Genetic evaluation and germplasm identification analysis on ITS2, trnL-F, and psbA-trnH of alfalfa varieties germplasm resources

In this study, genetic diversity and germplasm identification of 28 alfalfa germplasm cultivars materials were evaluated by analyzing their internal transcribed spacer 2 (ITS2), L-F, and A- H sequences to provide the innovative reference of alfalfa varieties genetic diversity and identify research. The results showed that the fragment average length of ITS2, L-F, and A- H sorting sequences were 455.7 bp, 230.3 bp, and 345.6 bp, respectively. The ITS2 sequence was too conservative to reflect the individual differences between intercultivars and intracultivars in the preliminary experiment. Furthermore, L-F and A- H sequence differences were relatively small between intercultivars but significant between intracultivars. Alfalfa cultivars were divided into four groups by sequence similarity clustering. Alfalfa cultivars L-F and A- H sequences have apparent differences, showing that chloroplast conservative sequences were independent evolution. Compared with L-F and A- H sequences of alfalfa cultivars, A- H sequence has abundant variation sites and can better reflect the differences between cultivars than the L-F sequence. Therefore, the A- H sequence can identify different alfalfa cultivars and establish the DNA sequence fingerprint.