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作物杂志,2020, 第1期: 55–60 doi: 10.16035/j.issn.1001-7283.2020.01.010

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

水稻苗期耐低温基因COLD1新功能标记的设计与验证

田孟祥1,宫彦龙2,张时龙1(),何友勋1,雷月2,余本勋1,余莉1,李佳丽2,张大双2,叶永印1   

  1. 1毕节市农业科学研究所,551714,贵州毕节
    2贵州省水稻研究所,550006,贵州贵阳
  • 收稿日期:2019-07-28 修回日期:2019-12-18 出版日期:2020-02-15 发布日期:2020-02-23
  • 通讯作者: 张时龙 E-mail:bjrice@163.com
  • 作者简介:田孟祥,主要从事水稻遗传育种研究,E-mail: tmengxiang@126.com
  • 基金资助:
    国家重点研发计划项目(2017YFD0100402);贵州省科技成果应用及产业化计划项目(黔科合成果[2018]4306);毕节市科技支撑计划项目(毕科合字[2016]20号)

Design and Verification of New Functional Marker of Chilling-Tolerance COLD1 Gene in Rice Seedling Stage

Tian Mengxiang1,Gong Yanlong2,Zhang Shilong1(),He Youxun1,Lei Yue2,Yu Benxun1,Yu Li1,Li Jiali2,Zhang Dashuang2,Ye Yongyin1   

  1. 1Bijie Institute of Agricultural Sciences, Bijie 551714, Guizhou, China
    2Guizhou Rice Research Institute, Guiyang 550006, Guizhou, China
  • Received:2019-07-28 Revised:2019-12-18 Online:2020-02-15 Published:2020-02-23
  • Contact: Shilong Zhang E-mail:bjrice@163.com

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摘要：

籼稻和粳稻在苗期耐低温基因COLD1的第4外显子存在1个功能性单碱基变异SNP2,粳型COLD1 Jap等位基因低温耐受性表现更强,具有重要的育种利用价值。通过籼粳杂交,可将粳型COLD1 Jap等位基因导入籼稻品种,提高其低温耐受力。为提高COLD1基因的选择效率,根据粳型COLD1 Jap与籼型COLD1 Ind基因存在的单核苷酸差异,结合扩增受阻突变体系PCR的技术原理设计功能标记。应用5个籼稻品种、5个粳稻品种、1个籼粳杂交F1个体以及1个籼粳杂交F2群体对功能标记进行检测验证。结果表明,所设计的功能标记可准确区分纯合粳型COLD1 Jap、纯合籼型COLD1 Ind和杂合基因型,其扩增带型与基因型完全一致,是一种鉴定COLD1基因的有效方法。该标记弥补了前人设计的衍生型酶切扩增多态性序列功能标记费用昂贵、操作复杂及费工费时等不足,可广泛应用于水稻COLD1基因的资源鉴定和分子标记辅助选择育种。

关键词: 水稻, 耐低温, COLD1基因, 功能标记

Abstract:

The nucleotide variation of SNP2 in the fourth exon of COLD1 gene associated with the difference of chilling tolerance at the seedling stage between indica and japonica rice. Individuals with japonica COLD1 Jap allele have higher chilling tolerance. Therefore, the COLD1 Jap allele of japonica rice could be introduced via hybridization, thereby enhancing the cold tolerance of indica rice. In order to improve the selection efficiency of COLD1 gene, a new functional marker for detecting the single nucleotide polymorphism SNP2 between COLD1 Jap and COLD1 Indwas designed based on the technical principles of amplification refractory mutation system PCR. Furthermore, the availability of the functional marker was evaluated using five indica varieties, five japonica varieties, one F1 individual and one F2 from the indica × japonica cross. The results showed that the designed functional markers could accurately identify homozygous COLD1 Jap, homozygous COLD1 Ind and heterozygous genotypes, and amplified bands were identical to their corresponding genotypes, suggesting that it was an effective method for identifying COLD1 gene using this marker designed in this study. The method is simple and inexpensive and could overcome the shortcoming of the COLD1 gene functional markers previously designed based on derived cleaved amplified polymorphic sequence. In conclusion, the new functional marker could be widely used in genetic resource identification and molecular marker-assisted selection breeding of rice COLD1 gene.

Key words: Rice, Chilling-tolerance, COLD1 gene, Functional marker

图1

COLD1基因功能标记设计策略 序列中字母下划线表示单核苷酸差异位点SNP2;省略号表示相同的碱基;箭头表示引物位置和扩增方向;方框内为引入的错配碱基"

表1

COLD1基因功能标记的引物序列"

引物Primer 序列（5′-3′）Sequence (5′-3′)
COLD1-O-F CATTTCCCCATGCCTTCTCC
COLD1-O-R CAACTGTCCCAACGATACGC
COLD1-I-F1 CCTGGCTTACAGGGAAATTGATGAGAT
COLD1-I-F2 CTGGCTTACAGGGAAATTGATGAGAC
COLD1-I-R GAGCTGCCTTTCCAATGTTTTGATGTTCT

图2

COLD1基因功能标记对水稻材料的检测 M:Ladder H1（100、200、300、400、500、600、700、800、900、1000bp）,下同;1-11分别为日本晴、9311、F1（9311/越光）、越光、空育131、稻花香2号、松粳9号、特青、明恢63、珍汕97和南京6号"

图3

使用功能标记对南京6号/稻花香2号F2群体16个植株的检测"

图4

使用4条引物对籼型COLD1Ind基因差异碱基的鉴定 1和4为越光;2和5为9311;3和6为F1（9311/越光）"

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