基于火龙果转录组测序的SSR标记开发及种质亲缘关系分析

SSR Markers Development and Genetic Relationship Analysis of Germplasm Based on Transcriptomic Sequencing of Pitaya

本研究利用MISA软件筛选火龙果转录组测序获得的108 127条Unigenes,共检测出7 622个EST-SSR位点,其发生频率为6.02%,平均每9.00 kb出现1个位点。单核苷酸重复类型占优势,占总EST-SSR位点的56.59%,其次是二核苷酸和三核苷酸,分别占28.4%和14.04%;其它特性重复类型数量较少,所占比例均不足1%。二核苷酸重复基元类型中以AG/CT、AC/GT为优势重复基元,分别占总SSR位点数目的25.37%和2.02%;三核苷酸重复基元类型以AAG/CCT为主,占总SSR位点数目的 3.13%。设计合成125对EST-SSR引物,并随机选取8份形态学差异明显的火龙果种质提取基因组DNA,进行PCR扩增,采用琼脂糖凝胶电泳和10%聚丙烯变性凝胶电泳检测方法对引物进行初步检测,筛选出32对扩增条带锐利清晰的引物。选取38份火龙果种质对筛选出的引物进行多态性检测,获得16对多态性较好的引物,共扩增出47个多态性位点,多态信息含量(polymorphism information content, PIC)范围为0.243～0.667,平均多态性信息含量(polymorphism information content, PIC)为0.459,平均观测等位基因数(number of alleles, Na)为3,平均香农信息指数(Shannon's information index, I)为0.891;利用引物C31931、C13719和C32141等8种引物组合可以有效区分38份火龙果种质,构建其DNA的EST-SSR指纹图谱;UPGMA聚类分析,以0.62为阈值,可将38份火龙果种质分为3类:第一类包括红肉与粉红肉种质,第二类为白肉种质,第三类为蛇鞭柱属的2个种质。本研究基于火龙果转录组测序序列开发了一批具有高度多态性潜力的SSR引物,该引物可有效地将38份火龙果种质区分开来。因此,基于火龙果转录组测序开发的EST-SSR标记,可为火龙果种质鉴定、亲缘关系分析及遗传图谱构建等提供更丰富的标记来源。

A total of 7 622 EST-SSR loci were detected in 108 127 Unigenes obtained by sequencing the transcriptome of pitaya fruit using MISA software. The frequency of EST-SSR loci was 6.02%, with an average of1 locus per 9.00 kb. The type of single nucleotide SSR was the most abundant, which counted for 56.59% of the total EST-SSR loci, followed by dinucleotide and trinucleotide, accounting for 28.4% and 14.4% of the total,respectively. The other types of SSR accounted for less than 1%. AG/CT and AC/GT were the predominant repeat types in dinucleotide repeat motifs, accounting for 25.37% and 2.02% of the total SSR loci, respectively.AAG/CTT was the predominant repeat type in trinucleotide repeat motifs, which counted for 3.31% of the total.Totally, 125 pairs of EST-SSR primers were designed and synthesized, and genomic DNA of 8 pitaya germplasms which distinguished obviously in phenotype was used to carry out PCR. Agarose gel electrophoresis and 10%de-naturing polyacrylamide gel electrophoresis were used for the preliminary detection of primers. The results showed that bands of 32 primer pairs were clear and sharp. 38 pitaya germplasms were selected to detect the polymorphism of the screened primers, from which 16 pairs of primers with good polymorphism were obtained. A total of 47 polymorphic sites were amplified, and the polymorphism information content(PIC) was 0.243~0.667 with the average of 0.459. The average observed number of alleles(Na) and shannon’s information index(I) were 3and 0.891, respectively. The combination of 8 primers, such as primers C31931, C13719 and C32141, could effectively distinguish 38 pitaya germplasms, and the EST-SSR fingerprint of DNA was constructed. The UPGMA dendrogram demonstrated that at a genetic distance of 0.62, the 38 germplasms could be divided into three groups.The first group contained the red pulp accessions and the pink pulp accessions, the second was the white pulp accessions, and the third only consisted of two accessions belonging to Seleniereus Meja-lantous. In this study, a group of SSR primers with high polymorphic potential was designed based on the sequencing of the transcriptome of Hylocereus polyrhizus. These primer could effectively distinguish 38 pitaya germplasms. Therefore, the EST-SSR markers developed based on the sequencing of pitaya transcriptome could provide more marker sources for the germplasm identification, phylogenetic relationship analysis and genetic map construction of pitaya.