SNP analysis of carotenoid biosynthesis related genes of red pummelo in Zhejiang province

Abstract:【Objective】Pummelo cultivation occupies an increasing important position in the citrus industry, which has formed as a characteristic advantageous industry in China. However, only a few excellent cultivars such as‘Guanximiyou'‘Shatianyou'and‘Yuhuanyou'are widely grown although we possess abundant pummelo germplasm resources in our country, moreover, some local pummelo resources are disappearing with ongoing rural development. One of the main distribution areas of the pummelo genetic diversity is in Zhejiang province, which has rich pummelo resources especially red flesh germplasms with different red degrees, such as reddish‘Sijiyou', pale red‘Mulanyou'and bright red‘Gusangyou'. However,the non-domesticated red flesh resources were rarely used in production or in new non-variety selection and were evendisappearing. Therefore, for their conservation and utilization, we investigated and collected the local red flesh pummelo resources in Zhejiang province and analyzed their genetic diversity.【Methods】Carotenoids are the main pigments in red citrus fruits. It shows a multi-enzyme catalyzed reaction in the cartotenoids biosynthesis pathway, and the phytoene synthase(PSY), phytoene desaturase(PDS), ζ-caro-droxylase(BCH) are six key enzymes which have been cloned in citrus or other plants. The primers for thesix carotenoid biosynthesis related genes were designed according to the reported genes in the Genebank,and the partial sequences of PSY, PDS, ZDS, LBCY, LECY and BCH were cloned from eleven local redpummelo cultivars in Zhejiang province by using RT-PCR, and then the SNPs in the obtained sequenceswere analyzed using the Seqman 7.1.0 and Clustal X 1.83 software. The genetic diversity of the six genepartials was analyzed using the neighbor-joining method in MEGA 4.0 software to accurately study the ge-netic diversity of the red pummelo germplasms in Zhejiang province.【Results】The 11 tested red fleshpummelo germplasms, 10 from Zhejiang province and one ‘(Hongrou miyou') from Fujian province,showed different red degree colors, accordingly they were divided into three classes. The first class includ-ed the reddish‘Mulanyou'‘Sijiyou'and‘Mabu wendan'. The pale red‘Qingtian hongxinyou'‘Yongjiahongxinyou'‘Wencheng hongxinyou'and‘Hongxin sijiyou'were more redder than the first class, and thethird class included‘Hongrou miyou'‘Gusangyou'‘Guannan hongyou'and‘Chuhongyou', whose fleshwere a bright red color. It had been reported that the color differences of the pummelo flesh could be as-cribed to the different types and contents of the carotenes. Lycopene and β-carotene were the two mainpigments in red pummelo, therefore, we cloned six key enzymes related to their accumulation in the carot-enoid biosynthesis pathway and detected their sequence variations in 11 red pummelo germplasms to ana-lyze their genetic diversity. The clustering based on the sequence variations of the key enzymes in the ca-rotenoid biosynthesis pathway may reflect the coloring differences. The length of PSY, PDS, ZDS, LBCY,LECY and BCH was respectively 348 bp, 521 bp, 426 bp, 429 bp, 403 bp and 459 bp; the number of SNPin PSY, ZDS, LBCY, LECYand BCH was respectively 2, 12, 2, 1 and 1; the polymorphism frequency was1SNP/174 bp, 1SNP/35.5 bp, 1SNP/214.5 bp, 1SNP/403 bp and 1SNP/459 bp while PDS was highly con-served and no SNP was detected. The eleven red pummelo cultivars were clustered into three groups.‘Guannan hongyou'‘Wencheng hongxinyou'‘Chuhongyou'and‘Mulanyou'were clustered in the firstgroup;‘Gusangyou'‘Yongjia hongxinyou'and‘Qingtian hongyou'were clustered in the second group;and the rest‘Hongrou miyou'‘Mabu wendan'‘Sijiyou'and‘Hongxin sijiyou'were clustered in thethird group.【Conclusion】The key enzymes in the carotenoid biosynthetic pathway showed SNP variationsin the coding region among the eleven red pummelo germplasm resources in Zhejiang province, and there-in the polymorphism frequency of ZDS was highest while the variation locuses of the other 5 genes werelower, which indicated the genes in the carotenoid biosynthetic pathway among pummelo cultivars wererelatively conserved, whereas the variations in the gene coding regions would result in different degrees ofred color. Based on the genetic diversity of the six genes partials,‘Guannan hongyou'‘Wencheng hongx-inyou'‘Chuhongyou 'and‘Mulanyou'were clustered into the first group;‘Gusangyou'‘ Yongjia hongx-inyou'‘ Qingtian hongyou'were clustered into the second group; and the rest‘Hongrou miyou'‘Mabuwendan'‘ Sijiyou'and‘Hongxin sijiyou'were clustered into the third group.