Genetic Diversity And Genetic Differentiation Of Aquilegia Yabeana Complex Based On Microsatellite Molecular Markers

Flower is the most important innovative trait of angiosperm.Aquilegia Linn,is becoming a new model plant for the evolution of petal structure because of the colorful flower and innovative petal spur structure.There are about 70 species in Aquilegia,distributed in the northern temperate,such as Europe,Asia and North America.Studies have shown that the speciation of Aquilegia in North America is more or less under the drive of pollinators.However,in China,there is a widely distributed spurless species named Aquilegia ecalcarata Maxim,which is the only one whose nectar spur has stopped developing in genus Aquilegia.The results of molecular phylogenetic studies based on three chloroplast fragments previously in our Lab showed that A.ecalcarata and its three closely related species(Aquilegia yabeana kitag,Aquilegia kansuensis(Bruhl)Erst comb.et stat.nov.and A.rocki Munz)formed a monophyletic group,which referred to as the Aquilegia yabeana Complex,and spurless traits experienced at least origin twice in this Complex.Because of the limitations of chloroplast sequences in phylogenetic studies,this thesis proposes a genomic SSR(simple sequence repeat,SSR,microsatellite)approach to explore the genetic diversities of 68 populations of Aquilegia yabeana Complex for following three questions:1)How is the genetic diversity andgenetic differentiation of these populations in A.yabeana Complex?2)what dynamic history had the populations of A.yabeana Complex experienced?3)Where and how did the spurless origin?Based on the screening of the whole genome of 4 individuals of A.yabeana Complex,47 SSR primers were designed.After pre-experiment for the efficient and polymorphism of all the primers,20 SSR molecular markers were chosen for further study.Based on this SSR molecular marker platform,68 populations(about 20 individuals per population)collected from the southwest,northwest,southnorth regions of China were systematically analyzed and the following results were gotten:1)Using the newly developed 20 microsatellites(hereinafter referred to as SSR),the genetic diversity and genetic differentiation of 68 populations(1368 individuals)of A.yabeana Complex were analyzed.In this study,380 allele were detected in 68 populations and the mean values of effective alleles(Ne)and Shannon's information index(I)were 6.012 and 1.989,respectively.The mean value of observed heterozygosity(Ho)and expected heterozygosity(He)were 0.332 and 0.805,respectively,and the genetic diversity of A.yabeana Complex was relatively high.Most of the loci did not deviate from the Hardy-Weinberg balance and linkage disequilibrium.The mean values of Fst,Gst and Rst were 0.487,0.470 and 0.518,respectively,implying relatively high genetic differentiation levels in A.yabeana Complex.2)The genetic structure of A.yabeana Complex showed by the results of PCoA analysis and Structure analysis were consistent with NJ cluster based on 20 SSRs'genetic distance:68 populations of A.yabeana Complex formed four groups,namely the Northeast group,Southnorth group,Southwest group and Northwest group.The genetic diversity and gene flow within these four groups are significantly different and the genetic diversity of in the Northeast group is the highest,and the diversity of the Northwest group is the lowest among these 4 groups.3)The average gene flow(Nm)of 20 SSR loci in 68 populations of A.yabeana Complex showed that the mean value of gene flow was 0.284(<1).According to the formula proposed by Ennos(1994),the ratio of pollen flow and seed flow among the population of A.yabeana Complex was calculated to be 4.76,which indicated that the gene flow of A.yabeana Complex was mainly pollen flow.And the order from high to low among these 4 groups is:the Northeast group(Am=0.8464)>Southeast group(Nm=0.3883)>Southwest group(Nm=0.2266)>Northwest group(Nm=0.1352),and all of the gene flow values within these groups are less than 1.The level of gene flow between any two groups showed that the largest gene flow(Nm=3.741)is between the Northeast group and the Southeast group,and the lowest level of gene flow(Nm=1.726)is between the Southwest group and Northwest group.4)Combined our data based on 20 SSRs in this work and previous cpDNA haplotype network and floral traits cluster in our Lab,it is inferred that the spurless trait in Southeast Group may originate from the population of A.kansuensis,which is distributed in Shaanxi Micang Mountain,and then migrate to the northwest and south respectively.Meanwhile,spurless trait in Southwest group originated at least three times,which probablely from the population of A.rockii which located in Bomi and Tibet,the population of A.rockii located in the Songpan of Sichuan,and the population of A.kansuensis located in Kangding of Sichuan or individulas of A.rockii in population located in Jiulong of Sichuan.Based on all of the results above,68 natural populations of A.yabeana Complex had been genetically differentiated and formed four groups genetically.And the genetic diversity,genetic differentiation and gene flow among the four groups were significantly diverged.By combining SSR data,cpDNA data and floral traits,we found the spurless trait in A.yabeana Complex has originated multiple times.We also inferred the direct location and sister populationin A.yabeana Complex for every origination of the spur lost event,which provides a foundation for the further study of the molecular mechanism of spurless in this Complex.