Genetic Differentiation Of The Endophytic Fungus Alternaria Oxytropis In Common Poisonous Oxytropis Spp.

Over the long-term evolution process,Oxytropis spp.may have been subjected to selection pressures or genetic drift.As a result,some Oxytropis spp.have formed symbiosis with Alternaria(sect.Undifilum)oxytropis,an endophytic fungus which produces a mycotoxin swainsonine(SW).Therefore,the host plants can become toxic,which has brought increasing harm to grassland animal husbandry.Previous studies have shown that the content of swainsonine in Oxytropis spp.in China is related to the levels of A.oxytropis,but the content is variable.It has also been poorly understood about what factors have led to such symbiosis between the Oxytropis spp.with A.oxytropis.It is still not clear whether the genetic diversity of A.oxytropis endophytic fungi is different among a variety of Oxytropis spp.,such genetic variation has any impact on the levels of swainsonine.Therefore,clarifying the genetic divergence background A.oxytropis can provide relevant information for the molecular systematic study its evolution.Such work can help in exploring the symbiotic relationship between A.oxytropis and its host,elucidating the occurrence mechanism of toxic Oxytropis spp.,and underpinning the control of toxic Oxytropis spp.to the livestock husbandry in China.In this study,plants of 19 common Oxytropis spp.were collected from five provinces in northwest China.First of all,in view of the lack of specific primers previously for real-time fluorescence quantitative PCR(q PCR)used in the determination of A.oxytropis levels in plants,a new q PCR primerset was designed based on the Swn K gene sequence,a key gene in the biosynthetic pathway of swainsonine.On this basis,the levels of A.oxytropis in the 19 Oxytropis spp.were accurately determined,and correlated with the levels of swainsonine,which provided a basis for the accurate identification of toxic Oxytropis spp.Secondly,102 A.oxytropis strains were successfully isolated and cultured from the leaves of 6 Oxytropis spp.,based on which the genetic diversity was determined by genome resequencing,and t swainsonine levels determined by LC-MS.The main results and conclusions were as follows:1.The levels of A.oxytropis in the 19 common Oxytropis spp.in China was determined,and its correlation to swainsonine levels in was analyzed.(1)Using the new primerset Swn K-q-S and Swn K-q-AS developed,the content of the endophytic fungus A.Oxytropis was carried out by q PCR.It solved the previous problem that A.Oxytropis in O.ochrocephala and O.kansuensis could only be semi-quantified.The new primerset developed have the following advantages:low detection limit,high amplification efficiency of 100.0%,and wider linear application range.(2)Among the 493 plant samples used for A.Oxytropis determination,191 samples have been identified for containing swainsonine.Therefore,Spearman correlation analysis showed that there was a significant positive correlation between the contents of A.oxytropis and swainsonine(r = 0.607,P < 0.001).2.The genetic background of A.oxytropis,isolated from six common species of Oxytropis spp.in northwest China was revealed.A.oxytropis was isolated and cultured from plant leaves,and the 102 strains were sequenced by genome resequencing.The average ratio of all samples to the reference genome was 98.17%,the average coverage depth was 19 X,and the genome coverage was 98.78%.A total of 566,574 high-quality SNP markers were obtained.Based on the high-quality SNP markers,the genetic background of A.oxytropis was analyzed,and the main results were as follows:(1)By correlation analysis of genetic diversity of all fungal samples of their host plant species,the results showed that the number of alleles(Ao)in all samples was 2;average expected number of alleles(Ae)was 1.6161;average Shannon Wiener index(SHI)was 0.5634;average polymorphism information content(PIC)was 0.2930;average MAF was 0.2652.Among the six groups,the genetic diversity of endophytic fungi samples from O.glabra was the highest.O.sericopetala,O.microphylla,O.falcata and O.sericopetala followed,O.ochrocephala had the lowest genetic diversity.This result was also consistent with the statistical result of Ao,that is,the more evenly alleles are distributed in the population,the higher the genetic diversity of the population.(2)Phylogenetic analysis,principal component analysis(PCA)and population structure analysis were used to analyze the genetic relationship of the 102 strains of A.oxytropis.The results showed that the 102 strains could be divided into two clades: the A.oxytropis strains from the host plants of O.falcata,O.sericopetala,O.microphylla and O.glabra were clustered into one large clade,indicating that the four populations were closely related;the endophytic fungi isolated from O.ochrocephala and O.deflexa were clustered into another large clade,indicating these two populations had the same genetic background.PCA showed that endophytic fungi samples collected from host plants O.deflexa and O.ochrocephala,O.glabra and O.falcata-B,O.falcata-C and O.falcata-D,O.microphylla,O.sericopetala and O.falcata-A were closely related to each other.The Admixture software was used to analyze the population structure of the samples.The results showed that the optimal number of the samples was K = 5,which indicated that all the samples could be divided into five different groups by their genetic structure.The majority of the A.oxytropis samples could be completely separated by showing a single background color,whereas some samples may have some gene introgression,consistent with the PCA analysis results.(3)Linkage disequilibrium analysis of the 102 A.oxytropis samples showed that there was no linkage in three populations,the endophytic fungi of O.falcata,O.glabra,and O.ochrocephala,respectively,with an attenuation distance of 0.In addition,the attenuation distance of A.oxytropis from O.sericopetala population was about 100 kb,indicating that there was some linkage compared with the above three populations,but the linkage intensity was not high.After Selective Sweep Analyses and annotation of the samples,we found that most gene functions annotated to the polymorphic loci between the sample groups were focused on metabolic processes,which might lead to variation in the A.oxytropis to produce swainsonine.Subsequent LC-MS results also confirmed that there were certain differences in the content of swainsonine between samples.3.Swainsonine content of the 102 A.oxytropis strains were measured by LC-MS and the difference was analyzed.The results showed that swainsonine levels of the A.oxytropis strains isolated from O.glabra was the highest,and those from O.sericopetala were the lowest.There were significant differences in swainsonine levels between the A.oxytropis samples of O.glabra from the ones from the other five hosts.This difference was also supported by the inference that most of the gene functions annotated to the polymorphic loci between A.oxytropis samples were enriched in the metabolic process.This suggested that the differences in the ability of A.oxytropis to produce swainsonine may be related to genetic differentiation.