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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (3): 703-718.doi: 10.3724/SP.J.1006.2023.24028

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

Development and validation of functional markers of GBSSI gene in proso millet

DING Min1(), DUAN Zheng-Yong1, WANG Yu-Zhuo1, XUE Ya-Peng1, WANG Hai-Gang2, CHEN Ling2, WANG Rui-Yun1,2,*(), QIAO Zhi-Jun2,*()   

  1. 1College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2Center for Agricultural Genetic Resources Research, Shanxi Agricultural University / Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture and Rural Affairs / Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops, Taiyuan 030031, Shanxi, China
  • Received:2022-01-22 Accepted:2022-06-07 Online:2023-03-12 Published:2022-07-07
  • Contact: WANG Rui-Yun,QIAO Zhi-Jun E-mail:yksin646@163.com;wry925@126.com;nkypzs@126.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-06-14.5-A16);Shanxi Agriculture Research System (Minor Grain Crop)(2022-03);National Natural Science Foundation of China(31271791);Natural Science Foundation of Shanxi Province(201901D11126)

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Abstract:

Functional molecular markers were designed based on nucleotide differences of known functional loci on Waxy (GBSSI), a regulation gene for amylose synthesis in proso millet. Genotypes and phenotypes of 270 proso millet accessions from Shanxi province were tested and verified by combining phenotypes and genotypes. The amylose content of 46 proso millet accessions with different genotypes was determined to explore the genetic effect of this gene and evaluate its breeding value. Waxy gene of proso millet existed in two forms (L and S), targeting 15 bp on S type gene. A functional marker was designed for InDel (RYW214), which could effectively distinguish 126 bp, 141 bp, and heterozygous (126/141 bp) types. The consistency between the marker and phenotypic identification was 93.30%, and the Pearson correlation index (r) was 0.745. Two CAPS markers (RYW215, RYW216) were designed for a single nucleotide insertion site and a SNP site in the L-type gene. Among 270 accessions, genotype S-15/LF accounted for the most (31.10%), S0/S-15/LF was the second (22.96%), and S-15/LY/LF was the least (0.74%). Genotype S-15/LC was absent. Among 46 proso millet accessions, the genotypes with the highest mean amylose content were S0/LY/LF (15.50%) and S-15/LF (0.58%). The mutant alleles of L type gene had little effect on amylose content, and the existence of wild type S0 was enough to guarantee amylose content in endosperm. The existence of mutant S-15 would lead to a qualitative decline in amylose content, and the amylose content of heterozygous genotype S0S-15 was slightly higher than that of homozygous genotype S-15.

Key words: proso millet, GBSSI gene, functional molecular marker, amylose content

Table S1

Proso millet accessions"

编号
Serial number		 统一编号
Unicode		 名称
Name		 来源
Accession donor		 品种类型
Variety type
1 00006548 太原1047 Taiyuan 1047 太原市 Taiyuan, Shanxi 地方品种 Local variety
2 00007523 A75-7 山西 Shanxi 地方品种 Local variety
3 00007526 A75-10 山西 Shanxi 地方品种 Local variety
4 00000832 鸭爪白 Yazhuabai 大同市 Datong, Shanxi 地方品种 Local variety
5 00000838 小红黍 Xiaohongshu 大同市 Datong, Shanxi 地方品种 Local variety
6 00000843 大白黍 Dabaishu 大同市 Datong, Shanxi 地方品种 Local variety
7 00000956 大青黍 Daqingshu 大同市 Datong, Shanxi 地方品种 Local variety
8* 00001263 大白黍 Dabaishu 阳泉市 Yangquan, Shanxi 地方品种 Local variety
9 00001275 小黑黍 Xiaoheishu 晋中市 Jinzhong, Shanxi 地方品种 Local variety
10 00001374 白鹅蛋糜子 Baiedanmizi 吕梁市 Lyuliang, Shanxi 地方品种 Local variety
11 00001514 黄硬黍 Huangyingmi 临汾市 Linfen, Shanxi 地方品种 Local variety
12* 晋黍1号 Jinshu 1 大同市 Datong, Shanxi 育成品种 Breed variety
13 晋黍2号 Jinshu 2 大同市 Datong, Shanxi 育成品种 Breed variety
14 晋黍3号 Jinshu 3 大同市 Datong, Shanxi 育成品种 Breed variety
15 晋黍4号 Jinshu 4 大同市 Datong, Shanxi 育成品种 Breed variety
16 晋黍5号 Jinshu 5 大同市 Datong, Shanxi 育成品种 Breed variety
17* 晋黍6号 Jinshu 6 大同市 Datong, Shanxi 育成品种 Breed variety
18 晋黍7号 Jinshu 7 大同市 Datong, Shanxi 育成品种 Breed variety
19 晋黍8号 Jinshu 8 大同市 Datong, Shanxi 育成品种 Breed variety
20 00009143 晋黍9号 Jinshu 9 大同市 Datong, Shanxi 育成品种 Breed variety
21 雁黍7号 Yanshu 7 大同市 Datong, Shanxi 育成品种 Breed variety
22 红黍子 Hongshuzi 阳泉市 Yangquan, Shanxi 农家种 Farm variety

Table 1

Detail of primers used in this study"

标记名称
Marker name		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 内切酶名称
Endonuclease name		 备注
Remark
RYW214 M5 GGACGTCAGCGAGTGGGACC Hunt et al. [31]
R11 CAGGCACACTGCTCCCAATG
M5QT1 CAAGTACGACGTGTCGACGG
R11QT1 AAGGACGATCTGGACGTCCT
M5QT2 AGTGCGTCGTCGGACTCTG
R11QT2 TCCTCCAGCCTGCCGACAAA
RYW215 int5Lf ATGTTTGAATGAATGCTCC ACC I Hunt et al. [31]
R3 TGGTAGTTGCTCTTGAGGTA
RYW216 M12 CGTGACCATCTCTTCCTGTA EcoN I Hunt et al. [31]
R12 CGACGACGAACTCTCAACAC

Fig. 1

Electrophoretic diagram of amplification products at InDel site of some proso millet accessions M: marker 500; 168: Erhongshu; 169: Dahongshu; 170: Xiaoheishu; 175: Yingdihuang; 176: Huangshuzi; 177: Laolaihong; 178: Bianmeishu; 183: Chengshuhong; 180: Gouweidan; 181: Baikulou; 182: Xiaohongruanmi; 184: Ruanbaimi."

Fig. 2

Electrophoretic patterns of amplification products at SNP (A/G substitution) site of some accessions M: marker 2000; 197: Yuza 1; 198: Dahuangmizi; 199: Xiaoqingmizi; 200: Dabaiyingmi; 203: Dahuangya."

Fig. 3

Electrophoretic diagram of amplification products of some materials at frame-shift mutation site M: marker 2000; 179: Jizhuahong; 180: Gouweidan; 181: Baikulou; 182: Xiaohongruanmi; 183: Chengshuhong; 184: Ruanbaimi; 185: Qianjinshu; 186: Zhenzhulianruanmi; 187: Bairuanmi; 188: Hongshuzi; 189: Jinhongshu; 191: Mizi."

Fig. 4

DNAMAN sequence alignment of 7 proso millet accessions amplified by 3 pairs of primers A, B, and C indicate primer M5/R11, M12/R12, and int5Lf/R3, respectively. 41: Baishuzi; 49: Shuzi; 60: Heimizi; 73: Baimizi; 80: Liushitianxiaohongshu; 87: Dabaishu; 89: Dabaishu."

Fig. 5

Waxy/non-waxy phenotype identification of proso millet accessions A: Non-waxy type; B: Waxy type."

Table 2

Genotype of 270 proso millet accessions"

基因型
Genotype		 LC LY LF LY/LF
S0 24 14 43 5
S-15 0 10 84 2
S0/S-15 3 15 62 8

Table 3

Amylose content in different genotypes of proso millet accessions"

基因型
Genotype		 种质份数
No. of accessions		 直链淀粉含量
Amylose content (%)		 变异区间
Range of variation (%)		 变异系数
Coefficient of variation (%)
S0/LC 4 13.13±2.28 10.14-15.63 17.40
S0/LF 3 16.28±5.66 11.60-22.57 34.76
S0/LY 3 12.47±2.66 9.42-14.33 21.36
S0/LY/LF 3 15.50±2.61 12.50-17.31 16.88
S-15/LF 6 0.48±0.32 0.10-0.84 66.05
S-15/LY 4 1.67±1.09 0.69-2.92 65.17
S-15/LY/LF 2 2.81±1.30 1.89-3.73 46.30
S0S-15/LC 3 1.85±0.60 1.38-2.53 32.75
S0S-15/LF 7 4.75±2.38 1.97-7.36 50.20
S0S-15/LY 6 4.43±2.53 1.59-8.21 57.08
S0S-15/LY/LF 5 4.84±3.43 1.31-9.03 70.87

Fig. 6

Multiple comparison of amylose content in different genotypes of proso millet accessions Different lowercase letters indicate significant differences at P < 0.05."

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