基于荧光SSR分析中国原产苹果属植物17个种的遗传多样性和遗传结构

Genetic diversity and population structure of 17 species of Malus Mill.native to China based on fluorescent SSR analysis

【目的】为探讨中国原产苹果属植物的遗传多样性水平以及种群间和种群内的遗传分化程度,利用荧光SSR分子标记,对新收集的中国原产苹果属植物17个种798份种质资源的遗传多样性和遗传结构进行分析。【方法】筛选19对多态性好的SSR引物检测苹果属植物17个种的多态性,利用GenAlEx 6.501计算遗传多样性指标、分析种群间的分子变异(AMOVA),利用GenepopV4和Fstat293分析种群间的遗传分化,使用STRUCTURE 2.3.4进行贝叶斯聚类并分析种群的遗传结构。【结果】19对SSR引物共检测出500个多态性等位基因,平均等位基因数为26.3,平均有效等位基因数为10.309,观察杂合度和期望杂合度的平均值分别为0.681和0.886,香农多样性指数为2.545。山荆子的观测等位基因数最多,为19.947;中国苹果的有效等位基因数和香农多样性指数最高,分别为9.585和2.418;13个种群内遗传变异占93%,而种群间的遗传变异仅占7%,遗传变异不显著(p>0.001)。楸子和中国苹果间的遗传分化系数最低,为0.006;变叶海棠和湖北海棠间的遗传分化系数最高,为0.253。【结论】17种苹果属植物的遗传多样性高于前人对苹果属植物野生种的研究。遗传分化主要存在于种群内,各种群间有基因交流,但同时又抵制了由于基因漂变而导致的种群间的遗传分化。新疆野苹果和山荆子可能参与了部分中国苹果的演化过程,除此之外,八棱海棠也可能参与了部分中国苹果的起源演化过程。

【Objective】The study aimed to analyze genetic diversity and population structure of newly collected 798 germplasm resources of 17 species of Malus Mill.native to China using fluorescent SSR molecular markers in order to provide references for germplasm collection and preservation of Malus and the study of the phylogenetic evolution of each species.【Methods】19 pairs of polymorphic SSR primers were screened to detect the polymorphism of 17 species of Malus Mill.GeneMapper3.0 was used to collect fluorescent labeling SSR data,and GenAlEx 6.501 was used to calculate the indexes of genetic diversity,such as number of polymorphic alleles(Na),number of effective alleles(Ne),observed heterozygosity(Ho),expected heterozygosity(He),fixed index(F)and shannon diversity index(I),and to analyze the molecular variation(AMOVA)among populations.The genetic differentiation among populations were analyzed by GenepopV4 and Fstat293.The Bayesian cluster was carried out using STRUCTURE 2.3.4 to analyze the genetic structure of populations.The characteristic number of allele variation frequency(population number)K=1-20,burn in cycle 100000 and MCMC repetition 100000 were set.The mixed model and related allele frequency were used to run different K values for 10 times,and the log function Lnp(D)of delta K and likelihood value was calculated.The optimal K value was determined by modeling the number of gene pools(k).Clumpp 1.1.2 software was employed to process the distribution coefficient Q value(the estimation coefficient between each individual in each group)obtained by 10 independent runs,and Ddistract 1.1 software was used to optimize the graph.【Results】500 polymorphic alleles were detected by 19 pairs of SSR primers,with an average allele number of 26.3 and effective allele number of 10.309.The average values of heterozygosity and expected heterozygosity were 0.681 and 0.886 respectively,and the Shannon index was 2.545.The genetic diversity of 17 species of Malus was studied.The observed allele number of Malus baccata was 19.947.The effective allele number and Shannon index of Malus domestica subsp.chinensis were 9.585 and 2.418,respectively.The heterozygosity of all populations was higher than 0.5,indicating that the new collection of germplasm resources of Malus had not been artificially selected with high intensity,and the genetic diversity was relatively high.The first three species with the largest difference between expected heterozygosity and observed heterozygosity were Malus yunnanensis,Malus ombrophila and Malus honanensis,the next one was Malus baccata,and Malus hupehensis had the smallest difference.The genetic diversity of Malus yunnanensis,Malus ombrophila and Malus honanensis was the highest,while that of Malus hupehensis was the lowest.There were more heterozygotes in the populations of Malus rockii,Malus hupehensis,Malus Komarovii,Malus transitoria,Malus toringoides,Malus yunnanensis,Malus ombrophila,Malus honanensis,Malus micromalus.The genetic diversity of 798 accessions of Malus Mill.in this study(He=0.886,I=2.545,Ne=10.309)was higher,among which Malus sieversii(He=0.814,I=2.041,Ne=6.054),Malus baccata(He=0.848,I=2.350,Ne=8.652),Malus toringoides(He=0.663,I=1.355,Ne=3.332)and Malus hupehensis(He=0.2628,I=0.4015,Ne=1.4375)had higher genetic diversity than those species in previous studies.Except for Malus honanensis,Malus yunnanensis,Malus ombrophila and Malus komarovii with only one accession in every group,the genetic variation in the remaining 13 populations accounted for 93%,while the genetic variation among populations accounted for only 7%,and the genetic variation was not significant(p>0.001).Different groups had different levels of gene exchange.The lowest genetic differentiation coefficient was 0.006 between Malus prunifolia and Malus domestica subsp.chinensis,followed by 0.007 between Malus prunifolia and Malus asiatica,and the highest genetic differentiation coefficient was 0.253 between Malus toringoides and Malus hupehensis.The analysis of population genetic structure of 798 accessions of 17 species showed that when k=2,△K got the maximum value,and when K=5,the rise of Lnp(D)slowed down,5 was an important clustering point.The genetic sources of wild apple species in Malus sieversii,Malus baccata,Malus rockii,Malus hupehensis,Malus halliana,Malus kansuensis,Malus Komarovii,Malus transitoria,Malus toringoides,Malus yunnanensis,Malus ombrophila,Malus honanensis were relatively narrow.Among the five cultivated species tested,only part of Malus domestica subsp.chinensis contained a large number of genes from group 1,and the other four cultivated species contained a small number of genes from group 1,but mainly from other wild species.【Conclusion】19 pairs of SSR primers were highly polymorphic,and had high transferability between populations.They could be used for the evaluation of genetic diversity and population structure of different species of Malus Mill.The genetic diversity of 17 species of Malus was higher than that of previous studies.There are abundant genetic variations among and within the species of Malus native to China,which was beneficial to the adaptation of different species of Malus to various ecological environments.Genetic differentiation mainly existed in populations,and there was gene exchange among different populations,but at the same time,the genetic differentiation among populations was resisted by gene drift.Malus sieversii and Malus baccata might be involved in the evolution of some of Malus domestica subsp.chinensis.In addition,Malus robusta might also be involved in the evolution of some of Malus domestica subsp.chinensis.