Study On Genetic Diversity And DUS Test Characteristics Of Alfalfa Germplasms

Alfalfa（Medicago spp.）is a widely distributed high-quality perennial forage with high yield,good quality,great economic and ecological value.Alfalfa germplasm resources are the basis for alfalfa breeding and improvement.However,the system of collection,preservation,and evaluation of alfalfa germplasm resources is insufficiently developed,which seriously limits the genetic improvement and development of new varieties of alfalfa.The distinctness,uniformity,and stability（DUS）test for new varieties of plants is an internationally used method for germplasm identification and evaluation of new varieties.DNA molecular marker as an auxiliary technology of plant new variety DUS testing provides a new basis for new variety identification.Therefore,it is important to develop DUS test guidelines suitable for the identification and protection of new alfalfa varieties in China,and to establish a new variety identification system for M.sativa and M.×varia Martyn.This study aims to evaluate the alfalfa germplasm resources and provide a basis for alfalfa genetic breeding and new variety approval.By studying the genetic diversity of morphological characteristics,agronomic traits,and molecular markers of 70 alfalfa varieties from multiple geographical origins,the richness and variability of alfalfa germplasm resources were revealed.The test characteristics and standard varieties suitable for DUS test of alfalfa in China were established,which providing technical support for the approval of new alfalfa varieties.The main results were as follows:1.There was abundant genetic variation in morphological characteristics among alfalfa germplasms.Among them the greatest coefficient of variation occurred for the white,yellow,and cream flower frequency characteristics.The Shannon index,Simpson index,and evenness index at the initial flowering stage were the highest.Principal component analysis and cluster analysis showed that the genetic variation among alfalfa germplasms mainly came from the natural height in autumn,winter,and spring,stem length,central leaflet characteristics,frequency of each flower color,and fall dormancy.The clustering results of morphological characteristics were not significantly related to the geographical origin of varieties,and the autumn dormancy level of foreign varieties was generally higher than that of domestic varieties.2.Correlation and PCA analysis of agronomic characteristics of alfalfa germplasm showed that plant height,total number of branches,leaf area index,light interception,and stem-leaf ratio all had a significant impact on alfalfa yield（P<0.05）.The agronomic characteristics with the highest gray correlation degree with annual yield was the total number of branches,followed by the stem-leaf ratio and light interception.Growing year,germplasm and their interaction significantly affected the total yield of alfalfa（P<0.05）.In the two-year experiment in the Hexi Corridor,the total yield of M.sativa‘WL319HQ’was the highest,reaching 15,729 kg ha^-1.According to the membership function analysis,among M.sativa germplasms,‘WL316’,‘WL354HQ’,‘Gannong No.7’,‘’Phabulous’,and‘AF2631’performed well,while among M.×varia Martyn germplasms,‘Tumu No.1’and‘Gongnong No.3’performed well.3.Developed 23 pairs of MTmi R-SSR markers that can stably amplify in M.sativa germplasms,detected a total of 90 alleles,of which 50 showed polymorphism.The expected heterozygosity（He）ranged from 0.15 to 0.83,with an average of 0.61,and the polymorphic information content（PIC）ranged from 0.14 to 0.8,with an average of0.55.By constructing a phylogenetic tree,M.sativa germplasms were divided into 5groups,but the clustering pattern was not significantly associated with geographical distribution.4.Determined 20 test characteristics suitable for DUS testing of new varieties of alfalfa,and screened out 21 example varieties with stable characteristics expression,covering 46 out of 61 expression states,with a coverage rate of 75%.Among them,the M.sativa variety‘Tianshui’appeared in 7 expression states with the highest frequency.This study conducted a systematic analysis of the morphological characteristics,agronomic characteristics and SSR molecular marker genetic diversity of alfalfa germplasms,and determined the test characteristics and example varieties for the DUS test guidelines of alfalfa（M.sativa and M.×varia Martyn）in China.It also established a new variety identification system for M.sativa and M.×varia Martyn,providing a scientific basis for the breeding,promotion,and protection of new alfalfa varieties.