Marker Assisted Selection for Complementation of Glu-A1b, Glu-B1i, and Glu-D1d alleles in Early Segregating Generations of Wheat

Mohsen Mohammadi^1*, Elham Mehrazar², Ali Izadi-Darbandi², Goodarz Najafian¹

¹Molecular Genetics Unit, Cereals Research Department, Seed and Plant Improvement Institute, P.O.Box 4119, Karaj, Iran

²Department of Agronomy and Plant Breeding Sciences, College of Aburaihan, University of Tehran, P.O.Box 3391653755, Tehran, Iran

^*Corresponding author: Mohsen Mohammadi (E-mail: mohsen@ualberta.ca)

Abstract

Marker assisted selection (MAS) is believed to revolutionize breeding practices through improved improve efficiency and precision of selection, but the implementation of the technology is restricted to limited public funding. Here we demonstrate MAS procedure with significant reduction of workload to fix alleles in early generations. In recent years, advancements in molecular genetics resulted in identification of DNA tags associated with specific alleles of high-molecular-weight glutenin subunits (HMW-GSs) loci involved in bread making quality i.e., Glu-A1, Glu-B1, and Glu-D1. In this study, we report on utilization of three molecular markers selecting for alleles attributed to high gluten strength i.e., Glu-A1b (2*), Glu-B1i (17+18), and Glu-D1d (5+10). Field grown segregating wheat plants at F₂ generation of an intercross (with allelic complementarity) were subjected to selection by using three molecular markers. Individuals deemed positive on the three markers were selected and the corresponding seeds were used to constitute F₃ generation. Nearly 15 promising heads per each F₃ family were tagged and the corresponding flag leaves were collected for marker analysis. The heads corresponding to flag leaves that were positive for the three markers were selected to proceed with F₄ generation. Marker analysis of DNA extracted from seedlings at F₄ generation revealed that F₄ individuals selected contain all three Glu-A1b (2*), Glu-B1i (17+18), and Glu-D1d (5+10).

Key words: wheat, marker assisted selection, HMW-GS, allele specific marker

Introduction

Decades of investment in biotechnology has resulted in tools i.e. tissue culture, doubled haploid production, transgenic organisms, and DNA tags to revolutionize breeding science. DNA tags play as landmarks guiding the selection of chromosomal segments of interest in breeding procedures. The use of DNA tags may facilitate accumulation of multiple genes of interest in a breeding material or so-called gene pyramiding. Tracking chromosomal segments and selections thereby in breeding materials by means of molecular markers and DNA tags is referred to as marker assisted selection (MAS) (Dubcovsky et al., 2004). However, this methodology has not yet been implemented in many public wheat breeding programs (Koebner and Summers, 2003). MAS would also improve efficiency and precision of conventional plant breeding (Collard and Mackill, 2008). In MAS procedure, gene of interest is naturally transferred through meiotic chromosomal recombination and therefore, public perception is not against implementation of such technologies programs. However, implementation of such technologies is delayed due to restricted public funding (Dubcovsky et al., 2004).

Bread making quality is associated with glutenin composition and properties (Bottomley et al., 1982; Huebbner and Wall, 1976). Glutenin properties control dough resistance and extensibility(Eagles et al., 2006) and gluten elasticity and extensibility have significant influence on dough viscoelasticity (Belderok, 2000) and bread making quality of wheat (Nieto-Taladriz et al., 1994). Gluten consists of high-molecular-weight glutenin subunits (HMW-GSs) and low-molecular-weight glutenin subunits (LMW-GSs) (Lindsay et al., 2000). HMW-GSs are encoded by loci Glu-A1, Glu-B1, and Glu-D1, collectively referred to as known as Glu-1 loci (Ammar et al., 2000; Payne et al., 1981; Payne et al., 1987). Allelic variations in Glu-A1, Glu-B1, and Glu-D1 were reported to have strong association with gluten strength (Payne et al., 1981; Payne et al., 1987). Traditionally, public breeding programs evaluate pure lines produced at the end of each cycle of breeding for examination of allelic polymorphism of HMW-GS by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) systems. However, this approach helps in detection of HMW-GS alleles rather than enabling breeders to play selecting role in pyramiding the high scoring alleles. In contrast to such approach, tracing of genes and alleles of interest in segregating populations by using molecular markers at DNA level enables breeders to precisely select for promising segregants with desired recombination. This communication reports the use of DNA tags for precise selection of segregants that possess high score alleles at Glu-1 loci i.e., Glu-A1b (2*), Glu-B1i (17+18), and Glu-D1d (5+10).

Materials and Methods

In this study we demonstrated MAS in early generations (F₂ and F₃) by selecting for high scoring alleles for improved bread making quality in a public wheat breeding program. Segregating wheat materials included F₂ generation from an intercross made between cultivars and breeding materials including, Parsi, Pishtaz, M-84-14, and M-83-17 as parental lines as demonstrated in Fig. 1A. M-83-17 is a breeding line obtained from a cross between Alvand and BACANORA-88. Alvand (pedigree: CF1770/1-27-6275) is a facultative wheat released for moderate and cold regions of Iran in 1995. Because 1-27-6275 is a landrace, Alvand is known to be moderately tolerant to drought, salinity, and frost. BACANORA-88 (pedigree: JUPATECO-73/(SIB)BLUEJAY//URES-81) is a spring wheat developed and released by CIMMYT in Mexico in 1988. Parsi named after Persian language (pedigree: Dove”S” / Buc”S” // 2* Darab) is a wheat cultivar released in 2011 by wheat program Iran for moderate region. Parsi is a product of backcross conventional breeding between a maternal parent named DOBUC 1 (pedigree: Dove”S” / Buc”S”) developed by CIMMYT/ICARDA in 1995 as the donor parent which shows moderate drought tolerance in Iran and a paternal recurrent germplasm from Iran named Darab. Darab (named after the local area of germplasm enhancement in Darab located at Fars province) is a relatively old red spring wheat cultivar released in 1980 for warm and dry regions of Iran. Pishtaz (equivalent to Frontier in English) is a modern spring cultivar released in 2002 for moderate region with terminal drought tolerance. Pishtaz is a progeny of a cross between Alvand and a Brazilian cultivar Aldan / Ias58. Aldan / Ias58 has shown durable rust and Karnal bunt resistance in India for over a decade. 20 M-84-14 is a breeding line obtained from a cross between Gonam (Ww33 / Vee”S”) and Niknejad. Gonam is a spring wheat cultivar developed by CIMMYT / ICARDA in 1989. And Niknejad (pedigree: F13471/Crow"s”) is a spring wheat cultivar developed by ICARDA and released in Iran in 1995 showing tolerance to limited water availability. The DNA diagnostic test for parental cultivars and breeding materials is given in Fig. 1B.

Forty F₂ individuals were selected and tagged in the field from which a segment of leaf was collected. DNA was extracted from leaf tissue using a rapid and small-scale DNA isolation by flash freezing the excised leaf sample from individual F₂ segregants in liquid nitrogen followed by grinding to find powder. Extraction was then performed in 2 mL sterile tubes. A quarter of the volume of 2 mL tube was filled with frozen leaf powder and 0.9 mL of preheated (up to 65ºC) CTAB buffer [2% (w/v) cetyl trimethyl ammonium bromide, 200 mM Tris/HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, and 1% (v/v) freshly added β-mercaptoethanol] was added. Samples were heated for 45 min at 65ºC. 900 µL Pre-cooled 24:1 (chloroform : isoamylalcohol) mixturewas added to the heated tubes. The extraction mixes were centrifuged at 12000 rpm for 15 min at 4ºC. The upper layer was then collected and transferred into a new tube and was supplemented with 700 µL of pre-cooled (-20ºC) isopropanol and 100 µL of 3 M ammonium acetate. DNA was precipitated by incubating at -20ºC for at least one hour. The precipitates were pelleted by centrifugation at more than 11000 rpm for 10 min at 4ºC. The pellets were washed with 300 µL of pre-cooled 70% (v/v) ethanol. The DNA pellets were dissolved in 70 µL of nuclease free water. MAS within F₂ and the successive F₃ generations was performed by DNA tags after a pre-selection made by breeder based on plant sand and ideotype.

For selecting for allele Glu-A1b (2*) and Glu-B1i (17+18) allele against other alleles at Glu-A1 and Glu-B1 loci, respectively, we have used primers reported by Ma et al. (2003). Glu-A1b allele was traced by using primer pairs Fwd: 5’-ATGACTAAGCGGTTGGTTCTT-3’ and Rev: 5’-ACCTTGCTCCCCTTGTCTTT-3’.  Glu-B1i allele was traced by using primer pairs Fwd: 5’- CGCAACAGCCAGGACAATT-3’ and Rev: 5’-AGAGTTCTATCACTGCCTGGT-3’. For selecting for Glu-D1d allele at Glu-D1 locus we have used primer pairs developed by Vjell (1998) i.e., Fwd: 5’-GCCTAGCAACCTTCACAATC-3’ and Rev: 5’-GAAACCTGCTGCGGACAAG-3’. The cycling conditions were as described by Ma et al. (2003) and Vjell (1998). DNA templates containing Glu-A1b, Glu-B1i, and Glu-D1d alleles are able to yield PCR products of sizes ~1319 bp, 669 bp, and 450 bp, respectively (Ma et al., 2003; Vjell, 1998).

PCR reaction mixes of 25 µL final volume contained 1 µl DNA, 2.5 µl 10X Taq buffer, 1.5 mM MgCl₂, 2.5 mM of each dNTP, 0.4 pmol of each primer, 1 unit of Taq polymerase. PCR amplifications for Glu-A1b, Glu-B1i, and Glu-D1d alleles consisted of an initial denaturing step of 94ºC for 4 min, followed by 35 cycles of denaturation at 94ºC for 30 s, annealing at 59ºC for 30 s, and extension at 72ºC for 90 s. The PCR reactions were terminated by a final extension of 72ºC for 10 min. PCR reactions were performed in an Eppendorf Mastercycler® (Eppendorf AG, Hamburg, Germany). For visualization, PCR products were run on 1% or 1.5% agarose gel electrophoresis followed by ethidum bromide staining. Imaging was performed by a UVItec Gel Document system (UVItec Limited, Cambridge, UK).

Results and discussions

Segregating wheat plant individuals at F₂ generation of an intercross made from four parental breeding materials were subjected to selection twice a cropping cycle (F₂ and F₃) once through breeder based on visual inspection and afterward on the basis of molecular markers. Breeder’s selection was exerted at F₂ generation resulting in 40 segregants. Thereafter, we traced HMW-GS alleles of interest by DNA tags to further screen the segregants based molecular data. Out of 40 progenies pre-selected by breeder, 15, 21, and 15 segregants demonstrated to possess Glu-A1b, Glu-B1i, and Glu-D1d high score HMW-GSs at Glu-A1, Glu-B1, and Glu-D1, respectively. Sixteen segregants were negative on all three markers tested. Seven segregants carried only one of the alleles. Four segregants were positive on two out of three markers tested. Only 12 segregants were positive on all examined markers. A representative gel picture of allele specific primers at F₂ generation is shown in Fig. 2A. Seeds from these 12 segregants were collected and were planted next cropping season each on 2 rows of 2 meters long to constitute 12 F₃ families. At F₃ generation, breeder’s selection was exerted to select six families from the 12 F₃ families. Nearly 15 promising heads per each family were tagged and the corresponding flag leaves were collected for marker analysis. The heads corresponding to flag leaves that were positive for the three markers were selected to proceed with F₄ generation. A representative gel picture of PCR products for seven F₂ derived F₃ families is shown in Fig. 2B. Once selected for those F₂:F₃ individuals deemed positive for the three markers, molecular analysis of DNA extracted from seedlings of selected plants at the next generation (F₄) revealed that F₄ individuals selected contain all three Glu-A1b (2*), Glu-B1i (17+18), and Glu-D1d (5+10) (Fig. 2C).

The availability of knowledge for mechanisms underlying aspects of end-use quality traits in wheat and the wealth of sequences for various alleles of the genes involved in end-use quality enabled the design and application of diagnostic DNA markers and implications thereof in wheat breeding programs (Gale, 2005). Selection is then possible for segregants and lines with positive tests on markers without the need for the direct assessment of traits. These breakthroughs further the efficiency and speed of the development of cultivars with improved quality in the future (Gale, 2005). Several MAS procedures have been implemented so far in breeding programs. Examples include the work reported by de Bustos et al. (2001), Radovanovic and Cloutier (2003), and Kuchel et al. (2007). MAS was applied on a backcross population by de Bustos et al. (2001) to improve glutenin quality Spanish wheat. Kuchel et al. (2007) have applied molecular markers for assisted breeding coupled with doubled haploid procedure for multiple loci in a back cross population targeting both disease resistance and end-use quality. They have used DNA tags to select for Lr34/Yr18, Lr46/Yr29, and glutenin subunits all at once and at haploid stage. They have concluded that integration of MAS for specific traits at the early generations of segregation substantially increase breeding gain in wheat. Radovanovic and Cloutier (2003) have combined benefits from MAS and doubled haploid procedure for HMW-GS. In our study, a composite cross with high allelic complementarity was made for the moderate climate region of Iran and then favorable Glu-1 alleles were captured from different parental lines at early generations i.e. F₂ and F₃ by means of DNA tags. A workflow similar to our strategy was reported by Ribaut and Bertan (1999), where the authors have fixed specific loci early in segregating generations while maintaining the rest of the genome as much segregating as possible. They named their strategy as single large-scale marker-assisted selection. In their study, they conducted MAS only once and selected plants homozygous for favorable alleles at target loci while exerting no selection pressure for genomic regions outside of the target region. Although the cost, time, and workload required for MAS procedures has been always a setback for MAS implementation, and solutions for reducing costs has been offered such as a rapid genotyping of a large number of samples coupled with direct staining of DNA with ethidium bromide without electrophoresis as reported by Gu et al. (1995), it is worth to note that MAS coupled with breeder’s interference and conscious selection made by breeders results in significant reduction of workload and reduces the number of segregants to be analyzed to a great extent. Our approach was a combination of conventional breeding and MAS where at F₂ generations we selected 40 individuals with our breeding experience as if we were not going to applied MAS pressure. Then the DNA samples of these 40 individuals were analyzed at the laboratory to confirm presence or absence of high scoring alleles. Individuals possessing three high score alleles at Glu-1 loci were selected. Thus far, we have generated genetic variation satisfying conventional breeding and molecular breeding. In F₃, we have exerted another round of breeder selection and molecular marker analysis to satisfy traits that are naturally being selected from conventional perspectives and to fix the Glu-1 genomic targets for high score alleles. Therefore, in contrast to what presented by Ribaut and Bertan (1999), our approach does not seem to require large populations, at least early in segregating generations, to achieve our goal of (haplotype) chromosomal segment engineering.

Acknowledgement

This work was financially supported by the Seed and Plant Improvement Institute, Iran under grant number 2-03-03-90018 to Dr. Mohsen Mohammadi.

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