Study On Genetic Diversity And Flower Volatile Components In Convallarira

The plant of the genus Convallaria is a perennial herb belonging to the Asparagaceae family.There are three species,Convallaria keiskei Miq.,Convallaria majalis Linn.,Convallaria montana Raf.,which are distributed in temperate regions of Europe,Asia and the Americas.The genus is mainly distributed in the undergrowth,the forest,or in the ditch.It is widely cultivated domestic and foreign because of its important role in horticulture and natural medicine.At present,the research of Convallaria is mainly focused on its chemical composition and horticulture.There is no systematic report on its genetic diversity and phylogentic studies.Resolving the population genetic relationship and phylogenetic history of species within the Convallaria genus is the necessary basis for further germplasm innovation and molecular breeding.In this study,SSR markers,comparative chloroplast genomics analysis,niche modeling(ENM),volatile component analysis and combined with population genetics theory and method were used to study the lineage differentiation of Convallaria genus and population dynamic history.The main research results are as follows:1.Microsatellite markers:16,228 simple sequences repeats were obtained for microsatellite primers developed using transcriptome data of the C.keiskei.Eighty pairs of primers were randomly synthesized for 12 populations of the Convallaria.PCR amplification of the population confirmed that 20 pairs of microsatellite primers had well amplification efficiency and polymorphism.These 20 pairs of microsatellite primers were used for population genetic analysis of 96 individuals in 12 populations of Convallaria.Based on the population genetic diversity of 20 pairs of microsatellite primers,the total genetic diversity Hs ranged from 0.372 to 0.608 with an average of 0.537.The observed heterozygosity HO ranged from 0.349 to 0.738 with an average of 0.650.The range of desired heterozygosity HE is 0.354-0.588 with an average of 0.518.Based on the PCoA principal component analysis of Nei's genetic distance,there are two main clusters of Convallaria genus.The STRUCTURE analysis based on Bayesian algorithm shows that the optimal grouping value K of all Convallaria genus populations is 2,indicating that Convallaria belongs to for the two lineages,which is East Asia lineage and North America lineage.We further detected there are two gene pools within the North American lineage when K=3.The IBD test shows that there is significant geographical isolation effect in the Convallaria group.2.Chloroplast genome:The chloroplast genomes of 5 individuals from 3 species of Convallaria were sequenced,aligned and annotated.Five complete chloroplast sequences with lengths ranging from 162,182 bp to 162,501 bp were obtained.Sequence lengths were ranging from 162,182 bp to 162,501 bp.ycfl is located in SSC/IRb at the boundary and a pseudogene was found in the SSC/IRa area.In the contraction expansion analysis of the boundary region,and the slight contraction amplification in IRa may be caused by gene translocation.Except for the differences in the LSC-IRa region,there were no differences in other locations,and the differences in the complete sequence of the five chloroplast genomes were analyzed,revealing a high degree of similarity(>90%identity)between the sequences.In the genomic sequence,the sequence of the inverted repeat region showed a lower difference than the presence of the LSC and SSC regions,indicating that the Convallaria chloroplast genome is conserved.Eighty-one loci(36 coding genes,32 intergenic regions and 13 intron regions)were screened in the individual sequences.The nucleotide diversity(Pi)values of these regions ranged from 0.00065(ycf2)to 0.08701(atpF-atpH).There are 11 nucleotide diversity Pi>0.02.Seventy-four forwards were detected in five Convallaria chloroplast genomes using REPuter software.There are 177 repetitions in 88 palindromes and 15 inverse repetitions.For all individuals,80.49%of the repeats ranged in size from 30-39 bp.The number of codons ranged from 54,060(C.majalis A69)to 54,165(C.montana A114).The data shows that the codon usage of the protein-coding genes in the five individual chloroplast genomes ranges from 50,973 to 51,385.The 30 codons have an RSCU value greater than one.In phylogenetic studies,C.monata showed a sister relationship to a clade of C keiskei and C majalis.In the chloroplast genome,Convallaria are closely related to Rohdea chinensis.3.Ecological Niche Modeling(ENM):The ENM of Convallaria shows that the current potential distribution area is consistent with the actual distribution area based on MAXENT software calculation(AUC value=0.954).Under the current climatic conditions,the potential distribution area of C.majalis is consistent with the actual distribution area.In Asia,C.keiskei is mainly distributed in China,Japan,and Korea.In China,C.keiskei distributes northeast,north and northwest of China(such as Gansu,Shaanxi and other regions.In North America,C.montana is distributed along the Ohio River and Lake Erie and Lake Ontario.In the last glacial period(LGM),the mapping simulation showed that C.majalis distributed in Europe moved to the eastern inland region.C.majalis in Europe shifts to the eastern inland in the last glacial period.C.montana in North America is distributed in the coastal plains.The distribution of C.keiskei in this period narrowed to the North China and East China regions.With the increase of greenhouse gas concentration and the rising temperature in the future(2050s),the potential distribution of Convallaria will change,and the suitable for growing area will shrink.The suitable area of Convallaria gradually shrinks and migrates to the northern high latitudes and inland areas,and the area of the suitable area is drastically reduced.4.Volatile constituents in C.keiskei:The volatile components were extracted and determinated using headspace solid phase microextraction(SPME)technique and GC-MS.The components obtained by GC-MS were combined with NIST11.L mass spectrometry.The database was compared and the results of NET>80 were selected for qualitative analysis.After preliminary research,a total of 23 compounds of 4 major classes were identified in C keiskei,including 4 alkane compounds,9 alcohol compounds,6 ester compounds and 4 other compounds.The volatile constituents of other plant flowers also indicated that the components were mainly alcohols and esters.