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一个存在与缺失变异调控甜瓜果实苦味
李娜, 尚建立, 周丹, 李楠楠, 王吉明, 马双武
0
(中国农业科学院郑州果树研究所)
摘要:
甜瓜(Cucumis melo L.)苦味物质严重影响其口感和品质。本研究拟通过正向遗传学的方法寻找控制甜瓜苦味形 成的分子基础。本文利用不苦的薄皮甜瓜品系 C69 和苦的薄皮甜瓜品系 C14 构建了一个包含 100 个单株的 F2群体。首先利用 2b-RAD 测序构建一个遗传连锁图谱。其次,结合群体的苦味性状进行全基因组的 QTL 定位和关联分析。然后,利用 2b-RAD 测序特有的技术优势进行群体的存在与缺失变异(PAV)的挖掘。最后,利用亲本的重测序信息确定控制苦味性状的关键基 因。F1 的果实表现出强烈的苦味,F2 群体中苦与不苦的单株分别为 81 和 19 个,符合 3:1 的分离比(?2=1.92,P=0.1659)。 利用 477 个 SNP 标记构建一张包含 10 个连锁群的连锁图谱,总长为 337.79 cM,标记间平均间隔 0.71 cM。全基因组 QTL 定 位在 8 号连锁群(对应 9 号染色体)检测到一个解释表型变异为 20%的甜瓜苦味 QTL。全基因组关联分析检测到 7 个 SNPs 与苦味性状相关,全部位于 9 号染色体苦味 QTL 的基因组区域。通过 PAV 分型分析仅发现一个特有的大片段缺失(21,707,702 bp 至 21,743,072 bp),位于 QTL 区域,且在所有的不苦株系中存在,而苦的株系中不存在。基于两个亲本材料的深度重测 序信息,发现这个 PAV 的区域更大,约为 62 kb,共涉及到 9 个连续的基因(MELO3C005601,MELO3C005602,MELO3C005603, MELO3C005604,MELO3C005605,MELO3C005606,MELO3C005607,MELO3C005608 和 MELO3C005609),其中 5 个是 细胞色素 P450 基因。构建的系统发育树表明,这 5 个细胞色素 P450 基因与参与葫芦素 C/B/E 合成的细胞色素 P450 基因簇 CYP81Q58、CYP81Q59 和 CYP712D8 在一个进化枝,可能行使类似的功能。F1 和 F2 群体的表型表明所用甜瓜材料的苦味主 要是由一个显性的基因位点控制。在 QTL 区域发现的特有 PAV 与群体中的苦味性状完全共分离,解释了苦味性状在群体中 100%的表型变异,说明这 9 个基因的缺失是不苦材料 C69 失去苦味的主要原因。PAV 涉及的 9 个连续的基因,其中 5 个是 细胞色素 P450 基因,为潜在的类似于黄瓜葫芦素 C 合成的基因簇的一部分。前人通过比较基因组学研究获得的两个控制葫 芦素 B 合成的 bHLH 转录因子 CmBr(MELO3C005610)和 CmBt(MELO3C005611)同在 9 号染色体,与本研究检测到的 PAV 紧密挨在一起。我们的研究结果为后续不苦甜瓜的育种提供了新的理论支撑和分子辅助育种目标。
关键词:  甜瓜  苦味  存在与缺失变异  2b-RAD  QTL
DOI:10.13430/j.cnki.jpgr.20190625001
投稿时间:2019-06-25修订日期:2019-11-12
基金项目:国家农作物种质资源平台(NICGR2015-016);国家作物种质资源保护和利用专项(2014NWB038);中国农业科学 院科技创新工程专项经费项目(CAAS-ASTIP-2018-ZFRI);国家现代农业产业技术体系建设专项(CARS-25)
A Presence-absence Variation Regulates Fruit Bitterness in Melon (Cucumis melo L.)
LI Na, SHANG Jian-li, ZHOU Dan, LI Nan-nan, WANG Ji-ming, MA Shuang-wu
(Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences)
Abstract:
Bitterness in melon (Cucumis melo L.) seriously affects fruit quality and marketability. The present study aimed to search for the molecular basis of bitterness in melon based on positive genetic strategy. An F2 segregating population was constructed using 100 individuals derived from a cross between the non-bitter female parent C69 and the bitter male parent C14. A genetic linkage map was constructed based on 2b-RAD sequencing, which was then combined with the phenotypic trait of fruit bitterness for QTL mapping and genome-wide association study, and a PAV was detected by the in-depth information of 2b-RAD sequencing. Key genes controlling fruit bitterness were determined by re-sequencing of the two parents. The F1 population showed strong bitterness in fruit, and the F2 population had 81 and 19 individuals with bitter and non-bitter fruit respectively, which approximated the 3:1 segregation ratio (?2=1.92,P=0.1659). A linkage map was constructed based on 477 SNPs distributed in 10 linkage groups. The total length of the linkage map was 337.79 cM, with an average distance of 0.71 cM between adjacent markers. Whole genome QTL mapping identified one QTL for bitterness, which explained 20% of the phenotypic variation on LG08 (corresponding to chromosome 9). Genome-wide association study detected seven SNPs significantly associated with bitterness in melon, which were located on the target QTL region for bitterness on chromosome 9. PAV analysis detected the presence or absence of a long fragment (from 21,707,702 bp to 21,743,072 bp on chromosome 9) in the QTL region, which explained the fruit bitterness or the lack of it in F2 population. The in-depth re-sequencing of the two parents were checked and it was found that the PAV region was larger, of about 62 kb, and was involved in nine consecutive genes (MELO3C005601, MELO3C005602, MELO3C005603, MELO3C005604, MELO3C005605, MELO3C005606, MELO3C005607, MELO3C005608 and MELO3C005609), of which five were cytochrome CYP450 genes. The constructed phylogenetic tree revealed that these five cytochrome P450 genes were in the same clade with the cytochrome P450 gene clusters CYP81Q58, CYP81Q59 and CYP712D8 involved in cucurbitacin C/B/E synthesis, and may function similarly. The phenotypes of F1 and F2 populations indicated that the fruit bitterness was controlled by one dominant gene in melon line C14. The PAV located in the QTL region co-segregated with the bitterness phenotype and explained 100% phenotypic variation in the F2 population, which suggested that the absence of the nine genes was the main cause for the non-bitterness of C69. The five cytochrome CYP450 genes are potentially part of the gene cluster of CuB biosynthesis pathway. Comparative analysis identified two bHLH transcriptional factors tightly linked with PAV in present study, which controlled the formation of CuB, CmBr (MELO3C005610) and CmBt (MELO3C005611) located in the consecutively linked regions in chromosome 9. The present results provide new theoretical support and target of molecular marker assisted breeding of melon.
Key words:  Cucumis melo L.  bitterness  presence-absence variation  2b-RAD  QTL

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