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作物学报 ›› 2021, Vol. 47 ›› Issue (4): 684-690.doi: 10.3724/SP.J.1006.2021.02035

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻氮高效、耐冷基因OsGRF4功能标记的开发及其利用

孙平勇1(), 张武汉1, 张莉2, 舒服1, 何强1, 彭志荣1, 邓华凤1,3,*()   

  1. 1湖南杂交水稻研究中心杂交水稻国家重点实验室, 湖南长沙 410125
    2湖南省核农学与航天育种研究所, 湖南长沙 410125
    3湖南省农业科学院, 湖南长沙 410125
  • 收稿日期:2020-05-22 接受日期:2020-08-19 出版日期:2021-04-12 网络出版日期:2020-09-10
  • 通讯作者: 邓华凤
  • 作者简介:E-mail: zlspy23@126.com
  • 基金资助:
    湖南省自然科学基金项目(2019JJ50427);湖南省自然科学基金项目(2020JJ5287);湖南省科技重大专项项目(2018NK1020-1);湖南农业科技创新资金项目(2020CX08);湖南农业科技创新资金项目(2019TD06)

Development and application of functional marker for high nitrogen use efficiency and chilling tolerance gene OsGRF4 in rice

SUN Ping-Yong1(), ZHANG Wu-Han1, ZHANG Li2, SHU Fu1, HE Qiang1, PENG Zhi-Rong1, DENG Hua-Feng1,3,*()   

  1. 1State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, Hunan, China
    2Nuclear Agriculture and Space Breeding Research Institute, Changsha 410125, Hunan, China
    3Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China
  • Received:2020-05-22 Accepted:2020-08-19 Published:2021-04-12 Published online:2020-09-10
  • Contact: DENG Hua-Feng
  • Supported by:
    Natural Science Foundation of Hunan(2019JJ50427);Natural Science Foundation of Hunan(2020JJ5287);Hunan Province Key Research and Development Program Project(2018NK1020-1);Hunan Agricultural Science and Technology Innovation Project(2020CX08);Hunan Agricultural Science and Technology Innovation Project(2019TD06)

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摘要：

通过分子标记辅助选择(molecular marker-assisted selection, MAS)培育氮高效水稻品种是减少氮肥使用量、发展绿色农业和可持续农业的一个重要途经。水稻OsGRF4基因编码生长调节因子蛋白, 编码区第487和488碱基由TC变异为AA, 导致丝氨酸突变为赖氨酸, 使得水稻具有氮高效利用、高产和耐冷的特性。为了提高OsGRF4基因在育种中的选择效率, 本研究根据功能SNP (single nucleotide polymorphism)位点设计和筛选出等位基因特异PCR (polymerase chain reaction)功能标记PF+DMR+PR和PF+XMR+PR。利用此功能标记对不同品种(品系)和川大粒/巨穗稻的F2群体进行基因型检测, 结合测序分析验证, 能准确快速鉴定OsGRF4的纯合显性、纯合隐性和杂合基因型, 且具有操作简单、成本低的特点。本研究开发的功能标记为通过MAS方法利用OsGRF4基因培育氮高效、高产和耐冷水稻新品种提供了技术支撑。

关键词: 水稻, 氮高效利用, 耐冷, OsGRF4基因, 等位基因特异性PCR

Abstract:

The use of molecular marker-assisted selection (MAS) to breed rice with high nitrogen-use efficiency is one of the most effective methods to reduce the amount of nitrogen fertilizer quantity and to develop the green and sustainable agriculture. The gene growth-regulating factor 4 (OsGRF4) encodes a growth regulatory factor protein, and the mutation in the coding region from the nucleotides TC to AA in 487 and 488 substituted the amino acid serine for lysine, which resulting in enhancing nitrogen-use efficiency, increasing grain yield, and improving chilling tolerance in rice. In order to improve the selection efficiency of OsGRF4 in rice breeding, an allele-specific PCR (AS-PCR) marker combination, PF+DMR+PR and PF+XMR+PR, was developed based on the single nucleotide polymorphism in the functional region of the OsGRF4 alleles. The functional marker was used to identify genotypes of different varieties and an F2 population derived from Chuandali/Jusuidao. The three different genotypes of OsGRF4 locus could be accurately distinguished, which was further confirmed by sequencing. This functional marker is simple to operate and low-cost, and could provide a technical support when using MAS to breed new rice varieties of high nitrogen-use efficiency, high yields, and increased chilling tolerance.

Key words: rice, high nitrogen-use efficiency, chilling tolerance, OsGRF4 gene, AS-PCR

图1

OsGRF4基因功能标记设计策略 SNP3为功能性多态位点, PF和PR为外引物, DR和XR为反向内引物, 分别与碱基AA和TC匹配。分别在DR和XR的3′端引入错配碱基C开发了DMR和XMR。箭头表示扩增方向。"

图2

不同引物组合对川大粒和巨穗稻的PCR 扩增效果 单数泳道为川大粒; 双数泳道为巨穗稻; M: 100 bp marker; 1, 2: PF+PR; 3, 4: PF+DR; 5, 6: PF+XR; 7, 8: PF+DR+PR; 9, 10: PF+XR+PR; 11, 12: PF+DMR; 13, 14: PF+XMR; 15, 16: PF+DMR+PR; 17, 18: PF+XMR+PR."

图3

功能标记对不同水稻品种OsGRF4基因型的检测 A: PF+DMR+PR引物组合; B: PF+XMR+PR引物组合; M: 100 bp marker; 1: 川大粒; 2: 巨穗稻; 3: NIL-OsGRF4; 4: NIL-Osgrf4; 5~24: 农香99, 玉针香, 湘晚籼17, 冈46B, BL122, 佳辐占, 农香18, 明恢86, 新银占, 玉柱香, R700, 丰源 B, 农香29, 南洋占, R299, 02428, C418, P7144, 农香16, CY016。"

图4

与OsGRF4共分离标记GL2-11对川大粒/巨穗稻部分F2群体基因型的检测 M: 100 bp marker; 1: 川大粒; 2: 巨穗稻; 3~24: F2分离单株。"

图5

功能标记对川大粒/巨穗稻部分F2群体OsGRF4基因型的检测 M: 100 bp marker; A: PF+DMR+PR; B: PF+XMR+PR; 1: 川大粒; 2: 巨穗稻; 3~24: F2分离单株。"

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