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作物学报 ›› 2023, Vol. 49 ›› Issue (3): 703-718.doi: 10.3724/SP.J.1006.2023.24028

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

糜子GBSSI基因功能标记的开发与验证

丁敏1(), 段政勇1, 王宇卓1, 薛亚鹏1, 王海岗2, 陈凌2, 王瑞云1,2,*(), 乔治军2,*()   

  1. 1山西农业大学农学院, 山西太谷 030801
    2山西农业大学农业基因资源研究中心 / 农业农村部黄土高原作物基因资源与种质创制重点实验室 / 杂粮种质资源发掘与遗传改良山西省重点实验室, 山西太原 030031
  • 收稿日期:2022-01-22 接受日期:2022-06-07 出版日期:2023-03-12 网络出版日期:2022-07-07
  • 通讯作者: 王瑞云,乔治军
  • 作者简介:E-mail: yksin646@163.com
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-06-14.5-A16);山西省现代农业产业技术体系建设(杂粮)项目(2022-03);国家自然科学基金项目(31271791);山西省自然科学基金项目(201901D11126)

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 Published:2023-03-12 Published online:2022-07-07
  • Contact: WANG Rui-Yun,QIAO Zhi-Jun
  • 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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摘要：

根据糜子胚乳直链淀粉合成调控基因Waxy (GBSSI)上已知功能位点的核苷酸差异设计功能分子标记, 并对山西省270份糜子资源进行基因型检测和基因分型。通过对粳糯性状的鉴定, 将表型与基因型结合对分子标记进行验证。对46份不同基因型糜子资源进行直链淀粉含量测定, 分析该基因的遗传效应, 评估其育种利用价值。GBSSI基因在糜子中以2种形式(L和S)存在, 针对S型基因15 bp的InDel位点设计了一个功能标记(RYW214), 该标记能有效区分126 bp、141 bp和杂合(126/141 bp) 3种带型, 粳糯性鉴定显示该标记结果与表型鉴定结果吻合度高, 达93.30%, Pearson相关性指数r为0.745。针对L型基因上的一个单核苷酸插入位点和一个SNP位点, 设计了2个CAPS标记(RYW215、RYW216), 该标记可对L基因准确分型。270份资源中, 共有11种基因型。其中, 基因型S-15/LF最多, 有84份(31.10%), 其次是S0/S-15/LF (22.96%), S-15/LY/LF最少(0.74%), 未发现S-15/LC。46份材料中, 直链淀粉含量较高的基因型为S0/LY/LF(15.50%), 最低为S-15/LF (0.58%)。突变基因型S-15会导致直链淀粉含量出现质的下降, 杂合基因型S0S-15直链淀粉含量稍高于纯合基因型S-15。

关键词: 糜子, GBSSI基因, 功能分子标记, 直链淀粉含量

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

附表1

糜子材料来源"

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

表1

试验所用引物明细"

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

图1

部分材料在InDel位点的扩增产物电泳图 M为marker 500; 168: 二红黍; 169: 大红黍; 170: 小黑黍; 175: 硬地黄; 176: 黄黍子; 177: 老来红; 178: 边梅黍; 183: 成熟红; 180: 狗尾蛋; 181: 白骷髅; 182: 小红软糜; 184: 软白糜。"

图2

部分材料在SNP (A/G替换)位点扩增产物电泳图 M为marker 2000; 197: 峪杂1号; 198: 大黄糜子; 199: 小青糜子; 200: 大白硬糜; 203: 大黄芽。"

图3

部分材料在移码突变位点的扩增产物电泳图 M为marker 2000; 179: 鸡爪红; 180: 狗尾蛋; 181: 白骷髅; 182: 小红软糜; 183: 成熟红; 184: 软白糜; 185: 千斤黍; 186: 珍珠连软糜; 187: 白软黍; 188: 红黍子; 189: 金红黍; 191: 糜子。"

图4

3对引物扩增7份材料的DNAMAN序列比对结果图 A、B和C分别代表引物M5/R11、M12/R12和int5Lf/R3。41: 白黍子; 49: 黍子; 60: 黑糜子; 73: 白糜子; 80: 六十天小红黍; 87: 大白黍; 89: 大白黍。"

图5

糜子粳糯性表型鉴定 A: 粳性; B: 糯性。"

表2

270份糜子基因型分型"

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

表3

各基因型糜子直链淀粉含量"

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

图6

不同基因型糜子直链淀粉含量多重比较 不同小写字母表示差异显著性(P < 0.05)。"

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