水稻Wx复等位基因基于PCR的功能标记开发与利用

摘要

Amylose content (AC) is one of the most important factors impacting rice eating quality, which was mainly determined by Wx locus.Multiple alleles,such as Wxa,Wxb,Wxin,and Wxmwcontribute to the variation of AC in rice varieties.Selecting rice varieties with low-medium AC through Marker-assisted selection (MAS) is an important way to improve rice eating quality. However, distinguishing the Single Nucleotide Polymorphisms (SNP) of Wx alleles requires the DNA sequencing or restriction enzyme digestion, which is time consuming and labor-intensive. In this study, we developed effective MAS markers by tetra-primer ARMS-PCR technology utilizing functional SNPs corresponding to the different alleles, moreover we put forward corresponding solving schemes on low amplification efficiency and inaccuracy extension through adjusting the location of delib-erate mismatch bases introduced. We easily differentiated the multiple alleles of Wx utilizing the markers Waxygt-ARMS2and Waxyac-ARMS2, showing that these markers have significant application value with the features of rapid operation and low cost. We further analyzed the genotype distribution of Wx allele in Liaoning Province rice varieties,and found all of the forty tested varieties carried the Wxballele with a narrow genetic diversity.The results of this study could provide theoretical basis and tech-nical support on both new rice varieties breeding and rice eating quality improvement utilizing Wx alleles corresponding to low-medium AC by MAS in Liaoning Province.%直链淀粉含量是影响稻米食味品质的重要因素, 主要由蜡质基因(Wx)调控, Wx 基因座存在 Wxa、Wxb、Wxin、Wxmw等多个复等位基因,通过分子标记辅助选择(marker-assisted selection, MAS)培育中低直链淀粉含量的水稻品种是提高稻米适口性的重要途径.Wx复等位基因的功能由单碱基核苷酸多态性(single nucleotide polymorphisms, SNP)决定,其检测依赖于测序或酶切,存在耗时多、费用昂贵等缺点.为提高选择效率,本文利用Wx复等位基因的SNP差异和四引物扩增受阻突变(tetra-primer ARMS-PCR)原理, 进行PCR功能标记的开发.引物设计过程中, 针对扩增效率低和非匹配的延伸2个技术难题, 提出了调整错配位点的解决方案, 成功开发了基于PCR技术的两对功能性标记Waxygt-ARMS2和 Waxyac-ARMS2, 可以准确区分上述复等位基因型, 且具有操作简单、耗费较低的特点.利用新开发标记对辽宁省育成的部分水稻品种进行Wx基因型检测的结果表明,40份辽宁育成品种均携带Wxb基因,遗传基础单一.上述结果为利用Wx基因位点的分子标记培育中低直链淀粉含量的水稻品种, 改良辽宁省水稻品种的食味品质提供了技术支撑.