Genetic Diversity Analysis Of Wild Milula Spicata In The Qinghai-tibet Plateau Based On ISSR Markers

As the only species of Milula Prain in Liliaceae,Milula spicata is a rare plant growing in dry alpine pastures and grasslands at an altitude of2,900-4,800 meters in the central Himalayas.It is a traditional characteristic condiment and wild vegetable for farmers and herdsmen in distributed areas,and it is rich in morphology in different communities In order to clarify the genetic diversity of wild Allium tuberosum in Qinghai-Tibet Plateau,this paper selected 165 samples(single plants)from 33 sample plots to study the morphological index and phenotypic diversity of wild Allium tuberosum,the establishment and optimization of ISSR-PCR reaction system of wild Allium tuberosum,and the genetic diversity of wild Allium tuberosum based on ISSR.(1)the phenotypic traits were measured by field investigation and laboratory test,and the phenotypic traits were analyzed by variance analysis and genetic variation analysis with DPSv18.10 software.The results showed that phenotypic traits of Allium spicatum were rich in genetic variation and diversity,and the diversity among populations was better than that within populations,which could be divided into four categories according to phenotypic traits.(2)The optimal ISSR-PCR system of wild Allium spicatum in Qinghai-Tibet Plateau was established by orthogonal design and single factor analysis.In the system of 20μL,2× PCR Master was 9 μ L;Template DNA 3 μ l;1 μ l primer;dd H2 O 7μL。 The procedure of PCR amplification is pre-denaturation at 94℃ for 5 min.Degenerate at 94℃for 20s;Annealing at 46.9-68.4℃ for 60s;Extend at 72℃ for 80s;39cycles,extending at 72℃ for 6 min;Store at 4℃.After obtaining the optimal reaction system,100 primers were screened by using the optimal reaction system and existing amplification,and 10 primers with good polymorphism and clear bands were selected.(3)Genetic diversity was analyzed by ISSR primers.At the species level of Allium spicatum,the observed allele number Na=1.4882;The effective allele number Ne=1.317;The expected heterozygosity h =0.3426;Shannon’s information index I = 0.5132;Polymorphic locus PPL=99.16%.The genetic differentiation coefficient(GST)of 33 populations Gst)=0.4537,indicating that the genetic variation index among populations is 45.37%,and the genetic variation index within populations is 54.63%,indicating that the genetic variation of Allium spicatum in Qinghai-Tibet Plateau is mainly within populations.At the same time,the gene flow coefficient is 0.6021,and the gene flow coefficient is less than 1,which indicates that there is a considerable degree of differentiation among 33 populations of Allium spicatum.Cluster analysis of Allium spicatum population by UPGMA showed that the genetic similarity coefficient of the population was between 0.750.93,and it could be divided into four categories when the genetic similarity coefficient Gx=0.81.The results of this study provide a reference for comprehensive understanding of genetic diversity information of Allium spicatum,innovative utilization of Allium spicatum germplasm resources,and enrichment of spice vegetable germplasm resources in China.