Haplotype Variation For Isoflavone Synthase Gene TpIFS And Development And Application Of Quantitative NIR Models For Isoflavone In Trifolium Pratense L.

Red clover is rich in isoflavones, which has lots of health value. The ability to synthesis isoflavones of red clover, however, can be directly affected by the activity of isoflavone synthase. We analysised the genetic diversity and haplotype variation of isoflavone synthase gene Tp IFS from red clover germplasm accessions which are from Europe, Asia, the Americas and Oceania. We also developed the near infrared spectrum models of several red clover isoflavones and total isoflavone. The main results are as follows:1. We identified the ploidy of 108 candidate germplasm accessions by using flow cytometry and chromosome count. The result showed that there were 10 tetraploid materials and 98 diploid materials.2. By sequencing, 414 Tp IFS sequences were obtained, including 20 bp of 5’UTR, 900 bp exon 1,123 bp introns, 675 bp exon 2 and 124 bp of 3 ’UTR. A total of 140 SNPs and 23 indel mutations were found by sequence analysis, and the nucleotide diversity index π = 0.00524. In the coding region,we found 85 SNPs, 14 indel sites. However, 55 SNPs and 9 indel mutations were found in noncoding region. In addition, 29 of the 140 SNPs were heterozygous. Neutral test results showed that, Tp IFS might be under a strong negative selection.3. 414 genetypes were divided into 350 haplotypes, haplotype diversity index is 0.9982. We found five major variation loci, and divided all plants into 36 haplotypes. There are no clear geographical boundaries or material properties within the 36 haplotypes.4. Near infrared spectrum models of several red clover isoflavones and total isoflavone were developed. Using the near infrared(NIR) model of total isoflavone, the isoflavone contents of 286 genetypes were determined.The average isoflavone content of all materials is 9.18 mg / g. There is no significant differences of total isoflavone content either within different origins or types.5. Haplotype and isoflavone content data suggests that Tp IFS has not been selected on purpose. Therefore, it has a high genetic diversity. Thus, the content of isoflavone in red clover can be improved numeriously. There is a number of germplasm with high isoflavone content in the tested populations, according to association analysis of candidate genes, ideal Tp IFS allelic variations can be identified.