水稻抗咪唑啉酮类除草剂基因ALS功能标记的开发与应用

摘要

选育和利用抗除草剂水稻品种具有重要的生产实践意义.通过筛选水稻资源,发现了抗除草剂材料金粳818,其ALS基因编码区第1880位碱基存在一个由G到A的碱基变异,导致丝氨酸突变为天冬酰胺,从而具有除草剂抗性.本研究基于该位点的碱基变异,设计了11条等位基因特异PCR (allelic-specific PCR,AS-PCR)引物,经过优化筛选,获得两个引物组合F1N (S1/S9)和F1M (S1/S10),将该标记命名为AS-ALS.利用F2群体及其亲本和杂交种,结合AS-ALS标记检测和除草剂抗性分析,结果表明感除草剂ALS-G等位基因型只能被F1N引物对有效扩增,抗除草剂ALS-A等位基因型只能被F1M引物对有效扩增,而杂合基因型能同时被两对引物F1N和F1M扩增,ALS-A纯合或杂合等位基因型都表现抗除草剂,ALS-G纯合基因型表现感除草剂.因此本研究开发的标记能有效区分除草剂抗性基因的3种基因型,基因型与表型完全对应.该标记用于回交育种,可以选择ALS-A杂合基因型单株,剔除ALS-G纯合等位基因型,在自交的F2保留ALS-A纯合基因型单株,连续自交,能快速获得除草剂抗性稳定的水稻材料.该除草剂抗性基因的功能标记还可用于咪唑啉酮类除草剂抗性资源筛选.%Breeding and utilization of herbicide resistant rice are significant to rice production.By screening the rice germplasm,we found the herbicide resistant material "Jinjing 818".An SNP mutation G to A was present in Acetolactate synthase (ALS) gene at 1880 bp position,leading to the alteration from serine (S,AGT) to asparagine (N,AAT),which confers herbicide resistance.In this study,11 allelic-specific PCR (AS-PCR) primers were designed based on the functional mutation.After optimized these primers,we obtained two primer combinations FIN (S1/S9) and F1M (S1/S10),named AS-ALS marker.Using this marker detected the genetic population,its parents,F1 hybrid,F2 and also rice collections,inbred lines,showing that the herbicide susceptibleness allelic ALS-G could be amplified by FIN,the herbicide resistance allelic ALS-A by F1M,and heterozygous genotype by F1N and F1M simultaneously.The genotype of those tested materials perfectly matched with the phenotype of herbicide resistance or susceptibleness.Aided by AS-ALS marker selection,the homozygous ALS-A pedigrees in multi-generation backcross or self-cross showed stable herbicide resistance.Therefore,the allelic-specific PCR functional marker AS-ALS can be used in herbicide breeding efficiently,also screening herbicide resistant rice germplasm.In conclusion,the AS-ALS marker developed in this research is inexpensive and effective in breeding practice.