玉米籽粒淀粉粒密度基因tw1的精细定位

doi:10.3864/j.issn.0578-1752.2018.07.002

摘要/Abstract

摘要： 【目的】淀粉粒密度影响籽粒容重，通过对一个玉米籽粒淀粉粒密度突变体Mrd进行鉴定和精细定位，为容重相关基因的克隆和功能验证奠定基础。【方法】以育种选系过程中发现的一个淀粉粒密度突变体Mrd为材料，利用近红外光谱分析仪检测其籽粒内部化学成分的变化，用扫描电镜观察授粉后18—45 d正常籽粒和突变籽粒中淀粉粒形态的差异；于2014—2016年分别在河南郑州和原阳以及海南三亚种植Mrd与B73的杂交组合及F₂和BC₁分离群体，并对其进行遗传分析；使用来自maizeGDB（http://www.maizegdb.org）的覆盖全基因组的1 000对SSR引物，通过集团分离分析法（bulked segregation analysis，BSA）筛选与目的基因紧密连锁的标记，实现目的基因的初步定位；并在该定位区间内开发新的标记，对从38 000 BC₁分离群体中筛选出的交换单株进行基因型分析，实现目的基因的精细定位；通过候选基因序列分析、功能预测和等位性测验确定首选候选基因。【结果】该突变体籽粒较正常籽粒体积变小，比重增加；细胞学和化学组份分析结果表明，与野生型籽粒相比，突变体籽粒中的粗蛋白含量降低，粗淀粉含量没有显著变化，淀粉粒形状不规则且变小、密度增加，可能是导致籽粒容重变大的原因；对授粉后不同天数籽粒内部淀粉粒结构的观察显示，突变体籽粒淀粉粒的密度比正常籽粒密度大，并随发育进程不断增加；对Mrd与B73的F₂及测交后代分离群体的遗传分析结果表明，Mrd籽粒突变是由单隐性基因（命名为tw1）控制的；该基因首先被定位在第6染色体的SSR标记umc1105和bnlg1154之间，物理距离为22 Mb；利用上述2个标记对BC₁群体进行交换单株筛选，并开发标记，将该基因定位于SSR标记B3和A47之间，物理距离为0.2 Mb；在该候选区段内有包含su2在内的3个候选基因，等位性测验结果表明，tw1与su2不是等位基因；候选基因序列分析和功能预测结果表明GRMZM2G042607编码的蛋白具有碳水化合物识别结构域，在种子中对碳水化合物的储藏起沉积作用，是tw1最可能的候选基因。【结论】实现了籽粒淀粉粒密度突变性状基因tw1的精细定位，并确定了候选基因为编码一种β-1,3半乳糖基转移酶的GRMZM2G042607。

关键词: 玉米, 容重, 淀粉粒密度, tw1, 精细定位

Abstract: 【Objective】Starch grain density affects maize kernel test weight. In this study, we use a mutant Mrd of maize to identify and fine map the gene controlling starch grain density, which are helpful for the cloning and function verification of the related grain test weight gene.【Method】The Mrd is a starch grain density mutant which was identified during maize breeding practices. Scanning electron microscopy and near infrared spectroscopy (NIR) analyzer were used to observe changes of chemical composition in the Mrd kernels. The segregation population F₂ and BC₁ were derived from the cross of Mrd and B73, which were planted in Zhengzhou and Yuanyang, Henan Province and Sanya, Hainan Province, and used for genetic analysis. BSA (Bulked Segregation Analysis) was used to identify linkage markers selected from 1 000 pairs of SSR primers from maizeGDB (http://www.maizegdb.org) of target gene. The BC₁ segregation population of 38 thousand individuals were used to fine map the target gene tw1. The candidate genes were sequenced and functional predicted by bioinformatics. Allelism test was performed for tw1 and su2. 【Result】Compared with the normal seed, the Mrd had smaller grain size and no change in grain length and increased specific gravity. The crude protein content in mutants decreased, the content of crude starch did not change significantly, but the Mrd has irregular shape and smaller starch grain, increased density, and increased grain test weight. Observations of the starch grain structure inside the kernel in different days after pollination showed that the density of the mutant kernel starch kernel increased with the progress of development and was always higher than that of the normal kernel. The genetic analysis of F₂and BC₁ population showed that grain test weight mutation was controlled by a single recessive gene tw1 which was firstly located on chromosome 6 between SSR marker umc1105 and bnlg1154. After screening and analysis the recombinant from the BC₁ segregation population, the gene was located between B3 and A47. There are three protein-coding genes in the 0.2 Mb candidate physical interval. Allelism test excluded su2 gene and sequence analysis of the other two candidate genes verified that GRMZM2G042607 should be the primary candidate gene of tw1, which encodes protein with a carbohydrate recognition domain and deposits deposition of carbohydrates in the seed. 【Conclusion】The tw1 gene is fine mapped and find the candidate gene, GRMZM2G042607, encodes beta-1,3 galactosyltransferase.

Key words: maize, test weight, starch grain density, tw1, fine mapping

孙粲然，张雪海，马指挥，郭战勇，汤继华，付志远. 玉米籽粒淀粉粒密度基因tw1的精细定位[J]. 中国农业科学, 2018, 51(7): 1233-1243.

SUN CanRan, ZHANG XueHai, MA ZhiHui, GUO ZhanYong, TANG JiHua, FU ZhiYuan. Fine Mapping of Grain Test Weight Gene tw1 in Maize[J]. Scientia Agricultura Sinica, 2018, 51(7): 1233-1243.

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