| The conservation,identification,and utilization of plant germplasm are closely related to molecular marker development and genetic diversity.The reduced genetic divergence among alfalfa cultivars makes identification based on traditional experience and intuitive phenotyping more difficult.Medicago sativa L.,as the most important and widely cultivated legume forage species,and with high nutritional and economic value,is important for the development and utilization of its germplasm resources to accelerate its breeding process.In the process of alfalfa breeding,the use of molecular markers can greatly improve the accuracy of variety identification and breeding efficiency.Long terminal repeat(LTR)retrotransposons are widely distributed in plant genomes and have been widely used for species variety identification,germplasm evaluation,and so on,based on their excellent polymorphism,stability,and high informativeness for various molecular markers developed.In this study,LTR retrotransposons were developed,characterized,analyzed,and applied based on Medicago truncatula and Medicago sativa genome data,and each of 40 domestic and international M.sativa accessions was evaluated in various dimensions of genetic diversity based on both LTR data combined with Inter-retrotransposon amplified polymorphism(IRAP)molecular markers.The main findings are as follows:1.LTR retrotransposons were identified,analyzed,and screened at the M.truncatula and M.sativa genomic levels,respectively.A total of 431 potential LTR retrotransposons,105 of the Gypsy family,96 of the Copia family,and 230 of the remaining unknown,were identified based on M.truncatula information.These retrotransposons were distributed across chromosomes with a Gypsy and Copia family ratio of 1.09.With the same method,a total of 2,283 potential LTRs identified based on the M.sativa information were screened,including 603 from Gypsy families,326 from Copia families,and 1,354 from the rest unknown,which yielded a Gypsy and Copia family ratio of 1.85.2.A large number of LTR retrotransposon intergenic amplified polymorphic(IRAP)markers were developed from the M.truncatula and M.sativa genome levels design,respectively,and 40 M.sativa accessions were subjected to genetic diversity analysis.A total of 69 pairs of IRAP primers were obtained following their chromosomal position information based on the M.truncatula LTR data.Genetic diversity evaluation and application of 40 Medicago sativa accessions in China and abroad were performed,and 37 primer sets with polymorphic combinations were screened to yield 325 total bands(TB),on average 8.8 per marker,and 268 polymorphic bands(PB).The percentage of polymorphic bands(PPB)varied from50% to 100% with a mean of 79.9% and the polymorphism information content(PIC)ranged from 0.34-0.88 with a mean of 0.69.M.truncatula among the 37 polymorphic primer pairs,the highest correlation was observed between C3-G4 and C6-G4,with a correlation coefficient reaching 0.95,indicating that the band patterns amplified from the two pairs were most similar and that the differences in the band pattern distribution were the smallest;the correlation coefficient between primers C2-C1 and C2-G2,C2-G4 was-0.31,indicating that the differences in band pattern arrangement were the largest between C2-C1 and the two pairs of primers,respectively.Using a threshold of 0.82 in cluster analysis(UPGMA)to classify the 40 accessions into four categories,the grouping results were relatively consistent with the different material geographic distribution information as well as the STRUCTURE analysis.Fifteen upstream and downstream primers were randomly selected based on the M.sativa LTR data,and random combinations yielded a total of 225 IRAP primer pairs.Screening resulted in 18 pairs of polymorphic primer combinations that amplified a total band count(TB)of 221,yielding an average of 12.3 per marker;a percentage of polymorphic bands(PPB)from 42.9% to 100%,with an average value of 79.8%,similar to that of the M.truncatula polymorphism ratio(79.9%);a polymorphism information content(PIC)ranging from 0.67 to 0.91,with an average value of 0.85,higher than the M.truncatula average PIC value(0.69).Among the 18 pairs of primer combinations,the correlation between primer F4-R10 and F4-R9 was the highest,and the similarity coefficient was 0.84,which indicated that the band patterns amplified by the two pairs of primers were the most similar,and the difference was the smallest;while the correlation between primer F4-R3 and F4-R9 was the worst(-0.19),and more varieties could be distinguished after combination.The correlation coefficient between primer F4-R3 and F3-R15 was-0.18,and the correlation coefficient between F4-R3 and F3-R14 was-0.13.Cluster analysis(UPGMA)with 0.79 as the threshold value could divide 40 tested materials into 4groups,and the grouping results were consistent with the geographical distribution information and STRUCTURE analysis of different materials.In this study,we first developed IRAP molecular markers based on LTR retrotransposons at the genomic level of M.truncatula and M.sativa,and evaluated their application in alfalfa germplasm resources.A large number of IRAP molecular markers can provide technical support for identification and protection of alfalfa varieties and genetic background analysis. |
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