中国农业科学 ›› 2018, Vol. 51 ›› Issue (8): 1421-1430.doi: 10.3864/j.issn.0578-1752.2018.08.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

玉米SLAF标记的开发及其在玉米果皮纤维素含量BSA分析中的应用

杜龙岗,王美兴   

  1. 浙江省农业科学院作物与核技术利用研究所,杭州 310021
  • 收稿日期:2017-12-06 出版日期:2018-04-16 发布日期:2018-04-16
  • 通讯作者: 王美兴,E-mail:wangmeixing1975@126.com
  • 作者简介:杜龙岗,E-mail:51968582@qq.com
  • 基金资助:
    浙江省农业(粮食)新品种选育重大科技专项(2016C02050-9-2)

SLAF-marker Development and Its Application in BSA Analysis of Cellulose Content in Pericarp of Maize Kernel

DU LongGang, WANG MeiXing   

  1. Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
  • Received:2017-12-06 Online:2018-04-16 Published:2018-04-16

摘要: 【目的】降低果皮纤维素是甜玉米品质改良的重要目标,然而玉米果皮纤维素含量调控的研究甚少,相关调控基因尚未定位。利用纤维素含量差异的重组自交系(RILs)群体,通过特异位点扩增片段(specific-locus amplified fragment-sequencing,SLAF)测序和分离混池分析(bulked segregant analysis,BSA)定位控制玉米果皮纤维素含量的染色体区段,鉴定调控玉米果皮纤维素含量的候选基因。【方法】以果皮纤维素含量显著差异的E327和G5-1为亲本,构建重组自交系(RILs)。对RILs群体进行果皮纤维素含量的测定,并根据纤维素含量的结果选择纤维素含量高、低的样本进行混池,用于SLAF标签的鉴定和BSA分析。在BSA分析中,首先对两混池和2个亲本DNA用HaeⅢ和Hpy166Ⅱ进行酶切,回收414—464 bp的酶切片段进行Illumina建库,并进行SLAF测序,然后根据多态性SLAF标签开发SNP标记,利用SNP标记对玉米果皮纤维素含量进行关联分析,鉴定调控甜玉米果皮纤维素含量的染色体区段。分析这些区段所包含的玉米基因,并找到它们对应的拟南芥同源基因,通过查阅拟南芥相关基因功能研究的文献,进一步鉴定控制玉米果皮纤维素含量的候选基因。【结果】两亲本和2个混池SLAF建库测序得到的SLAF符合预期,基于SLAF测序数据,鉴定了73 786个多态性SLAF标签,这些SLAF标签均匀分布在玉米的10条染色体上。在这些多态性标签中得到了523 395 SNP位点信息。通过关联分析,调控果皮纤维素变异的基因被定位到玉米基因组的6个染色区段,都位于玉米的第5染色体上。在这些区段上,一共有47个玉米基因。通过进一步的研究分析,在这些关联的染色体区段最终确定了9个候选基因。【结论】定位到调控玉米果皮纤维素的含量的基因,表明此方法可以用于基因定位。

关键词: 玉米, 果皮, 纤维素, SLAF测序, BSA分析

Abstract: 【Objective】 Reducing cellulose content in pericarp is one of the important goals for quality improvement of sweet maize. However, the research focusing on cellulose content in pericarp of maize is limited and the related gene has not been identified. In order to map the chromosome regions and candidate genes controlling cellulose content in pericarp of sweet maize, specific-locus amplified fragment-sequencing (SLAF-seq) and bulked segregant analysis (BSA) were conducted in this study. 【Method】 A recombinant inbred line (RIL) population was constructed using E327 and G5-1 as parents. In F6 generation of RILs, the cellulose contents in pericarp of each line were detected. According to cellulose content results, the lines with relatively high and low cellulose contents in pericarp were selected for SLAF tags identification and BSA. For BSA, DNA was extracted from two bulked populations as well as two parent lines and digested with HaeⅢ and Hpy166Ⅱ restriction enzymes. After digestion, the 414-464 bp DNA fragments were recovered and used for Illumina library construction which were subsequently sequenced by 2nd generation sequencing technology. Based on the polymorphic SLAF tags developed in this analysis, the SNPs-cellulose content association analysis was performed to map the chromosome regions associated with cellulose content in pericarp of sweet maize. Then, the genes located on the associated regions were found. To further reduce the number of candidate genes, the genes in Arabidopsis homological to these genes were identified and annotated. Based on the published works related on the functional analysis of these Arabidopsis genes, we further identified the candidate genes regulating cellulose content in pericarp of sweet maize. 【Result】The sequencing results of Illumina libraries were consistent with expected. As a result, 73 786 polymorphic SLAF tags were identified, which were uniformly distributed on 10 chromosomes. A total of 523 395 SNPs in those polymorphic tags were identified from the SLAF-sequencing data. Genes responsible for cellulose content in pericarp were mapped onto 6 chromosome regions of maize genome via association analysis and all these 6 chromosome regions were located on the 5th chromosome. In total, 47 gene loci in those regions and 9 genes in these associated regions were identified as candidate genes in further analysis. 【Conclusion】Through SLAF-sequencing based bulking segregated analysis, the cellulose content related genes in pericarp of sweet maize were mapped, suggesting that this method can be applied in gene mapping for other traits.

Key words: maize, pericarp, cellulose, specific-locus amplified fragment-sequencing, bulked segregant analysis