基于转录组测序的玫瑰及其近缘种遗传多样性分析与指纹图谱构建

Genetic diversity analysis and fingerprint construction of Rosa rugosa and its related species based on transcriptome sequencing

【目的】分析玫瑰及其近缘种之间的遗传多样性并构建指纹图谱,为玫瑰种质资源鉴定与开发利用奠定基础。【方法】在玫瑰的传统品种、杂交繁育品种和国内外引进品种中各选择1种,取其鲜嫩叶片进行转录组测序;基于测序得到的玫瑰转录组数据,使用MISA在reads覆盖的基因组数据中查找玫瑰的SSR位点,并根据SSR位点两端的保守序列使用Primer 3.0设计引物。选取10种玫瑰的DNA作为试验材料,筛选设计、合成后的引物。以48份玫瑰及其近缘种的DNA作为试验材料,利用筛选出的峰值较好的引物进行TP-M13-SSR PCR,并对其扩增产物进行毛细管电泳检测,应用GeneMarker 2.2.0(SoftGenetics,USA)读取毛细管电泳数据并用Excel进行整理;使用POPGEN VERSION 1.32计算筛选引物的观测杂合度、期望杂合度、Nei’s遗传多样性指数、观测等位基因数、有效等位基因数、Shannon信息指数,并用CERVUS version 3.0计算多态性信息含量。利用Powermarker计算玫瑰及其近缘种各种质之间的遗传距离;采用NTSYSpc 2.10e计算每2个种质之间的遗传相似性系数,并绘制UPGMA聚类树状图。最后采用引物与基因型组合的方式构建玫瑰及其近缘种的指纹图谱。【结果】基于玫瑰样品转录组测序数据,使用MISA共检测出48796个SSR位点,分布于139712条Unigene中,碱基重复类型数量最多的为二核苷酸重复和三核苷酸重复,分别为20628和12828个。使用Primer 3.0以SSR位点两端的保守序列为依据初步设计并合成了144对引物;以10个玫瑰品种的DNA作为模板筛选引物,共筛选出峰值较好的28对引物。以48份玫瑰及其近缘种的DNA为试验材料,28对引物在48份试验材料中均能扩增出峰型良好、多态性高的DNA片段;28对引物的观测杂合度、期望杂合度、Nei’s遗传多样性指数、观测等位基因数、有效等位基因数、Shannon信息指数和多态性信息含量的平均值分别为0.4101,0.7505,0.7011,4.607个,3.5116个,1.3442和0.6526。大多数供试样品间的遗传距离为0.6000~0.8000;聚类分析结果显示,在遗传相似系数为0.571时,48份玫瑰及其近缘种被分为两类。运用核心引物法筛选出的4对核心引物可将48份试验材料全部区分开,并构建了其指纹图谱。【结论】开发并筛选出28对多态性较好的SSR引物,可用于后续玫瑰的遗传多样性分析、遗传图谱构建、遗传稳定性鉴定等方面。

【Objective】The genetic diversity of Rosa rugosa and related species was analyzed and its fingerprint was constructed to provide foundation for the identification,development and utilization of R.rugosa germplasm resources.【Method】Firstly,the fresh leaves of one traditional R.rugosa variety,one hybrid variety and one imported variety were selected for transcriptome sequencing.Based on the transcriptome data obtained by sequencing,MISA was used to find R.rugosa SSR loci in the genomic data covered by reads,and Primer 3.0 was used for primer design according to the conserved sequences at both ends of the loci.Secondly,the DNA of 10 R.rugosa kinds was selected to screen the designed and synthesized primers.Then,the DNA of 48 R.rugosa and related species were selected for TP-M13-SSR PCR with be-tter peak primers,and the amplified products were detected by capillary electrophoresis.GeneMarker 2.2.0(SoftGenetics,USA)was used to read the capillary electrophoresis data.Excel was used to organize the electrophoresis data.The observed heterozygosity,expected heterozygosity,Nei’s diversity index,observed number of alleles,effective number of alleles and Shannon’s diversity index were calculated by POPGEN VERSION 1.32.The polymorphism information content was calculated by CERVUS version 3.0.Then,Powermarker was used to calculate the genetic distances between each quality of R.rugosa and its relatives.The genetic similarity coefficient between each 2 accessions was calculated using NTSYSpc 2.10e and the UPGMA cluster dendrogram was drawn.Finally,primers and genotype combinations were used to construct fingerprints of R.rugosa and related species.【Result】Based on transcriptome sequencing data of R.rugosa samples,a total of 48796 SSR sites were detected in 139712 Unigenes by MISA.The most frequent base repeat types were dinucleotide repeats(20628)and trinucleotide repeats(12828).A total of 144 pairs of primers were designed and synthesized based on the conserved sequences at both ends of the site using Primer 3.0.DNA from 10 R.rugosa species was used as a template to screen primers,and a total of 28 pairs of primers with better peaks were selected.The 28 pairs of primers were able to amplify DNA fragments with good peak type and high polymorphism in 48 samples of R.rugosa and related species.The mean values of observed heterozygosity,expected heterozygosity,Nei’s diversity index,observed number of alleles,effective number of alleles,Shannon’s diversity index and polymorphism information content of 28 pairs of primers were 0.4101,0.7505,0.7011,4.607,3.5116,1.3442 and 0.6526,respectively.The genetic distances of most samples were between 0.6000 and 0.8000.The cluster analysis showed that 48 R.rugosa and their relatives were divided into two groups with genetic similarity coefficient of 0.571.The four pairs of core primers selected by the core primer method could distinguish all 48 test materials and construct their fingerprints.【Conclusion】The 28 SSR primers with good polymorphism selected in this study can be used for the analysis of genetic diversity,construction of genetic map and identification of genetic stability in R.rugosa.