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

Abstract:【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 Malusand 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 100 000 and MCMC repetition 100 000 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 al- lele number of 26.3 and effective allele number of 10.309. The average values of heterozygosity and ex- pected heterozygosity were 0.681 and 0.886 respectively, and the Shannon index was 2.545. The genet- ic 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 andMalus 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 ofMalus rockii, Malus hupehensis, Malus Komarovii, Malus transitoria, Malus toringoides, Malus yunna- nensis, Malus ombrophila, Malus honanensis, Malus micromalus. The genetic diversity of 798 acces- sions of Malus Mill. in this study (He=0.886, I=2.545, Ne=10.309) was higher, among which Malus siev- ersii (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=0.262 8, I=0.401 5, Ne=1.437 5) had higher genetic diversity than those species in previous studies. Except for Malus honanensis, Malus yunnanen- sis, Malus ombrophila and Malus komarovii with only one accession in every group, the genetic varia- tion 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 Ma- lus prunifolia and Malus domestica subsp. chinensis, followed by 0.007 between Malus prunifolia andMalus asiatica, and the highest genetic differentiation coefficient was 0.253 between Malus toringoidesand 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. chinen- sis 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 Ma- lus 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.