基于引物“随机组合”构建观赏桃SSR指纹图谱

Construction of SSR fingerprint for ornamental peach based on primers “random combination”

关键词:  观赏桃, 种质, SSR标记, 分子身份证, 引物组合法

Key words:  ornamental peach, germplasm, SSR marker, molecular ID, primer combination method

摘要:

近年来,我国观赏桃新品种日渐繁多、名称混乱、市场难以监管,同时用以区分品种的SSR指纹图谱的构建方法在研究界无统一的科学标准,尤其是构成最终引物组合的核心引物的确定,具体操作流程层出不穷、五花八门。为探索筛选SSR指纹图谱核心引物的科学方法,同时构建观赏桃SSR指纹图谱,该研究选用35对已报道的SSR引物对22份观赏桃种质进行试验。结果表明:通过PCR扩增与分析,多态性较高的8对引物——候选引物总共扩增出31个多态性条带,变幅为3～5个,PIC值变幅为0.458～0.668,MI值变幅为1.374～3.340。采用“随机组合”法对8对引物进行C¹₈、C²₈、C³₈…依次分析,得到区分能力最强的3种不同的最少引物组合方式——候选组合,并能区分出18份种质,从中发现区分能力最强的3种引物组合方式并不都是由引物PIC值、alleles数量或MI值等多态性指标最高的引物组成,而是由互补性最强的引物组成。选用组合内各引物多态性条带总数最多的组合方式“4-3”(BPPCT001+BPPCT015a+BPPCT017+BPPCT025)为22份观赏桃种质构建了指纹图谱。基于此,通过常规多态性指标筛选候选引物可以确定出单对引物鉴别能力最强的少量引物; 通过“随机组合”筛选候选组合可以进一步确定出引物之间互补性最强的几种组合方式; 根据组合内各引物的多态性条带总数确定最终核心引物可以确定出可扩容性最大的引物组合。该研究最终建立了候选引物——候选组合——核心引物组合“三步法”确定SSR指纹图谱核心引物组合的科学方法,不仅为22份供试观赏桃种质构建了SSR指纹图谱,也为其它作物SSR指纹图谱的构建提供了新的思路。

Abstract:

In the recent years, the nomenclature of the cultivars of ornamental peach in China is falling into mess under the rapidly increasing of new cultivars. This makes it more and more hardly to deal with the market regulation. Meanwhile, there is no statistically unified scientific standard for the methods of constructing SSR fingerprint in research community, especially for filter the core primers that consisted of the finally unique group, the concrete operating procedures are plenty, variable and mess. In order to explore the statistically unified scientific method for filtering the core primers of the finally unique group and construct the SSR fingerprint for experimental ornamental peach, we selected 35 pairs of SSR primers reported previously to test on 22 ornamental peach germplasms. After PCR amplification and analysis firstly, 8 pairs of primers among them with high polymorphism called “candidate primers” were detected 31 polymorphism bands in total, ranging from 3-5 per pairs of primer. And the 8 pairs of primers gained 0.458-0.668 in PIC values and 1.374-3.340 in MI values correspondingly. Secondly, we analyzed C¹₈、C²₈、C³₈…successively by “random combinations” method, obtained 3 different primer combinations consisting with lest of primers called “candidate combination”, which possessed the strongest discrimination ability. All of the three could distinguish 18 accessions. And the 3 best primer groups were not consisted with the series of primers which possessed high leaves of PIC values, MI values or high numbers of alleles and so on, but a series primers which possessed an high leaves of complementarity. Lastly, the combination “4-3”(BPPCT001+BPPCT015a+BPPCT017+BPPCT025), which possessed the most polymorphism bands in total of the primers among groups, was finally chosen to establish the fingerprint for the 22 ornamental peach germplasms tested. The rerults showed that to filter candidate primers according to routine polymorphic index could select the few pairs of primers which possessed the strongest discrimination ability per pairs of primer; to filter candidate combination according to “random combination” could furtherly select the few compound modes which possessed the strongest complementarity; to filter core primer combination according to numbers of the bands in total of primers among groups could finally select the unique group which possessed the largest expandable sex. Therefore, “3 steps” for candidate primers-candidate combination-core primer combination was advocated as a scientific method to fitter the core primers of SSR fingerprint. It did constructed the SSR fingerprint for the 22 ornamental peach germplasms tested, and would provide a new approach to construct SSR fingerprints for other crops.