基于黄花蒿低覆盖度高精度基因组SSR分子标记开发

Development of Genomic SSR Molecular Markers with Low Coverage and High Precision in Artemisia annua Linn.

不同产地黄花蒿品质差异较大,因此开发黄花蒿特异性分子标记对于筛选鉴定优质种源具有重大意义。本研究结合形态标记及化学计量学对相同环境下不同种源黄花蒿的特征进行统计分析;再利用Krait软件对黄花蒿不同覆盖度CCS数据及全基因组进行SSR位点数据挖掘,使用Primer Premier 3.0软件设计引物,筛选出具有多态性的引物并鉴定种源。结果表明,相同培养环境下黄花蒿的形态特征及有效成分含量具有显著性差异;同时,黄花蒿1X CCS数据能够有效反映基因组SSR的特征,二者的6种SSR重复类型的重复次数集中于5~13次,长度大多分布于10~30 bp。以1X CCS数据筛选出的SSR位点为基础,设计筛选得到373187对引物,随机挑选的20对引物用于有效性检测,14对引物扩增出条带,其中引物S1能在材料间扩增出特异性条带,可用于对不同产地黄花蒿进行区分。本研究以1X CCS数据为基础,开发黄花蒿SSR标记,结合形态学标记及有效成分含量,能够有效区分不同种源的黄花蒿,为黄花蒿遗传多样性分析、资源鉴定保护、基因功能研究等方面提供了丰富的分子标记。另外低覆盖度CCS具有测序量小,无需拼接组装,读长长,精确度较高且成本较低等优势,可作为开发SSR标记的基础数据,为具有超大基因组的物种提供了一种新的思路。

The quality of Artemisia annua from different germplasm varies greatly,so the development of specific molecular markers of A.annua is of great significance for screening and identifying high-quality provenances.In this study,morphological markers and stoichiometry were combined to analyze the characteristics of A.annua from different provenances in the same environment.Krait software was used to mine the CCS data of different coverage levels and the whole genome for SSR loci.Primer Premier 3.0 software was used to design primers,screen out polymorphic primers and identify the provenances.The results showed that the morphological characteristics and content of effective components of A.annua were significantly different in the same culture environment.At the same time,the 1X CCS data could effectively reflect the characteristics of genomic SSR,and the repeats of the6 SSR repeat types were concentrated in the range of 5~13,and the length of most of them was 10~30 bp.Based on the SSR loci screened by 1X CCS data,373187 pairs of primers were designed and screened,20 pairs of primers were randomly selected for validity detection,and 14 pairs of primers were amplified.Among them,primers S1could amplify specific bands between materials,which could be used to distinguish A.annua from different habitats.In this study,based on 1X CCS data,SSR markers were developed in A.annua.Combined with morphological markers and active component content,SSR markers could effectively distinguish A.annua from different provenances,providing abundant molecular markers for analysis of genetic diversity,resource identification and protection,and gene function research of A.annua.In addition,CCS with low coverage has the advantages of small sequencing amount,without splicing and assembly,long reading length,high accuracy and low cost,which can be used as support data for developing SSR markers and provide a new idea for species with large genomes.