| Low-molecular-weight glutenin subunits (LMW-GS), encoded by the genes at Glu-3 loci (Glu-A3,Glu-B3 and Glu-D3) on the short arms of wheat group 1 chromosomes, play an important role in determining the processing quality of end-use products of common wheat. Traditionally, sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is used to determine allelic compositions of LMW-GS in wheat. However, difficulties in resolving the multi-gene families and the overlapping fractions of LMW-GS and gliadins hinder their routine use, particularly for testing large populations in the early generations of wheat breeding programs. Therefore, it is important to develop functional markers to discriminate different LMW-GS protein alleles. In the present study, the gene sequences of the LMW-GS submitted in GenBank were analyzed and used to design locus-specific primers for the isolation of the genes at Glu-A3 and Glu-B3 loci, and allele-specific STS markers were developed for different alleles defined by protein mobility, and the novel allelic variants at Glu-B3 locus were also cloned from the common wheat related species. The main results obtained in this study are summarized below.1. Three Glu-A3 genes with 17 allelic variants were cloned from the six near-isogenic lines (NILs) of Aroona and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by protein electrophoretic mobility, designated as GluA3-1, GluA3-2 and GluA3-3, and their deduced amino acid sequences were designated as AP1, AP2 and AP3, respectively. The GluA3-1 gene had seven allelic variants or haplotypes, designated as GluA3-11, GluA3-12, GluA3-13, GluA3-14, GluA3-15, GluA3-16 and GluA3-17. The GluA3-2 gene had four allelic variants, designated as GluA3-21, GluA3-22, GluA3-23 and GluA3-24. The GluA3-3 gene had six allelic variants, designated as GluA3-31, GluA3-32, GluA3-33, GluA3-34, GluA3-35 and GluA3-36. All the three genes contained a complete coding sequence. The deduced amino acid sequences of the seven allelic variants of GluA3-1 and two of GluA3-2 (GluA3-23 and GluA3-24) contained an uninterrupted open reading frame (ORF), whereas the others belonged to pseudogenes with one or two stop codons in the middle of the ORF. The molecular weight of AP1 ranged from 40.9 (AP1-4) to 44.8kDa (AP1-5), AP2 ranged from 34.1 (AP2-2) to 34.5 kDa (AP2-4), and AP3 ranged from 37.8 (AP3-1) to 39.3 kDa (AP3-3). AP1 and AP3 started with ISQQQQ- at the N-teminus, and AP2 started with MDTSCIP-. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. These markers were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.2. Four Glu-B3 genes with complete coding sequence were obtained from the eight near-isogenic lines (NILs) of Aroona and Cheyenne with different Glu-B3 alleles (a, b, c, d, e, f, g, h and i) defined by protein electrophoretic mobility and designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4, and their deduced peptides were designated as BP1, BP2, BP3 and BP4, respectively. The GluB3-1 gene had five haplotypes or allelic variants, designated as GluB3-11, GluB3-12, GluB3-13, GluB3-14 and GluB3-15. The GluB3-2 gene had three allelic variants, designated as GluB3-21, GluB3-22 and GluB3-23. GluB3-3 had four allelic variants, designated as GluB3-31, GluB3-32, GluB3-33 and GluB3-34. GluB3-4 had five allelic variants, designated as GluB3-41, GluB3-42, GluB3-43, GluB3-44 and GluB3-45. GluB3-1 and GluB3-2 were cloned from the varieties with Glu-B3 a, b, e, f and g, and GluB3-3 was from the varieties with Glu-B3 c, d, h and i, and GluB3-4 was from all nine materials. All the deduced amino-acid sequences of the four genes contained a single ORF encoding the peptides with a typical LMW-GS sequence stucture. BP1, BP2 and BP3 were characterized with the amino acids MENSHIP- in the N-terminal domain, with predicted molecular weights ranged from 39.0 kDa (BP1-5) to 44.6 kDa (BP3-1). BP4 started with METSHIP- in the N-terminal domain with predicted molecular weights of 39.0 kDa (BP4-5) or 39.8 kDa (BP4-1, BP4-2, BP4-3 and BP4-4). Ten allele-specific PCR markers were developed based on the SNPs presented in the allelic variants to discriminate the Glu-B3 protein mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated on 161 wheat varieties and advanced lines with different Glu-B3 alleles, thus confirming that the markers can be used in marker-assisted breeding for improving wheat processing quality.3. Seven multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, Glu-A3b + Glu-A3e, Glu-B3c + Glu-B3d, Glu-B3b + Glu-B3g and Glu-B3h + Glu-B3i. These multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 and Glu-B3 alleles, confirming that they can be efficiently used in marker-assisted breeding.4. Sixteen novel allelic variants of GluB3-1, GluB3-3 and GluB3-4 were isolated by the primer sets LB1F/LB1R, LB3F/LB3R and LB4F/LB4R from common wheat related species, i.e. T. durum, T. dicoccum, T. dicoccoides, Ae. longissima, Ae. searsii, Ae. bicornis and Ae. speltoides, and designated as GluB3-16, GluB3-35 GluB3-36, GluB3-37, GluB3-46, GluB3-47, GluB3-48, GluB3-49, GluB3-410, GluB3-411, GluB3-412, GluB3-413, GluB3-414, GluB3-415, GluB3-416 and GluB3-417, respectively. In detail, GluB3-16 was cloned from T. dicoccoides by LB1F/LB1R, and its deduced amino acid was 39.2 kDa in molecular weight. GluB3-35, GluB3-36 and GluB3-37 were isolated from T. durum and T. dicoccum by the primer set LB3F/LB3R, and the molecular weight of their decuced peptides were 44.5 kDa (GluB3-36) or 44.6 kDa (GluB3-35 and GluB3-37). The others distributed in the 7 species and were cloned by LB4F/LB4R with the molecular weight ranged from 38.6 (GluB3-414) to 42.5 kDa (GluB3-413) in their deduced proteins. All the deduced amino-acid sequences of the 16 new allelic variants contained a single open reading frame (ORF), and characterized with a typical LMW-GS sequence structure.5. Phylogenetic analysis and multiple sequence alignments were performed based on the Glu-B3 genes cloned in the present study. The results suggested the gene GluB3-4 may be derived from T. dicoccum and T. dicoccoides, and its diploid ancestor may be from Ae. longissima or Ae. bicornis. GluB3-1 and GluB3-2 may be originated from T. dicoccoides, and GluB3-3 originated from T. dicoccum or T. durum directly. The results surpported the hypothesis that common wheat had more than one tetraploid ancestor.
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