Association Analysis For Hawthorn (C. Pinnatifida Bge.) Leaf Flavones Content

In this study,87 hawthorn (C. pinnatifida Bge.) accessions were come from National Hawthorn Germplasm Nursery in Shenyang Agricultural University. Flavones content including vitexin-rhamnoside, vitexin, rutin, hyperin and quercetin were detected by HPLC. Using SRAP and SSR technology to conduct the genom-wide association analysis and discover the significant associated SRAP and SSR markers. Designing the primer to conduct the Chalcone Synthase genes (CHS) clone and sequencing for 87 hawthorn resources, after that, screened the SNPs (Single Nucleotide Polymorphisms) among the result of sequencing, and then conduct the association analysis between the SNPs and phenotypic traits of hawthorn flavonoid content and discover the significant associated SNPs. The results were as follows:1. The content of 5 flavonoid monomers in the year of 2014 and 2015 were detected by using HLPC system. The peak appearance time and area were defined for the fiavonoes content in quality and quantity. The result showed that vitexin-rhamnoside content was the highest among 5 flavonoid monomers in the yeae of 2014 and 2015 and then followed by hyperin, rutin, vitexin, and quercetin.2. The hawthorn genomic DNA was extracted by using modified CTAB method. 87hawthorn genomic DNA were amplificated by SRAP and SSR, Using STRUCTURE 2.0 to conducted the group structure analysis for 87 hawthorn resources, and they were separated into 4 groups.3. The result of association analysis between SRAP and the phenotypic traits of hawthorn flavones content showed that the marker combination me22eml2 was significantly associated with the vitexin-rhamnoside content (P<0.01), the contribution rate was 8.02%; 7 marker combinations were significantly associated with the vitexin content, me3em8 and me20em7 has the highest contribution rate,33.65%; m22e12 was associated with the phenotypic traits of rutin(P<0.05), the highest contribution rate was7.28%; me20em7 and mel0eml2 were significantly associated with hyperin content, the highest contribution rate was 42.78%; me7em3 was associated with quercetin content and the highest contribution rate was 7.5%. The result of SSR association analysis showed that 7 marker combinations were associated with the vitexin-rhamnoside content and the highest contribution rate was 7.87%which came from CHe01; EB106307 and 7UB40868-288 was significant associated with vitexin content among 14 marker combinations (P<0.01) and the highest contribution rate was 16.46%; 8UB24166-191 was significant associated with the hyperin content among 7 marker combinations and the highest contribution rate was 9.02%.4. The hawthorn RNA were extracted by using modified CTAB method.Full length of hawthorn candidate gene CHS (Chalcone Synthase) ORF (Open Reading Fragment) was 1170bp, and it can be translated into 389 amino acids. Using the software of Clustal to conduct sequence alignment for 87 CHS genes and the sequence polymorphism showed that 71 SNPs were discovered, no Indels were found, the frequency SNPs was 1 SNP per 16.5bp, the rate of synonymous mutation and non-synonymous mutation was 1.45:1.5. Based on the candidate gene association analysis between 71 SNPs and the phenotypic traits of hawthorn flavonoid content, the result showed that in total of 8 SNPs were significantly associated with 5 flavonoid monomers content, especially S12, S14 and S18 were significantly associated with vitexin content both in the year of 2014 and 2015, the result can lay a gand they can be considered as the most important SNPs for the research in future, Thus considerable allelic variation present in CpCHS gene can be exploited for improving hawthorn leaves flavonoid content.