Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (22): 4402-4415.doi: 10.3864/j.issn.0578-1752.2024.22.002

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

Development and Identification of Molecular Markers for Oil-Related Functional Genes and Polymerization Analysis of Excellent Alleles in Soybean

WU ChuanLei1(), HU XiaoYu1, WANG Wei1, MIAO Long1, BAI PengYu1, WANG GuoJi1, LI Na1, SHU Kuo1, QIU LiJuan2(), WANG XiaoBo1()   

  1. 1 College of Agriculture, Anhui Agricultural University, Heifei 230036
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic lmprovement (NFCRl). Mlinistry of Agriculture and Rural Affairs/Key Laboratory of Crop Gene Resource and Germplasm Enhancement, Ministry of Agriculture and Rural Affairs, Beijing 100081
  • Received:2024-04-27 Accepted:2024-05-28 Online:2024-11-16 Published:2024-11-22
  • Contact: QIU LiJuan, WANG XiaoBo

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Abstract:

【Objective】Polymerizing soybean high oil genotypes aims at breeding varieties with higher oil content to improve economic efficiency and nutritional value. It is of great significance to increase agricultural output, reduce processing costs and meet global demand for vegetable oil growth.【Method】Glyma.18G027100 C2 gene family was identified by bioinformatic analysis method at the whole genome level. A total of 66 soybean C2 gene family members were identified, named GmC2-01.1-GmC2-20.2 according to chromosome position. Tissue pattern analysis revealed that 7 genes were highly expressed in grains among 66 C2 family genes (GmC2-03.6, GmC2-02.7, GmC2-07.2, GmC2-18.1, GmC2-18.4, GmC2-19.1 and GmC2-20.2). In order to analyze the effect sites of these genes on soybean oil content, SNP sites in the coding regions of these genes were obtained from SFGB database. Correlation analysis of oil content in two years showed that GmC2-18.1 has SNP loci that significantly affect oil content. The genetic diversity of GmC2-18.1 coding region was analyzed by 12 extreme materials. There was a G/A mutation at 2 038 273 bp in coding region of Wm82.a2.v1 version, which regulated seed oil content. It was preliminarily speculated that this gene played a role in seed development or nutrient accumulation. Then, SNP/InDel molecular markers were developed for GmC2-18.1-G/A gene combined with InDel natural allelic variation site 225 bp upstream of the start codon of GmSWEET39, T/C natural allelic variation site at 8 381 058 bp in coding region of GmST1, A/C natural allelic variation site at the third exon of 41 854 422 bp in coding region of GmMFT. 1 200 soybean germplasm resources from three ecological regions in China were identified by markers in 2 years.【Result】Analysis of variance showed that GmC2-18.1-G, GmSWEET39-Deletion, GmST1-T and GmMFT-A significantly increased oil content by 1.72, 1.95, 1.58 and 2.06 percentage points (P<0.01). The results showed that the average oil content of soybean seeds carrying GmC2-18.1-G, GmSWEET39-Deletion, GmST1-T and GmMFT-A high-oil allele type (PFAT-1) was 22.89%, which increased by about 4.5% compared with that carrying GmC2-18.1-A, GmSWEET39-Insertion, GmST1-C and GmMFT-C low-oil allele type (PFAT-14). 5 percentage points, the contribution rate to oil content is about 21.69%. 【Conclusion】Based on the markers developed above, 115 PFAT-1 high oil alleles were screened.

Key words: soybeans, bioinformatic analysis, molecular markers, oil content, gene aggregation

Table 1

Primer sequences used for PCR detection and desired fragment size"

基因
Gene		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 目的基因片段大小
Fragment size (bp)		 参考文献
References
GmSWEET39 SWEET39InDel-F CTTAAAGTTCTCAAACTTATCCTCC 158 [8]
SWEET39InDel-R GACCAGAAATCAAAAAGAAACAAGG
GmST1 ST1dCAPS-F GCCGTGATGTCGCATGATATCTCATGTTGTTCTTGTTCCTAACAAGC 132 [9]
ST1dCAPS-R AGGAGTTTGAAGCTGTTGAGTCT
GmMFT MFTdCAPS-F CAGCGAAGCAACACTGTTTCCACTTATTTTCCAGAAGC 211 [10]
MFTdCAPS-R AACATGATGTTTTAGCCCGAAG
GmC2-18.1 C2-18.1CAPS-F CTCCATGTCATAAGGTCTACATC 1151 [4]
C2-18.1CAPS-R CAGCAGTGTGTTTACATACC

Fig. 1

Phylogenetic tree of the C2 gene family"

Fig. 2

Gene distribution of the C2 gene family on soybean chromosomes"

Fig. 3

Analysis of the GmC2-18.1 gene and the tissue expression pattern of homologous genes a: Analysis of tissue expression patterns of GmC2-18.1 homologous genes; b: Analysis of tissue expression patterns of GmC2-18.1 gene"

Fig. 4

Screening of gene SNP sites for differences in oil content a: GmC2-02.7; b: GmC2-03.6; c: GmC2-07.2; d: GmC2-18.1; e: GmC2-18.4; f: GmC2-20.4. A, C: Low oil genotype; G, T: High oil genotype"

Table 2

The information of extreme material varieties"

编号 Numbers 品种名称 Variety name 来源 Source 油分均值 Average oil content (%)
1 Sprite87 国外Abroad 25.61
2 Hobbit87 国外Abroad 25.12
3 吉密豆2号Jimidou 2 中国东北Northeast China 24.95
4 吉密豆3号Jimidou 3 中国东北Northeast China 24.85
5 辽08012 Liao 08012 中国东北Northeast China 23.14
6 吉林20号Jilin 20 中国东北Northeast China 24.98
7 大黄豆Dahuangdou 中国黄淮Huanghuai, China 18.23
8 大青豆Daqingdou 中国黄淮Huanghuai, China 18.37
9 平顶黄Pingdinghuang 中国黄淮Huanghuai, China 18.82
10 泰州白花乌甲2 Taizhoubaihuawujia 2 中国南方Southern China 18.96
11 早黄豆-1 Zaohuangdou 中国南方Southern China 18.87
12 七月黄-1 Qiyuehuang-1 中国南方Southern China 18.09

Fig. 5

Genetic diversity analysis of GmC2-18.1 coding region"

Fig. 6

Development of molecular markers for soybean oil genes a：InDel marker developed for GmSEWWT39, Insertion is 9 bp insertion, Deletion is 9 bp deletion; b: dCAPS marker developed for GmMFT, 1-6 is G variant type, 7-8 is A variant type; c: dCAPS marker developed for GmST1, 2 and 7 are T variant type, 1, 3, 4, 5, 6, 8 are C variant type; d: CAPS marker developed for GmC2 -18.1, 1-10 is A variant type, 11-20 is G variant type"

Fig. 7

Correlation analysis between genotype and seed oil content"

Table 3

Joint analysis of the genetic effects of oil content related genes at multiple sites of oil content traits"

GmSWEET39 GmST1 GmMFT GmC2-18.1 数量
Number		 聚合优异等位基因类型
PFAT		 范围
Range		 油分
Oil content (%)		 标准偏差
SD		 标准误差
MD		 P值
P value
缺失Deletion T A G 115 PFAT-1 8.88 22.89 1.72 0.16 -
缺失Deletion T C G 6 PFAT-2 2.68 21.04 1.00 0.41 0.274
缺失Deletion T A A 409 PFAT-3 8.69 21.67 1.61 0.08 <0.01
缺失Deletion T C A 194 PFAT-4 8.66 20.32 1.19 0.09 <0.01
缺失Deletion C A A 61 PFAT-5 5.77 21.59 1.46 0.19 <0.01
缺失Deletion C A G 12 PFAT-6 7.67 21.53 2.58 0.74 0.252
缺失Deletion C C A 61 PFAT-7 6.91 19.03 1.71 0.22 <0.01
缺失Deletion C C G 5 PFAT-8 4.96 19.84 2.34 1.05 <0.01
插入Insertion T A G 6 PFAT-9 3.59 20.64 1.40 0.57 0.056
插入Insertion T A A 30 PFAT-10 5.83 20.90 1.62 0.30 <0.01
插入Insertion T C A 32 PFAT-11 4.95 19.53 1.24 0.22 <0.01
插入Insertion T C G 9 PFAT-12 5.59 19.28 1.78 0.59 <0.01
插入Insertion C C G 8 PFAT-13 4.67 19.43 1.77 0.63 <0.01
插入Insertion C C A 61 PFAT-14 6.69 18.37 1.89 0.24 <0.01
插入Insertion C A A 9 PFAT-15 4.89 20.70 1.54 0.51 <0.01
插入Insertion C A G 2 PFAT-16 3.78 19.17 2.67 1.89 0.075
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