

89.3

310

0.00

100.

0053

90.6

70.

002.

672.

000.

0016

.00

0.00

4.00

1.00

4.00

0.00

0.00

0.00

0.67

0.00

0.00

546.

6716

.00

31.3

321

.33

7.00

5.00

0.00

21.0

012

.00

13.0

00.

000.

000.

0018

.00

100.

0010

0.00

556.

670.

676.

672.

6728

.00

8.00

0.00

0.00

5.00

10.0

00.

000.

000.

0010

.00

0.00

0.00

5699

.33

99.3

310

0.00

97.3

394

.00

97.0

010

0.00

100.

0010

0.00

100.

0099

.00

100.

0010

0.00

96.6

710

0.00

100.

0057

5.33

1.33

0.00

0.00

0.00

3.00

0.00

0.00

0.00

3.00

2.00

3.00

6.00

7.33

0.00

0.00

582.

000.

000.

670.

002.

000.

003.

000.

000.

005.

001.

000.

002.

001.

330.

000.

0059

4.67

0.00

2.00

0.67

2.00

0.00

4.00

0.00

0.00

9.00

1.00

0.00

5.00

2.00

0.00

0.00

600.

000.

001.

332.

670.

004.

002.

000.

000.

002.

000.

000.

003.

004.

670.

000.

0061

10.0

00.

008.

001.

3320

.00

1.00

1.00

34.0

00.

000.

001.

000.

007.

0010

.00

0.00

0.00

6226

.00

6.67

8.67

4.67

22.0

07.

0014

.00

39.0

00.

000.

0021

.00

0.00

8.00

14.0

00.

000.

0063

1.33

0.00

0.67

0.67

4.00

8.00

14.0

00.

000.

000.

0012

.00

0.00

1.00

2.00

0.00

0.00

642.

670.

001.

330.

000.

001.

000.

001.

000.

001.

000.

000.

001.

000.

000.

000.

0065

17.3

30.

003.

330.

671.

001.

000.

000.

000.

005.

001.

000.

000.

000.

6710

0.00

0.00

6630

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3.33

10.0

05.

3319

.00

3.00

13.0

00.

002.

0027

.00

17.0

00.

0011

.00

4.67

0.00

0.00

6783

.33

32.6

738

.67

32.0

023

.00

5.00

12.0

00.

002.

0037

.00

27.0

00.

0036

.00

23.3

30.

0043

.85

6892

.67

30.0

073

.33

65.3

366

.00

39.0

041

.00

0.00

36.0

073

.00

51.0

052

.00

0.00

36.6

710

0.00

76.1

569

10.6

789

.33

100.

0018

.67

47.0

021

.00

9.00

41.0

01.

0080

.00

87.0

029

.00

74.0

030

.67

0.00

26.1

570

1.33

71.3

317

.33

16.6

710

.00

70.0

072

.00

75.0

025

.00

3.00

25.0

033

.00

0.00

76.6

70.

0019

.23

7160

.67

0.67

8.67

6.67

1.00

5.00

87.0

015

.00

91.0

07.

001.

003.

000.

0010

.67

100.

005.

3872

2.00

56.6

70.

6712

.00

1.00

0.00

14.0

00.

002.

003.

000.

001.

002.

003.

330.

000.

0073

51.3

30.

005.

3314

.67

39.0

01.

001.

008.

000.

0096

.00

33.0

064

.00

18.0

035

.33

100.

0014

.62

740.

0039

.33

0.00

0.67

0.00

0.00

14.0

024

.00

5.00

0.00

12.0

00.

001.

003.

330.

000.

00

(Con

tinu

ed )


Loc

usH

MW

se

calin

s

Popu

latio

nsV

arie

ties

Mon

tale

gre

Vila

Po

uca

Alv

ã oPa

drel

aC

ente

nico

Gim

onde

Lam

ego

Lam

as

de Ô

loM

alha

das

Mor

gade

Cep

eda

Vila

r D

ouro

Edr

alA

tilho

Mar

der

Pica

sso

750.

000.

000.

000.

000.

004.

002.

0017

.00

29.0

00.

000.

000.

000.

000.

670.

000.

0076

29.3

30.

000.

000.

000.

002.

0015

.00

2.00

0.00

0.00

0.00

1.00

2.00

2.00

0.00

0.00

7799

.33

97.3

332

.67

26.6

741

.00

27.0

019

.00

1.00

3.00

60.0

033

.00

53.0

026

.00

50.0

010

0.00

40.7

778

2.00

2.67

0.00

0.00

0.00

14.0

08.

0032

.00

36.0

00.

0016

.00

0.00

0.00

0.00

0.00

0.00

7936

.00

6.67

0.00

2.00

0.00

0.00

2.00

11.0

00.

000.

002.

000.

000.

000.

0010

0.00

0.00

800.

000.

0014

.00

46.0

075

.00

3.00

22.0

07.

009.

0024

.00

0.00

0.00

0.00

13.3

30.

000.

0081

2.67

0.00

100.

0092

.67

94.0

052

.00

19.0

010

0.00

86.0

096

.00

100.

0010

0.00

100.

0097

.33

100.

0010

0.00

826.

000.

0034

.67

16.6

721

.00

3.00

6.00

0.00

0.00

15.0

014

.00

0.00

0.00

26.0

00.

000.

0083

8.67

0.00

3.33

1.33

0.00

0.00

79.0

00.

004.

008.

002.

000.

008.

0036

.00

0.00

0.00

8499

.33

98.6

74.

670.

000.

0010

0.00

10.0

00.

000.

000.

000.

000.

000.

000.

0010

0.00

0.00

8537

.33

40.0

00.

000.

000.

008.

0010

.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

860.

002.

674.

670.

000.

003.

000.

000.

000.

000.

000.

000.

000.

000.

670.

000.

0087

0.00

0.00

12.0

03.

330.

000.

003.

000.

001.

006.

003.

003.

001.

001.

330.

000.

0088

0.00

0.00

38.6

712

.67

24.0

00.

003.

000.

004.

0014

.00

0.00

29.0

07.

0010

.67

0.00

0.00

890.

000.

0010

0.00

95.3

390

.00

0.00

1.00

100.

0080

.00

98.0

010

0.00

100.

0010

0.00

94.6

70.

0010

0.00

900.

000.

0028

.00

49.3

313

.00

0.00

52.0

00.

0064

.00

1.00

0.00

0.00

16.0

029

.33

0.00

0.00

910.

000.

005.

334.

000.

000.

004.

000.

000.

000.

000.

000.

001.

002.

000.

000.

0092

0.00

0.00

0.00

0.00

1.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

930.

000.

000.

000.

001.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

0094

0.00

0.00

0.00

0.00

0.00

0.00

1.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

950.

000.

000.

000.

000.

000.

001.

000.

000.

000.

000.

000.

000.

000.

000.

000.

0096

0.00

0.00

0.00

0.00

0.00

0.00

2.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

970.

000.

000.

000.

670.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

000.

00

Tabl

e 2.

(C

ontin

ued)


to 100% for most populations studied. The bands 68, 69, 70 and 77 are also very common. The most common allelic pattern for the ω -secalins throughout the collection studied was 52-56-68-77-81-89. In addition, the very low frequency registered from the band 90 is worthy of note, with the maximum frequency (5.33%) to be obtained for the band 91 in the ‘ Alv ã o ’ population.

The mean values of the genetic variation indices and comparison of genetic dissimilarity indices among the populations and varieties studied are shown in Table 3 and 4, respectively.

All the populations studied showed a high level of polymorphism. The mean value of the genetic variation indices of the four secalin polymorphic loci ranged from 0.67 to 0.78. ‘ Morgade ’ and ‘ Vilar Douro ’ populations showed the highest genetic variation and ‘ Centenico ’ the lowest. In relation to the studied varieties, lower average values of genetic variation, ranging from 0.57 to 0.58, were obtained. Concerning the studied loci, Sec-1 , Sec-2 , Sec-3 and Sec-4 encoding respectively the 40k γ -secalins, 75k γ -secalins, HMW secalins and ω -secalins, a high level of polymorphism was found, the ω -secalins presenting the highest variation index. The 40k γ -secalins was always the lowest polymorphic group, showing no polymor-phism (values of H � 0) for rye varieties studied.

Regarding the genetic dissimilarity indices among the populations, ‘ Vilar Douro ’ and ‘ Edral ’ showed the closest relationship with a genetic dissimilarity value of 0.0821. The highest value (0.5484) was obtained for the ‘ Edral ’ population and ‘ Marder ’ variety. The varieties obtained a higher degree (0.3627) of genetic dissimilarity than the

‘ Cepeda ’ in relation to the fourteen populations studied its presence was not detected.

Regarding the 75k γ -secalins, both populations and varieties showed a very high frequency for bands 25, 26 and 28. ‘ Cepeda ’ , ‘ Edral ’ and ‘ Atilho ’ populations, and ‘ Marder ’ variety showed a frequency of 100% for the allelic constitution 25-26-28. The variety ‘ Picasso ’ revealed a unique allelic constitution presenting a fre-quency of 99.23 and 96.15% for the bands 25 and 27, respectively.

A total of 21 alleles were found for the 40k γ -secalins. The presence of the band 41 in virtually all populations and varieties, is noteworthy. It achieves maximum fre-quency in about nine of the fourteen populations studied and in the two varieties. Only ‘ Malhadas ’ did not show this band, but achieved the maximum frequency for the band 40. Moreover, the bands 40 and 42 must be indicated as measured values of frequency ranging from 98 to 100% for some populations: ‘ Alv ã o ’ (99.33% for band 40), ‘ Gimonde ’ (100% for band 42), ‘ Malhadas ’ and ‘ Morgade ’ (100% for band 40) and ‘ Vilar Douro ’ (98% for band 42).

The ω -secalins is the group of proteins that showed higher polymorphism, being observed a total of 47 alleles. Especially notable is the presence of the bands 52 and 56 in all populations and varieties studied, with frequencies very close and equal to 100%. Only the ‘ Padrela ’ popula-tion does not present as the most frequent pattern 52-56, with a frequency of 81.33% for the band 51 and 97.33% for the band 56. Also noteworthy are the bands 81 and 89 which also showed frequencies very close or equal

Table 3. Genetic variation indices for the rye populations and varieties studied.

Populations

H

Mean HMW secalins 75k γ -secalins 40k γ -secalins ω -secalins

Montalegre 0.75 0.89 0.70 0.50 0.93Vila Pouca 0.73 0.85 0.69 0.49 0.90Alv ã o 0.77 0.84 0.68 0.66 0.92Padrela 0.74 0.86 0.67 0.53 0.92Centenico 0.67 0.84 0.67 0.26 0.92Gimonde 0.68 0.84 0.69 0.31 0.89Lamego 0.75 0.87 0.68 0.53 0.92Lamas de Ô lo 0.76 0.90 0.67 0.59 0.90Malhadas 0.69 0.88 0.68 0.31 0.90Morgade 0.78 0.87 0.69 0.66 0.92Cepeda 0.75 0.89 0.68 0.53 0.91Vilar Douro 0.78 0.89 0.68 0.68 0.88Edral 0.77 0.87 0.67 0.68 0.88Atilho 0.72 0.84 0.67 0.46 0.93Mean 0.74 0.87 0.68 0.51 0.91Varieties

Marder 0.58 0.76 0.67 0.00 0.91Picasso 0.57 0.67 0.74 0.00 0.88Mean 0.58 0.71 0.70 0.00 0.90


populations (0.2762). Considering only the populations studied, the highest value of genetic dissimilarity indices (0.5338) was obtained between ‘ Vila Pouca ’ and ‘ Malha-das ’ , these being the most distant populations studied.

Cluster analyses with presence/absence of storage pro-tein alleles, using the unweighted pairwise group methods with arithmetical average (UPGMA), were performed with different algorithms in the program NTSYS-pc ( ROLPH 2000). The results were used to generate a dendro-gram displaying the hierarchical associations among all populations and varieties studied. Results were similar to the different trees and only the one based on NEI (1972) is shown (Fig. 4).

DISCUSSION

Portuguese rye populations maintained by farmers have a large genetic variability detected using SDS-PAGE of rye storage proteins. The present study represents one of the fi rst monitoring activities of variability in rye populations made using the study of storage proteins. Most of the studies carried out in different countries focused on the variability and relationships among rye populations used allozymes ( PEREZ DE LA VEGA and ALLARD 1984; RAMIREZ et al. 1985; ADAM et al. 1987; CARNIDE et al. 1997) and/or random ampli fi ed polymorphic DNA (RAPD) and inter- simple sequence repeat (ISSR) markers ( MATOS et al. 2001; PERSSON et al. 2001).

The results obtained showed a wide range of alleles and a high level of polymorphism. The fourteen populations studied here presented a mean value of the genetic variation indices of the four secalin polymorphic loci that ranged from 0.67 to 0.78. Regarding the varieties studied, the genetic variation ranged from 0.57 to 0.58, contrasting with the values obtained for the populations. In parallel, all loci, Sec-1 , Sec-2 , Sec-3 and Sec-4 encoding the 40k γ -secalins, 75k γ -secalins, HMW secalins and ω -secalins, respectively showed a high level of polymorphism. For both populations and varieties studied, ω -secalins showed the highest variation index, while the 40k γ -secalins group was always less polymorphic. None particular association was founded with different groups of bands highlight the same locus of tightly linked genes (data not shown).

The genetic variation observed in the present study is higher than reported by PERSSON and BOTHMER (2002) using allozyme patterns. They studied the genetic diver-sity amongst landraces of rye ( Secale cereale L.) from northern Europe and obtained lower variability indices for the landraces in relation to the populations studied here, resembling more the values obtained for rye varieties.

Using isozyme loci and PCR markers (ISSRs and RAPDs), MATOS et al. (2001) analyzed a larger number of loci and described phylogenetic relationships of 10 rye landraces and cultivars from the north of Portugal and Ta

ble

4. C

ompa

riso

n of

gen

etic

dis

sim

ilar

ity

indi

ces

amon

g th

e po

pula

tion

s an

d va

riet

ies

stud

ied

base

d on

SD

S-P

AG

E a

naly

sis .

Mon

tale

gre

Vila

Po

uca

Alv

ã oPa

drel

aC

ente

nico

Gim

onde

Lam

ego

Lam

as

de Ô

loM

alha

das

Mor

gade

Cep

eda

Vila

r D

ouro

Edr

alA

tilho

Mar

der

Pica

sso

Mon

tale

gre

0.00

00V

ila P

ouca

0.19

660.

0000

Alv

ã o0.

4000

0.32

540.

0000

Padr

ela

0.38

060.

3890

0.19

070.

0000

Cen

teni

co0.

3160

0.29

100.

1443

0.13

370.

0000

Gim

onde

0.33

710.

2871

0.47

060.

5095

0.38

050.

0000

Lam

ego

0.30

390.

2828

0.40

800.

3626

0.26

620.

2838

0.00

00L

amas

de

Ô lo

0.47

750.

3583

0.28

250.

2810

0.16

370.

2554

0.21

920.

0000

Mal

hada

s0.

4883

0.53

380.

1740

0.27

960.

2923

0.43

810.

3177

0.31

600.

0000

Mor

gade

0.33

140.

3616

0.14

570.

2317

0.12

270.

4691

0.33

950.

2337

0.28

230.

0000

Cep

eda

0.39

780.

3094

0.15

760.

2021

0.12

390.

3547

0.34

450.

1508

0.29

760.

1295

0.00

00V

ilar

Dou

ro0.

3998

0.39

540.

3051

0.30

520.

1498

0.23

570.

2775

0.11

210.

3975

0.18

230.

1772

0.00

00E

dral

0.44

460.

3662

0.24

650.

2819

0.13

830.

3034

0.28

490.

0892

0.38

430.

1928

0.14

360.

0821

0.00

00A

tilho

0.41

990.

3312

0.23

260.

2250

0.10

780.

2693

0.17

990.

1063

0.30

960.

1762

0.16

320.

0858

0.11

400.

0000

Mar

der

0.24

370.

3390

0.42

860.

4198

0.34

550.

5356

0.43

230.

5362

0.47

060.

3875

0.45

150.

4616

0.54

840.

4550

0.00

00Pi

cass

o0.

5168

0.44

080.

2829

0.32

350.

2204

0.50

260.

4492

0.33

140.

4356

0.34

550.

3113

0.26

760.

2708

0.21

090.

3267

0.00

00


provide reasons for the high levels of genetic variation found. Rye is a species that usually outcrosses and consequently, most varieties are mixtures of genotypes.

Contamination by foreign pollen during multiplication may have changed the genetic identity of rye populations, which emphasizes the need for extended efforts to main-tain the genetic integrity of the rye germplasm ( CHEBOTAR et al. 2003). All populations were subjected to a multi-plication process between 1995 and 2000 (Table 1). More-over, the abandonment of traditional practices of crop production and declining production of rye in Portugal since the 1960s, owing to lower economic viability, may have led to the abandonment of local cultures and conse-quently to loss of genetic identity of different popula-tions of rye. In contrast, the rye varieties studied showed values of genetic variation below those obtained for the rye populations, mainly due to the fact that they are sub-ject to different selective pressures and breeding.

Using the SDS-PAGE methodology, SHEWRY et al. (1983a) showed similar results to those presented in this study: high genetic variation in secalins and the presence of high variation in the band patterns of all four groups of secalin polypeptides. They also reported that, because of the intravarietal heterogeneity, the analysis of secalin polypeptide patterns is of limited potential value for the varietal identifi cation of single seeds of rye, which is in contrast to the situation verifi ed with wheat and barley

from Brazil. The values reported were similar to those reported by PERSSON and BOTHMER (2002), resembling those obtained for the varieties, being lower compared to the populations studied.

Comparing our results with those obtained by AKHAVAN et al. (2010) using microsatellites, minor differences were obtained for the populations studied. Likewise, BOLIBOK-BR A GOSZEWSKA (2009) reported a large genetic diversity for rye, using diversity arrays technology (DArT) markers.

In addition, although the values of genetic variation shown are higher, it should be noted that there is no basis for direct comparison of the results obtained in this study because no previous studies of variability using the char-acterization of storage proteins of rye are available. More-over, most of the diversity was observed within populations (0.74) in contrast with the variation revealed between populations (0.28). This situation is completely consistent with the studies mentioned above for other molecular markers ( MATOS et al. 2001; PERSSON and BOTHMER 2002; AKHAVAN et al. 2010).

High levels of heterozygosity and heterogeneity are characteristic for open-pollinating species and, as expected in an open-pollinating crop, the genetic diversity was higher within than between populations. The fact that rye ( Secale cereale L.) is an open-pollinating species and traditional varieties of rye are panmictic populations may

Coefficient0.08 0.16 0.24 0.32 0.40

Montalegre

Vila Pouca

Marder

Alvão

Centenico

Morgade

Cepeda

Padrela

Lamas de Ôlo

Vilar Douro

Edral

Atilho

Picasso

Malhadas

Gimonde

Lamego

Fig. 4. UPGMA dendrogram of the genetic distances for the populations and varieties studied.


‘ Lamas de Ô lo ’ , ‘ Morgade ’ , ‘ Cepeda ’ , ‘ Vilar Douro ’ , ‘ Edral ’ and ‘ Atilho ’ populations and a possible relationship to their geographical origin, is evident with the exception of the ‘ Montalegre ’ and ‘ Vila Pouca ’ populations, there seems to be a relationship between the group formed in the den-drogram and in the map.

Crossing the data relative to years of multiplication and geographic origin, only ‘ Alv ã o ’ and ‘ Centenico ’ popula-tions seem to have a close genetic relationship associated to the two factors. The varieties studied, ‘ Marder ’ and ‘ Picasso ’ did not show any clear relationship between them.

Conclusions

The allelic identifi cation of rye storage proteins in Portuguese rye populations revealed high diversity. Although other methods such as polymerase chain reac-tion (PCR) provide interesting tools for the type of study reported here, if high defi nition gels can be obtained the use of 1D SDS-PAGE continues to be a valuable, effi cient and economical method. Analysis of storage protein alleles is well known to be a powerful tool for genotyping genetic resources and SDS-PAGE of secalins provides an easy tool for allelic identifi cation and many genotypes can be distin-guished. However, due to the high intravarietal hetero-geneity, the analysis of secalin polypeptide patterns is of limited potential value for the rye varietal identifi cation.

The high genetic diversity found in the Portuguese populations can be valuable to search for useful new alleles for crop improvement. However, due to the increas-ing abandonment of traditional agricultural practices and the decline of production of rye in the north of Portugal in detriment to crops with higher economic advantage, this diversity may be at risk.

Of the cereal fl ours, only wheat and rye can be used successfully in production of leavened bread. This work provided important knowledge about the polymorphism of rye storage proteins, which are an important fac-tor for rye bread-making quality, namely in the dough-mixing step. In addition to their nutritional importance, rye seed proteins also infl uence the utilization of the grain in food processing. This work contributes to a better understanding of the frequencies on Portuguese rye ger-mplasm and the results obtained will allow a more effi -cient management and conservation of these resources, and their effi cient use in breeding programs. Our study may assist in deciding on which ‘ population cultivars ’ are important to conserve. It may also be possible to reduce stored populations by collecting material closely related to one unit. However, it must be emphasised that fi nal decision on genetic rye resources cannot be exclusively based on molecular markers. Although rye is inferior in several ways to the predominant cereal crops (wheat, rice and maize), it will continue to be an important crop for

( AUTRAN 1975; SHEWRY et al. 1979). We share the view that the analysis of storage proteins of rye is of limited potential for varietal identifi cation. Although some pop-ulations studied present very characteristic bands, the observed high polymorphism and high rates of genetic variation calculated for the loci encoding rye storage proteins, make varietal identifi cation very diffi cult. Two-dimensional electrophoresis (IEF � SDS-PAGE) of the most common pattern of the ‘ Montalegre ’ population confi rms the previous complexity of secalins observed by one dimensional (1D) electrophoresis (Fig. 5).

The presence/absence of storage protein alleles was used to generate phylogenetic trees showing relationships of the rye populations with the different algorithms in the program NTSYS-pc ( ROLPH 2000). Results were similar to the different trees; only the one based on NEI (1972) is shown (Fig. 4). Discrimination of rye populations into groups can be revealed by cluster analysis in the dendrogram.

The dendrogram resulting from UPGMA of the four storage protein loci shows that the populations were dis-tributed into a large number of groups. Analyzing the populations for the year of multiplication, it is not possible to see any clear relationship between them. For example, the closest populations ‘ Edral ’ and ‘ Vilar Douro ’ (0.0821) underwent processes of multiplication in 1997 and 1999, respectively. The closer relations with equal years of multiplication are between the populations ‘ Padrela ’ and ‘ Malhadas ’ (0.2796) for the year 2000, ‘ Alv ã o ’ and ‘ Centenico ’ (0.1443) for the year 1999, ‘ Lamas de Ô lo ’ and ‘ Cepeda ’ (0.1508) for the year 1996 and ‘ Atilho ’ and ‘ Morgade ’ (0.1762) for the year 1995.

Regarding the geographical origin of populations, we can observe the closeness between the populations ‘ Vilar Douro ’ and ‘ Edral ’ (0.0821), ‘ Alv ã o ’ and ‘ Centenico ’ (0.1443) and ‘ Morgade ’ and ‘ Cepeda’ (0.1295). Further-more, the formation of a central extended group in the den-drogram composed of the ‘ Alv ã o ’ , ‘ Padrela ’ , ‘ Centenico ’ ,

3 10

HMW secalins

γ-secalins

ω-secalins

SDS-PAGE

IPG

Fig. 5. Two-dimensional IEF (3-10) � SDS-PAGE pattern of ‘ Montalegre ’ population.


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