Pollination Biology And Application Of Molecular Markers In Lespedeza Bicolor

Lespedeza species are an important ecological and economic-type native shrub species in China.In recent years,it has received special attention on its strong adaptability and wide use.Compared with developed countries,utilization of this genus is still in a low level in China.To carry out genetic improvement for Lespedeza genus,it is necessary to unfold detailed researching in genetic background and phylogenetic relationship between them.In this study,24 provenances in Lespedeza bicolor and 10 species in Lespedeza were collected and regarded as tested materials.Population genetic structure of L.bicolor and relationship of some Lespedeza species were investigated using the methods of pollination biology,molecular markers and DNA sequencing.This study firstly developed the SSR-specific primers of Lespedeza genus by combining ISSR markers and chromosomal walking technique.The results are as follows:1) Pollen viability,stigma receptivity and breeding system for L.bicolor were carried out.The optimum liquid medium for pollen germination of L.bicolor was screened and rapid staining method was found.The changes of pollen viability stored in different conditions suggested that the pollen vitality can last 7 days under 4℃and dry conditions can last 7 days.Results of hybrid index(OCI), pollen-ovule ratio(P/O) and artificial pollination indicated that the breeding system of L.bicolor belonged to outcrossing,part self-compatibility and pollinator-dependence.As an entomophilous plant, the high frequency of visits can significantly increase the seed setting rate of L.bicolor.2) The SRAP reaction system was established and optimized.Senventeen pairs of primer combinations were selected from 484 sets of SRAP primer combinations.Taken 248 individuals as materials,a total of 464 bands were amplified by using 17 pairs of primer combinations,among them 360 bands were polymorphic;the percentage of polymorphic bands was 87.28%.At provenance level, average number of alleles and effective alleles on per locus was 1.881 and 1.568.The Nei's gene diversity index was 0.331,and Shannon's information index was 0.491.Genetic distance between species ranged from 0.1029 to 0.3434,and genetic identity of inter-provenance ranged from 0.709 to 0.902.The coefficient of genetic differentiation was 0.4157,which indicated that 58.4%variabilities occurred in provenance of L.bicolor.The gene flow among provenances(Nm) was 0.7028.According to the UPMGA cluster analysis based on the genetic distance,24 provenances of L.bicolor were divided into five groups.In accordance with cluster figure,10 kinds of Lespedeza were divided into three categories,while L.fomosa showed a higher independence.3) Chromosomal walking and ISSR technologies were used in developing SSR primers.According to sequencing results of ISSR PCR amplification,6 pairs of newly designed primers were successfully applied to chromosomal walking.Of the 20 obtained SSR primers,10 primers showed high polymorphisms.All of them can amplify in other species of Lespedeza.At the same time,new SSR primers were utilized to the research of the relationship between 10 Lespedeza species.UPGMA cluster analyses based on Nei's genetic distance dividedthe 10 species into three main groups.4) Phylogenetic trees of Lespedeza genus were constracted based on sequencing results.Nine ITS sequences and 10 trnL-F sequences were amplified and sequenced.The results of sequence analysis showed that ITS sequence had 61 information sites,accounting for more than 10 percent of the sequencing length.Compared with ITS sequencing results,trnL-F sequence only provided less than 1% information sites.Phylogeny trees of Maximum Parsimony(MP) and Neighbor-Joining(NJ) were constructed based on two fragments respectively.For single fragment,phylogenetic trees constructed by different methods were largely consistent and there was only slightly different in bootstrap values.As a whole,NJ tree reflected more clear than the MP tree in the relationships between subgroups. Comparison analysis phylogenetic tree showed that the tapnology of trees based on two fragments are almost identical,but the cluster location of L.floribunda was obvious differences,which clustered with L.bicolor and other Lespedeza species in phylogenetic tree based on ITS sequence,as contrast,it is independent in trnL-F phylogenetic tree.