He ZH

Wang L

Xia XC

Zhao X

English

Sydney eScholarship

 1Characterization of low-molecular-weight glutenin subunit genes at Glu-B3 and Glu-D3 loci and development of functional markers in common wheat  Wang L1, Zhao X1,2, He Z1,3, Xia X1,* 1Institute of Crop Science, National Wheat Improvement Centre/The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences (CAAS), Zhongguancun South Street 12, Beijing 100081, China; 2Wheat Research Institute, Henan Academy of Agricultural Sciences, Nongye Road 1, Zhengzhou, Henan, 450002, China; 3International Maize and Wheat Improvement Centre (CIMMYT) China Office, c/o CAAS, Zhongguancun South Street 12, Beijing 100081, China *Author for correspondence: Xianchun Xia, Email address: xiaxianchun@caas.net.cn   SUMMARY  Four genes with 17 allelic variants at Glu-B3 locus and 6 genes with 12 variants at Glu-D3 locus were isolated in common wheat using the technique of in-silico cloning in combination with PCR amplification. The relationship between the Glu-B3 and Glu-D3 alleles defined by protein mobility at SDS-PAGE and the allelic variants were determined, and 10 STS markers for different Glu-B3 alleles and 7 markers for different Glu-D3 genes were developed based on the allelic variations among different gene haplotypes. The functional STS markers were validated by wheat varieties and advanced lines with different Glu-B3 and Glu-D3 alleles.   INTRODUCTION  Low-molecular-weight glutenin subunits (LMW-GS) showed large effects on dough extensibility, and thus influence the quality of end-use products of wheat, particularly for Asian noodles[1, 2, 3]. Compared with high-molecular-weight glutenin subunits (HMW-GS), the extensive allelic variations of LMW-GS and their overlapping mobility with the abundant gliadin proteins[4] make it difficult to discriminate the role of individual alleles. Characterization of LMW-GS genes at DNA level and development of functional markers is therefore very important for the discrimination of different LMW-GS alleles in wheat breeding.  MATERIALS AND METHODS  Aroona (Glu-B3b) and its seven near-isogenic lines (NIL), Aroona-B3a (Glu-B3a), Aroona-B3c (Glu-B3c), Aroona-B3d (Glu-B3d), Aroona-B3f (Glu-B3f), Aroona-B3g (Glu-B3g), Aroona-B3h (Glu-B3h), Aroona-B3i (Glu-B3i) and Cheyenne (Glu-B3e) with different Glu-B3 alleles defined by protein mobility were used to isolate Glu-B3 genes. The Aroona and its NILs were kindly provided by Dr Marie Appelbee at SARDI Grain Quality Research Laboratory, Australia. Cheyenne was provided by Dr. Baoyun Li at China Agricultural University. Eight common wheat cultivars (Tasman, Chinese Spring, Silverstar, Sunco, Aroona, Norin61, Hartog, and BT2288A) carrying five common Glu-D3 alleles (a, b, c, d, and e)[5] were used to amplify Glu-D3 genes. Chinese Spring and its nulli-tetrasomic lines N1A-T1D, N1B-T1A, N1B-T1D and N1D-T1B, kindly provided by Prof. R. A. McIntosh at the Plant Breeding Institute Cobbitty, University of Sydney. Besides the eight NILs and Cheyenne, 20 varieties from our laboratory collection and 141 wheat varieties and advanced lines from CIMMYT with different Glu-B3 alleles detected by SDS-PAGE were employed to validate the allele-specific markers.   Gene-specific primers for cloning Glu-B3 and Glu-D3 genes were developed according to the description of Zhang et al.[6, 7]. Eight reference genes with complete coding region, including seven LMW-GS genes (GenBank accessions AB119006, AB164415, AB164416, AB262661, Y14104, AB062852 and AJ007746) located on the short arm of chromosome 1B and one (AY542898) with high similarity to Glu-B3 available in GenBank were used for cloning Glu-B3 genes (http://www.ncbi.nlm.nih.gov). Seven reference genes X13306, AB062851, AB062865, AB062872, AB062873, AB062875 and AY223396 available in GenBank (www.ncbi.nlm.nih.gov) were employed for the isolation of Glu-D3 genes. The PCR fragments with expected sizes were recovered from agarose gel, followed by cloning into the pGEM-T Easy Vector, and the recombinant clones with expected size were sequenced after PCR test. All the sequencings were performed by the Sangon Biotechnology Co. Ltd (Shanghai, China). Each PCR and sequence procedure was repeated 3-6 times to avoid any technical errors. Sequence analysis and characterization were performed using software DNAMAN (http://www.lynnon.com). Allele-specific PCR markers were designed based on the allelic variants of Glu-B3 and Glu-D3 genes following the method of Zhang et al. [6,]. These markers were validated with wheat varieties and advanced lines from CIMMYT, Australia and France with different protein mobility alleles.      2 RESULTS   Four Glu-B3 genes with complete coding sequence were obtained, designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4, respectively. GluB3-1 had five allelic variants, GluB3-11, GluB3-12, GluB3-13, GluB3-14 and GluB3-15 that were present in the NILs and varieties with Glu-B3 alleles a, b, e, f and g, respectively. Compared with GluB3-12, four, one and two triplet nucleotides (CAA) deletion at the position 319-330 were found in the coding region of GluB3-11, GluB3-13 and GluB3-14, leading to four, one and two glutamines absence in the glutamine-rich repetitive domain, respectively. GluB3-15 had a 63-bp deletion at the position 292-354, resulting in twenty-one amino acids deletion in the deduced peptide. GluB3-2 contained three allelic variants GluB3-21, GluB3-22 and GluB3-23. GluB3-21 was present in the NIL with the allele Glu-B3a, GluB3-22 in the genotypes with Glu-B3 alleles b, e, and f, and GluB3-23 in those with Glu-B3g. Compared with GluB3-21, both GluB3-22 and GluB3-23 had a 3-bp deletion (CAA) at the position 378-380 in the coding region, leading to a glutamine deletion in the repetitive domain of the deduced peptide. GluB3-22 had two SNPs at the positions 671 and 1246 with the latter leading to an amino acid mutation from serine to asparagine in the C-terminal conserved region. GluB3-3 was amplified from the NILs containing Glu-B3c, d, h and i, with four allelic variants, designated as GluB3-31, GluB3-32, GluB3-33 and GluB3-34, respectively. Compared with GluB3-31, 4-5 SNPs were found in the coding region of GluB3-32, GluB3-33 and GluB3-34, and a double base substitution in GluB3-33. In addition, GluB3-34 had a 3-bp (CAA) deletion at the position 603-605 and a 3-bp (CAA) insertion at 651-653, leading to a glutamine deletion and insertion, respectively. GluB3-4 contained five allelic variants designated as GluB3-41 (in the genotypes with Glu-B3a, c and d), GluB3-42 (Glu-B3b), GluB3-43 (Glu-B3e and h), GluB3-44 (Glu-B3f and g) and GluB3-45 (Glu-B3i). Compared with GluB3-41, three SNPs were detected in the coding region of GluB3-42, GluB3-43 and GluB3-44, and six SNPs and a 21-bp deletion in GluB3-45. Ten allele-specific PCR markers were designed based on the SNPs presented in the identified allelic variants for the discrimination of Glu-B3 protein mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated with the 8 NILs and Cheyenne, and 161 wheat varieties and advanced lines with different Glu-B3 alleles, indicating they may be useful in marker-assisted breeding to improve wheat quality. The detailed information of the Glu-B3 genes and functional markers associated with different Glu-B3 alleles are available at our lab upon request.  Six Glu-D3 genes, designated as GluD3-1, GluD3-2, GluD3-3, GluD3-4, GluD3-5 and GluD3-6, respectively, were identified in the eight cultivars[8, 9]. Computer program DNAMAN was used to predict the protein outputs from these genes. GluD3-1 showed two allelic variations, one amplified from cultivars containing b and c alleles (designated GluD3-1bc) and the other from a, d, and e alleles (designated GluD3-1ade). Compared with GluD3-1bc, a 3-bp deletion was found in the coding region of N-terminal repetitive domain of GluD3-1ade， leading to a glutamine deletion at the 116th position. GluD3-2 had three variants in the eight cultivars, which were designated as GluD3-2ae2, GluD3-2be1 and GluD3-2cd, respectively. In comparison to GluD3-2ae2, a single nucleotide polymorphism (SNP) was detected for GluD3-2be1 in the signal peptide region, resulting in an amino acid change from alanine to threonine at the 11th position; and 11 mutations were found at GluD3-2cd, with five in upstream region, four in coding region and two in downstream region, respectively. GluD3-3 had two allelic variations, designated as GluD3-3abe and GluD3-3cd, encoding LMW-m and LMW-i types of glutenin subunits, respectively. Compared with the GenBank GluD3 genes, nucleotide sequences of GluD3-2ae2 and GluD3-2cd were the same as X13306 and AB062875, respectively. GluD3-2be1 and GluD3-1ade had only one-base difference from U86027 and AB062865, respectively. GluD3-1bc gene was not found in the GenBank database, indicating a newly identified GluD3 gene variation. GluD3-3 was a new gene different from any other known Glu-D3 genes. GluD3-4 showed three allelic variants or haplotypes at the DNA level in the eight cultivars, which were designated as GluD3-41, GluD3-42 and GluD3-43. Compared with GluD3-42, a single nucleotide polymorphism (SNP) was detected for GluD3-43 in the coding region, resulting in a pseudo-gene with a nonsense mutation at the 119th position of deduced peptide, and a 3-bp insertion was found in the coding region of GluD3-41, leading to a glutamine insertion at the 249th position of its deduced protein. The coding regions for GluD3-5 and GluD3-6 showed no allelic variation in the eight cultivars tested, indicating that they were relatively conservative in common wheat. Based on the 12 allelic variants of three Glu-D3 genes identified in this study and three detected previously, seven STS markers were established to amplify the corresponding gene sequences in wheat cultivars containing five Glu-D3 alleles (a, b, c, d and e). The seven primer sets M2F12/M2R12, M2F2/M2R2, M2F3/M2R3, M3F1/M3R1, M3F2/M3R2, M4F1/M4R1 and M4F3/M4R3 were specific to the allelic variants GluD3-21/22, GluD3-22, GluD3-23, GluD3-31, GluD3-32, GluD3-41 and GluD3-43, respectively, which were validated by amplifying 20 Chinese wheat cultivars containing alleles a, b, c and f based on protein electrophoretic mobility. These markers will be useful to identify the Glu-D3 gene haplotypes in wheat breeding programs.  REFERENCES  1.  Cornish GB, Burridge PM, Palmer GA, Wrigley CW (1993) Mapping the origins of some HMW and LMW glutenin subunit alleles in Australian germplasm. Proceedings of the 42nd Australian Cereal Chemistry Conference, Sydney, pp 255–260 2.  Gupta RB, Shepherd KW (1990) Two-step one-dimensional SDS-PAGE analysis of LMW subunits of glutenin. I. Variation and genetic  3control of the subunits in hexaploid wheats. Theor Appl Genet 80: 65–74 3.  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Characterization of low-molecular-weight glutenin subunit genes at Glu-B3 and GluD3 loci and development of functional markers in common wheat

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Characterization of low-molecular-weight glutenin subunit genes at Glu-B3 and GluD3 loci and development of functional markers in common wheat

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