浙江红肉柚类胡萝卜素合成酶基因片段的SNP分析

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

【目的】对浙江红肉柚类资源进行遗传多样性分析,为其搜集保护和合理利用提供理论依据。【方法】采用RTPCR技术从11份浙江红肉柚类资源中克隆6个类胡萝卜素合成主链上的关键酶基因片段,分析所得基因片段中的SNP位点,对浙江红肉柚类资源的遗传多样性进行精确研究。【结果】在11份供试材料中克隆到的八氢番茄红素合成酶(PSY)、八氢番茄红素脱氢酶(PDS)、ζ-胡萝卜素脱氢酶(ZDS)、番茄红素β环化酶(LBCY)、番茄红素ε环化酶(LECY)、β-胡萝卜素羟化酶(BCH)的基因片段长度分别为348、521、426、429、403、459 bp,其中PSY、ZDS、LBCY、LECY、BCH的基因序列在供试材料中分别有2、12、2、1、1个单核苷酸位点发生变异,多态性频率分别为1 SNP/174 bp、1 SNP/43.3 bp、1 SNP/213 bp、1 SNP/403 bp和1 SNP/459 bp,而PDS基因序列在11份供试材料中高度保守,无核苷酸位点变异。利用Mega软件NJ聚类法对6个基因片段的核苷酸序列进行遗传多样性分析显示,11个样品聚为3大类群。‘官南红柚’‘文成红心柚’‘处红柚’‘木兰柚’亲缘关系较近,归于第一类;‘古磉柚’‘永嘉红心柚’‘青田红柚’归于第二类;‘红肉蜜柚’‘麻步文旦’‘四季柚’和‘红心四季柚’归于第三类。【结论】浙江红肉柚类资源果肉中类胡萝卜素合成主链上的关键酶基因在编码区除PDS外均存在SNP变异,其中ZDS的单核苷酸多态性频率较高,PSY、LBCY、LECY和BCH 4个基因片段的SNP变异位点较少,说明类胡萝卜素生物合成基因在柚品种间总体较为保守,但这些变异可能是导致果肉红色深浅程度的变因。根据6个基因片段核苷酸序列的遗传多样性分析,‘官南红柚’、‘文成红心柚’、‘处红柚’和‘木兰柚’,‘古磉柚’‘永嘉红心柚’和‘青田红柚’,‘红肉蜜柚’‘麻步文旦’‘四季柚’和‘红心四季柚’,分别归于亲缘关系较近的同一类群。

【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.