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【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RT-PCR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)和β-胡萝卜素羟化酶(BCH)的基因片段长度分别为348 bp、521 bp、426 bp、429 bp、403 bp和459 bp,其中PSY、ZDS、LBCY、LECY和BCH的基因序列在供试材料中分别有2、12、2、1和1个单核苷酸位点发生变异,多态性频率分别为1SNP/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均外存在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个基因片段的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】 Genetic diversity of red meat pomelo resources in Zhejiang Province was analyzed to provide a theoretical basis for its conservation and rational utilization. 【Method】 The key enzyme genes of six main carotenoid genes were cloned from 11 red meat pomelo resources of Zhejiang province by RT-PCR. The SNPs in the gene fragments were analyzed. The genetic diversity of resources is precisely studied. 【Result】 The results of phytoene synthase (PSY), phytoene dehydrogenase (PDS), zeta-carotene dehydrogenase (ZDS), lycopene The lengths of gene fragments of LBCY, LECY and BCH were 348 bp, 521 bp, 426 bp, 429 bp and 403 bp, respectively 459 bp, in which the sequence of PSY, ZDS, LBCY, LECY and BCH in the tested materials were 2,12,2,1 and 1 single nucleotide site variation, the polymorphic frequencies were 1SNP / 174 bp, 1 SNP / 43.3 bp, 1 SNP / 213 bp, 1 SNP / 403 bp and 1 SNP / 459 bp, respectively. However, the PDS gene sequences were highly conserved among 11 tested materials with no nucleotide variation. Genetic diversity analysis of nucleotide sequences of six gene fragments using Mega software NJ clustering showed that 11 samples clustered into three major groups. Guannan red pomelo, Wencheng red pomelo, Department of red pomelo, Magnolia pomelo closer genetic relationship attributed to the first category; Gujian pomelo, Yongjia red pomelo, Qingtian red pomelo attributed to the second category; red meat pomelo, Four seasons grapefruit and pomelo attributed to the third category. 【Conclusion】 There are SNP variations in the coding region of carotenoid in the flesh of red meat pomelo resources in Zhejiang Province except for PDS. The single nucleotide polymorphism of ZDS is higher, and the frequencies of PSY, LBCY , LECY and BCH 4 gene fragments SNP loci less, indicating that carotenoid biosynthetic genes in the pomelo variety is more conservative, but these variations may be the reason leading to the extent of the flesh-red variable. According to the analysis of the genetic diversity of the nucleotide sequences of 6 gene fragments, Guannan red pomelo, Wencheng pomelo, Pomelo and Mulan pomelo, Gujian pomelo, Yongjia pomelo and Qingtian pomelo, red meat pomelo, Step pomegranate, grapefruit and grape four seasons pomelo, respectively, belong to the same taxa closer to the same group.