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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (1): 18-25.doi: 10.3724/SP.J.1006.2019.84078

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Molecular identification of a new soybean germplasm Zhonghuang 608 lacking of 7S globulin alpha' subunit

Jun-Ying LI1,2(),Ru-Jian SUN2,3,Zhong-Feng LI2,Zhong-Yan WEI2,Yu-Long REN2,Jun WANG1,*(),Li-Juan QIU2,*()   

  1. 1 College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
    2 Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture, Institute of Crop Sciences, Chinese Academy of Agricultural Science, Beijing 100081, China / National Key Facility for Gene Resources and Genetic Improvement, Beijing 100081, China
    3 Hulun Buir Institution of Agricultural Sciences, Zhalantun 162650, Inner Mongolia, China
  • Received:2018-06-02 Accepted:2018-10-08 Online:2018-11-06 Published:2018-11-06
  • Contact: Jun WANG,Li-Juan QIU E-mail:13051379117@163.com;wjsoybean_2008@163.com;qiulijuan@caas.cn
  • Supported by:
    The study was supported by the National Key R&D Program for Crop Breeding(2016YFD0100201);the National Soybean Germplasm Resources Platform.(2016-004)

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Abstract:

The content of alpha' subunit of 7S globulin has an important effect on the nutritional quality and processing characteristics in soybean. In this study, using polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, Zhonghuang 608 (ZH608), an α' subunit deletion mutant, was identified from the Zhongpin 661 (ZP661) EMS mutant library. Zhonghuang 608 was crossed to the soybean variety Dengke 1, and self-pollinated to establish an F2 segregating population composed of 210 individuals. Genetic analysis showed that the alpha' subunit deletion trait in ZH608 was controlled by a single recessive nuclear gene. By linkage analysis we mapped the locus between SSR10-1489 and SSR10-1612 on chromosome 10. Coincidentally, in the mapping region Cgy-1 (Glyma.10G246300) was annotated as an alpha' subunit synthesis-related gene according to the Williams 82 reference genome. Further sequence analysis showed that the mutant had a single base change (G 84 to A 84) in the first exon of Cgy-1, which resulted in the premature termination of the amino acid sequence. Based on the newly discovered SNP mutation, a co-dominant molecular marker was developed, and used to detect the genotypes of those F2 individuals. The results indicated that the Cgy-1 genotype cosegregated with the alpha' subunit phenotype in the F2 segregating population of Zh608 and Dengke 1. This study not only provides new materials for quality improvement, but also offers technical support for molecular breeding in soybean.

Key words: soybean, alpha' sunbunit, mutant, 7S globulin, dCAPS marker

Table 1

Primers for amplifying the Cgy-1 by PCR"

引物名称
Name		 引物序列
Sequence of primer (5'-3')		 PCR产物长度
PCR product size (bp)		 退火温度
Annealing temperature (℃)
Cgy1-A Forward: AGCCCAAAACATTCACCAAC 1598 58
Reverse: AGGACTGTTGAGCTTGAGTGC
Cgy1-B Forward: CGCCATACCCGTTAACAAAC 1702 58
Reverse: TTGTGGCAGGACATTGCTAC

Table 2

Primers and restriction endonuclease of the dCAPS marker"

标记
Marker		 突变位点
SNP		 酶
Enzyme		 引物序列
Sequence of primer (5'-3')		 退火温度
Tm (℃)		 PCR片段长度
PCR product
Size (bp)		 酶切后片段长度
Product size after digestion by Taq I
GM7S-1 G/A Taq I F: TCTCATTTGGCATTGCGTATCG
R: TTGACCTTCTTCGCATTCT		 55 158 158 bp/(138 bp+20 bp)

Fig. 1

Phenotype of the α' protein subunit mutant by SDS-PAGE a: protein profiles of the α' subunit deletion mutant ZH608 and the wild-type ZP661 by SDS-PAGE. b: α' subunit mutant trait of ten individuals derived from ZH608 validated by SDS-PAGE (lanes 1-8)."

Table 3

Genetic analysis of the α' subunit deletion mutant in an F2 segregated population of ZH608 and Dengke 1"

表型
Phenotype		 观测值
Observed value		 期望值
Excepted value		 χ2(3:1) P值
P-value
突变体(α'亚基缺失) Mutant (α' subunit deletion) 64 53 1.43 0.08
野生型 Wild type 146 157
共计 Total 210 210

Fig. 2

Genetic and physical mapping of the α' subunit deletion mutant Zhonghuang 608 a: the candidate gene Cgy-1 was mapped to between SSR marker SSR10-0982 and SSR10-1638 by using 210 F2 individuals. b: the candidate interval was reduced to SSR10-1489 and SSR10-1619 by SSR polymorphic markers. c: the mutation of eighty-fourth bases at the first exon of Cgy-1 gene in the mutant Zhonghuang 608, resulted in pre-terminating of encoding amino acid. Blue boxes, white boxes, and black lines represent exons, 5' UTR/3' UTR and introns, respectively."

Fig. 3

SDS-PAGE (a) and Western blot (b) results of mutants and wild type a: the result of SDS-PAGE showed that the mutant ZH608 lacked α' subunit. b: in antibody hybridization results showed that the wild type (ZH608) had two bands and the mutant (ZP661) lacked the α' subunit band."

Fig. 4

A agarose detection for amplified products and enzyme products a: the single fragment was detected for the amplified PCR product of both the wild type and the mutant by the primer GM7S-1. b: the PCR product of ZH608 was cleaved into two smaller fragments by the restriction enzyme Taq I."

Fig. 5

Genotyping of some F2 individuals by the dCAPS marker P1 is the female parent, and P2 male parent. G, A, and H represent the wild type, mutant, and heterozygous genotype, respectively."

Fig. 6

Distribution of 11S/7S ratio for materials with and without α' subunit in F2 population"

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