基于BSA-SSR技术的高丹草低氢氰酸性状目的片段的筛选与鉴定  

Screening and identification of target fragments with low cyanide traits of sorghum-sudangrass hybrid using BSA-SSR

作　　者：吴国芳 于肖夏[1] 于卓[1] 杨东升[1] 卢倩倩 WU Guo-fang;YU Xiao-xia;YU Zhuo;YANG Dong-sheng;LU Qian-qian(Agronomy College,Inner Mongolia Agricultural University,Hohhot 010019,China)

机构地区：[1]内蒙古农业大学农学院,内蒙古呼和浩特010019

出　　处：《草业学报》2021年第7期82-92,共11页Acta Prataculturae Sinica

基　　金：国家自然科学基金面上项目(31772664)资助。

摘　　要：采用BSA-SSR技术对高丹草低氢氰酸含量性状DNA目的片段进行了筛选和鉴定。结果表明,利用筛选出的9对SSR适宜引物PCR扩增找到了与高丹草低氰酸性状相关的SSR目的片段26个,经对PCR产物回收、部分纯化及测序分析,得到了这些低氰DNA片段的碱基序列。经与BLASTn数据库序列比对发现,有2个低氰目的片段TF8和TF16分别与高粱叶绿体磷酸核糖激酶XM_021458168.1和高粱克隆BAC 88M4 AY661656.1基因同源性高达95.4%和97.0%,其片段大小分别为101和586 bp。但在DNA碱基上有一定变异,TF8片段发生了3次碱基替换和1次插入,导致丙氨酸和甘氨酸替换成半胱氨酸,苏氨酸替换成丙氨酸,甘氨酸缺失;TF16片段缺失了1个碱基A,导致丙氨酸缺失。据此推测TF8和TF16这2个低氰目的片段具有调控低氢氰酸含量性状的作用。该研究结果为深入开展高丹草低氢氰酸含量性状主效QTL精细定位及标记辅助育种等研究奠定了基础。In this study,bulk segregant analysis using simple sequence repeat markers(BSA-SSR)was used to screen and identify DNA fragments linked to low hydrocyanic acid content of sorghum-sudangrass hybrids.By PCR amplification using nine pairs of suitable SSR primers screened,26 SSR target fragments related to low cyanide traits in sorghum-sudangrass hybrids were discovered.After PCR product recovery,partial purification,and sequence analysis,the base sequences of these DNA fragments linked to low cyanide content were obtained.Sequence comparison using the BLASTn database showed that two‘low cyanide’target fragments,TF8(101 bp)and TF16(586 bp)had,respectively,95.4%and 97.0%homology with sorghum phosphoribulokinase chloroplastic gene XM_021458168.1 and sorghum clone BAC 88M4 gene AY661656.1,but there was no mention of hydrocyanic acid regulation in the functional annotation of these two genes.However,there are a number of variations in the DNA base sequences.The TF8 fragment has three base substitutions and one insertion,resulting in the replacement of an alanine and a glycine with cysteine,a threonine with alanine,and deletion of a glycine.Fragment TF16 has one base A missing,resulting in alanine deletion.Based on these results it is inferred that TF8 and TF16,two low cyanide target fragments,regulate the expression of a low hydrocyanic acid content trait.These results lay a foundation for further research on the detailed mapping and marker-assisted breeding of the major QTLs for low hydrocyanic acid content of sorghum-sudangrass hybrids.

关 键 词：高丹草 低氢氰酸含量性状 BSA-SSR技术 目的片段筛选 序列比对鉴定 

分 类 号：S544.9[农业科学—作物学]