Genetic Evolution Of Typical Domestic Oxytropis Spp. And The Relationship With Endophyte And Swainsonine Content

Oxytropis spp.is a potential germplasm resource to maintain the ecological balance of grassland,which is widely distributed in grassland and the desert areas in western China.Some of these plants were found to be toxic because of the toxic component,an alkaloid--swainsonine（SW）.Consuming those toxic Oxytropis forbs cause livestock poisoning,resulting in serious harm to the grassland environment and the economic development of agriculture and animal husbandry.At present,studies have proved that endophytic fungi（Undifilum oxytropis）symbiotically associated with the plants can produce swainsonine in most of the toxic Oxytropis spp.in China.However,it is not yet clear which biotic or abiotic factors can influence the symbiosis and biosynthesis of swainsonine.Therefore,we determined the genetic differentiation background of 19 Oxytropis spp.,revealed the correlation among the genetic differentiation background,geographical distance,environmental factors,endophytic fungi（Undifilum oxytropis）,and swainsonine.Such results can provide certain theoretical basis for reducing the harm of toxic Oxytropis spp.to animal husbandry.However,there is no systematic study on this aspect.In this study,plants from 19 common Oxytropis species in west China were collected,and genetic diversity analyzed by SLAF-seq.Correlation analysis and GWAS analysis were carried out.The main research contents and results were as follows:1.To reveal the genetic evolutionary background of the natural population of Oxytropis spp.A total number of 196 individuals from 19 species of Oxytropis spp.in west China were sequenced by the SLAF-seq technology.It generated 7,473,950 high quality SNP markers,with the average heterozygosity of the population being of 5.37%.The genetic background of Oxytropis spp.species was then investigated based on the developed SNP,and the main research results are as follows:（1）It showed that the genetic diversity among 19 Oxytropis spp.was different,and the average number of alleles Ao,the number of expected alleles Ae,the observed heterozygosity Ho,the expected heterozygosity He,Nei’s diversity index h,the fragrance index I,and the polymorphism information content PIC were 1.207,1.999,0.038,0.060,0.066,0.092,and 0.048,respectively.Among them,the genetic diversity of O.microphylla（Pall.）DC.and O.stracheyana Benth.was higher,while the genetic diversity of O.merkensis was the lowest.This may be due to that the complex topography and changing climatic conditions of the Tibetan Plateau region of O.microphylla and O.stracheyana,which led to the evolution of higher levels of genetic diversity between groups in order to adapt to more complicated environment.At the same time,the multiploidization phenomenon of O.stracheyana may also be one of the important factors affecting its high genetic diversity.The lower level of genetic diversity of O.merkensis may be related to the least number of samples,the smallest population,and the single source of the sample.Studies have shown that genetic drift plays a more rapid role in small populations,and generally small populations tend to have a lower level of genetic diversity.（2）AMOVA analysis showed that the between-species variation（71.37%）was the main source of genetic variation of Oxytropis spp.,and there was significant genetic differentiation between species(F_st=0.71370>0.25),the group inbreeding coefficient was F_is=-0.19506<1.This indicated that the reproduction of Oxytropis spp.in the natural state is dominated by out-crossing,and factors such as inefficient seed transmission,geographical isolation,and interference of human activities might increase the genetic differentiation between groups.（3）The results of group gene flow analysis showed that:the gene exchange among these,e.g.O.microphylla（Pall.）DC.and O.stracheyana Benth,O.stracheyana Benth and O.falcata Bunge,O.melanocalyx Bunge and O.giraldii ULbr,O.bicolor and O.myriophylla,O.psamocharis and O.ochrantha,O.microphylla（Pall.）DC.and O.sericopetala was frequent,and the groups can become interconnected units to promote the evolution of species.Gene flow Nm<1 among most species,and average gene flow Nm=0.1002<1 among Oxytropis spp.species,indicating a low degree of gene exchange among Oxytropis spp.,and genetic drift may cause genetic differentiation between species.（4）The results of population genetic structure indicated that the analysis of the phylogenetic relationship between the Oxytropis spp.species and individuals and the population genetic structure by phylogenetic analysis,population genetic structure analysis and principal component analysis（PCA）showed that 19 species of Oxytropis spp.were divided into two major clusters with boostrap=93%.The admixture analysis of population structure showed that the best taxonomic group K equals to 15,and similarity of the genetic background between O.microphylla（Pall.）DC.and O.sericopetala,O.glacialis Benth.ex Bunge and O.stracheyana Benth.,O.falcata Bunge and O.stracheyana Benth,O.bicolor and O.myriophylla,O.psamocharis and O.ochrantha,O.melanocalyx Bunge and O.giraldii Ulbr was observed visually,indicating that there may be gene introgression.2.To analyze the relationship between endophyte（U.oxytropis）and swainsonine of Oxytropis spp.（1）We first confirmed the limitations of qPCR for quantitative detection of endophytic fungi.In particular,this method is not applicable to the O.ochrocephala and O.kansuensis Bunge.We speculated that the applicability of this method is closely related to the sample selected by the experiment.The studies were first carried out on the detection of endophyte in 8 species.（2）LC-MS was used to detect swainsonine levels in the 19Oxytropis spp.The results showed that there are significant differences in the Oxytropis spp.species from different collection ranges and geographical conditions.We speculated that this variability may be related to the collective influences from the geographical locations,the climatic environment,and soil microbes of the samples.It is also the first time that we conducted toxicity studies in O.microphylla（Pall.）DC.,O.melanocalyx Bunge,O.ochrantha Turcz.,O.stracheyana Benth.,O.giraldii Ulbr.,O.ramosissima Kom.,O.myriophylla（Pall.）DC.,and O.kansuensis Bunge.In the experiment,we divided the 19Oxytropis spp.species according to previously reported the toxicity limits and chemical types,and classified their toxicity.（3）Variation analysis showed that the content of endophytic fungi was significantly different among species of Oxytropis spp.,and correlation analysis between swainsonine and endophyte showed that there was a significant positive correlation between them（r=0.652,p<0.01）,indicating there was a close relationship between the infection of endophytic fungi and swainsonine.3.To explain the relationship among genetic distance,geographical distance,altitude,environmental factors,endophytic fungi and swainsonine in Oxytropis spp.（1）Mantel and Spearman tests were carried out to analyze the correlation of several factors at the species level.The results showed that the content of swainsonine and endophyte had significant correlation with certain environmental factors,altitude and geographical distance.On the other hand,the geographical environment factors may have certain effects on the endophyte infection and the biosynthesis of swainsonine.There was a significant positive correlation between plant genetic distance and geographical distance,7 environmental factors and altitude,indicating that geographical environment and altitude gradient differences may have a certain impact on genetic variation among Oxytropis spp.（2）The correlation between the genetic distance and the content of swainsonine and endophytic fungi was very low,which may be related to the grouping analysis according to the species and sampling point in the correlation analysis.In addition,GWAS analysis was able to correlate to 38 SNP sites that were significantly related to the content of swainsonine,of which 22 SNP sites were compared with various functional databases for annotation.In the following studies,based on the transcriptome database in O.ochrocephala obtained previously,we will carry out further reasearch on hose 22 markers to help understand the biosynthesis pathway in swainsonine.