基于极端混合池（BSA）全基因组重测序的甘蓝型油菜有限花序基因定位

doi:10.3864/j.issn.0578-1752.2018.16.001

摘要/Abstract

摘要： 【目的】适合机械化收获是当今油菜育种改良和遗传研究的重要目标。该研究以一个自然变异产生的油菜有限花序（denterminate inflorescence 1，di1）突变体为研究对象，通过分析有限花序的遗传模式，开展有限花序性状的基因定位和克隆，以期发掘候选基因，为培育适合机械化收获的油菜新品种提供新思路和新材料，为揭示油菜有限花序遗传机制奠定基础。【方法】以一个稳定遗传的有限花序突变株系FM8与野生型自交系FM7开展正反交，观察F1和F2后代的花序形态，分析有限花序性状的遗传模式。在F2群体中挑选20个有限花序单株和20个野生类型单株构建混合池，对混合池和亲本开展20×和10×覆盖度的全基因组重测序，定位有限花序性状的关联区间。根据关联区间对应到拟南芥基因组的共线性区段和基因注释信息，预测候选基因，并对候选基因进行同源克隆，发掘序列变异，筛选关键基因。【结果】油菜有限花序突变性状表现为初花期主花序和侧枝花序顶部形成一个或若干个顶生花，花序无限生长受阻，导致结角期主枝和侧枝有封顶特征即有限花序。有限花序突变株系与野生型正反交F1均表现为野生型，F2代无限花序与有限花序的分离比符合13﹕3，说明有限花序的遗传受2对隐性基因和1对隐性上位抑制基因互作控制。对混合池及亲本开展全基因组重测序，得到30 123个单核苷酸多态性（SNPs）标记和107 636个插入缺失标记（InDels）标记，用于有限花序性状的全基因组定位。定位结果共检测获得7个显著关联区间，分布于油菜A08、A09、A10、C08和C09共5条染色体。其中，A10染色体上的关联区间峰值最高，是控制有限花序性状的主效位点。并且，A10染色体关联区间内的14.36—15.07 Mb的区域与C09染色体2个关联区间显示高度同源性。候选基因预测发现位于A08、A09、A10、C08和C09的5个关联区间包含有8个候选基因，包括TERMINAL FLOWER 1（TFL1）、FLOWERING LOCUS C（FLC）、ATBZIP14（FD）、MULTICOPY SUPPRESSOR OF IRA1 4（FVE）和SCHLAFMUTZE（SMZ）。基因序列分析表明di1突变体TFL1、FVE和SMZ的基因编码区存在序列变异，并导致蛋白序列变异。【结论】油菜有限花序突变由2对隐性基因和1对隐性上位抑制基因互作控制。与有限花序性状显著关联的区间有7个，其中，位于染色体A10和C09的关联区间具有高度同源性。TFL1、FVE和SMZ被推断为有限花序性状的候选基因。

关键词: 甘蓝型油菜, 有限花序, 基因定位, 关联区间, 候选基因

Abstract: 【Objective】Mechanical harvesting has been one of the major goals of rapeseed breeding and genetic research worldwide. A natural and novel rapeseed mutant with determinate inflorescence (di1) was identified in this study. Genetic analysis, gene mapping, candidate gene prediction and gene cloning were used to elucidate the genetic control of determinate inflorescence.【Method】For genetic analysis, reciprocal crosses were performed between determinate inflorescence line FM8 and wild type FM7, and the inflorescence morphology was observed for F₁ and F₂ progenies. Two pools with 20 di1 F₂ lines and 20 wild type lines were constructed. For gene mapping of determinate inflorescence, 20× and 10× depth of whole genome re-sequencing were conducted for the two pools and parental lines, respectively. The associated loci were aligned to the genome of A. thaliana for synteny blocks searching. Potential candidate genes for determinate inflorescence were predicted by annotation analyses of genes within the physical boundaries of the associated regions. Gene cloning was used to identify polymorphisms and screen candidate gene(s). 【Result】The di1 mutant showed a single fruiting body or a cluster of fruiting bodies at the top of the inflorescence axis, and the growth of inflorescence was hampered. The F₁progenies from the reciprocal crosses were indeterminate, and the trait segregation of indeterminate inflorescence and determinate inflorescence among F₂ progenies fit the 13:3 segregation ratio, assuming that the determinate inflorescence was controlled by two pairs of recessive duplicate genes interacting with one pair of recessive epistatic inhibitor genes. Whole genome re-sequencing of two pools and two parental lines identified 30123 homozygous SNPs and 107636 homozygous InDels. Seven significantly associated loci were mapped on chromosomes of A08, A09, A10, C08 and C09. Of which, the locus on chromosome A10 not only exhibited the highest peak, but also showed homologous with the two loci on chromosome C09 by synteny analysis. Genes of TERMINAL FLOWER 1 (TFL1), FLOWERING LOCUS C (FLC), ATBZIP14 (FD), MULTICOPY SUPPRESSOR OF IRA1 4 (FVE) and SCHLAFMUTZE (SMZ) were predicted as potential candidate genes. Gene cloning identified coding region polymorphisms and protein polymorphisms for genes of TFL1, FVE and SMZ.【Conclusion】The determinate inflorescence of di1 mutant was controlled by two pairs of recessive duplicate genes interacting with one pair of recessive epistatic inhibitor genes. Seven loci were significantly associated with determinate inflorescence. Of which, the loci on chromosomes A10 and C09 were homologous. TFL1, FVE and SMZ showed coding region polymorphisms and protein polymorphisms, and were deduced to be candidate genes for determinate inflorescence.

Key words: Brassica napus, determinate inflorescence, gene mapping, associated regions, candidate genes

张尧锋，张冬青，余华胜，林宝刚，华水金，丁厚栋，傅鹰. 基于极端混合池（BSA）全基因组重测序的甘蓝型油菜有限花序基因定位[J]. 中国农业科学, 2018, 51(16): 3029-3039.

ZHANG YaoFeng, ZHANG DongQing, YU HuaSheng, LIN BaoGang, HUA ShuiJin, DING HouDong, FU Ying. Location and Mapping of the Determinate Growth Habit of Brassica napus by Bulked Segregant Analysis (BSA) Using Whole Genome Re-Sequencing[J]. Scientia Agricultura Sinica, 2018, 51(16): 3029-3039.

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