基于cpInDel标记和cpSSR标记的柑橘属及近缘属植物亲缘关系

Genetic Relationship Among Citrus and Its Relatives as Revealed by cpInDel and cpSSR Marker

【目的】通过对柑橘叶绿体基因组变异位点的研究,从胞质层面揭示柑橘类果树的遗传进化,为柑橘种质资源的收集、评价和利用提供参考。【方法】以甜橙叶绿体高变区序列为参考,利用已发表的二代基因组测序数据对代表性的柑橘属及其近缘属材料进行De Novo组装,获得叶绿体基因组高变区组装序列,通过序列比对,发掘代表性材料的差异位点,针对差异位点设计cpInDel引物。对甜橙叶绿体基因组两个及以上核苷酸重复构成的SSR位点进行定位分析,找出代表性材料间有差异的位点,针对差异位点设计cpSSR引物。选择在分类学和/或起源研究上有代表性的柑橘属及近缘属材料48份,利用新开发的cpInDel、cpSSR标记和已报道的cpSSR标记进行扩增检测、电泳谱带分析和聚类分析。【结果】本研究新开发4个cpInDel标记和13个cpSSR标记。扩增检测显示,这些标记在48份试验材料中均能扩增出较为清晰的多态性条带;但cpInDel标记扩增出的条带更为单一,类群区分时,仅通过单个标记便能实现对某一类群或几个类群的区分,具有更好的区分效果。聚类分析显示,cpInDel和cpSSR标记得到了比较类似的结果,但在小类群关系以及部分材料的归类上存在差异。二者均揭示枸橼类柠檬-藜檬-来檬杂种的胞质来源并非香橼,枳杂种的胞质也并非枳,显示宽皮柑橘类黄柑的代表性品种‘旺苍皱皮柑’有不同于宽皮柑橘类其他品种的胞质来源,‘资阳香橙’和‘韩国香橙’胞质差异明显。针对宽皮柑橘类、甜橙-酸橙类和宜昌橙-大翼橙类3个小类群的相互关系,cpInDel显示它们关系较近,而cpSSR则将它们归为独立的类群;cpInDel标记将印度野橘归于真正的枸橼类,cpSSR将其单独聚为一类。【结论】叶绿体基因组分子标记分析能从胞质角度揭示不同于核基因组的柑橘属及近缘属植物的亲缘关系,但若与核基因组分子标记分析相结合,能更全面地揭示材料间的亲缘关系,更准确地鉴定柑橘种质资源。

【Objective】The genetic diversity analysis of the chloroplast genome was performed to reveal the genetic evolution of citrus from the cytoplasmic level,so as to provide a reference for the collection,evaluation and utilization of citrus germ plasm resources.【Method】Taking the hypervariable region of Citrus sinensis chloroplast genome as reference,De Novo assemblies were carried out with next-generation sequencing data of representative Citrus and its related genera materials.The assembled sequences were aligned to discover site-variations,and cpInDel markers were designed on the basis of variation sites.SSR sites composed of two or more nucleotide repeats were located in the chloroplast genome of sweet orange,and SSR sites with length variations were adopted to design cpSSR markers.The newly developed markers and reported cpSSR markers were used to detect genetic diversity in 48 samples of Citrus and its relatives that are representative in taxonomy and/or origin through band typing and clustering analysis.【Result】4 cpInDel markers and 13 cpSSR markers were developed successfully and they could produce clear polymorphic bands in all tested samples.The band pattern of cpIndel was relatively simple.Single marker could distinguish a specific genus or several genera.At species and/or above taxa level,the cpInDel markers gave more convenient and accurate discrimination effect.Similar clustering results were obtained with cpInDel and cpSSR markers,and minor differences were found in the relationship between the lower-rank taxa and the classification of a few genotypes.The cytoplasmic origins of P.trifoliate hybrids were found to be different from P.trifoliata,and those of lemon,limonia and lime hybrid were different from C.medica by both markers.Wangcang zhoupigan,a representative variety of C.speciosa,had a different cytoplasmic origin from other mandarin varieties.Ziyang xiangcheng and Korean xiangcheng,both classified as C.junos,might have different cytoplasmic origin.cpSSR markers classify mandarin,sweet orange and sour orange,ichang papeda and honghe papeda into three clusters,representing C.reticulata,C.sinensis and Papeda,while cpInDel markers showed that they were closely related and classified into one cluster.Indian wild mandarin were clustered into C.medica by cpInDel,and were put into a single cluster by cpSSR.【Conclusion】Molecular marker analysis of chloroplast genome could reveal the genetic relationship of Citrus and its related genera and discover a different relationship from the view of nuclear genome.However,if combined with markers from nuclear genome,more comprehensive and accurate indentification and phylogenetic relationship revealing should be obtained for the citrus germplasm resources.