基于SLAF-seq技术的抗杨树叶锈病SNP位点开发

戴美丽; 方乐成; 尹佟明; 李小平

doi:10.3969/j.issn.1000-2006.201806020

戴美丽,方乐成,尹佟明,李小平

南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (02) : 73-78.

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南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (02) : 73-78. DOI: 10.3969/j.issn.1000-2006.201806020

研究论文

基于SLAF-seq技术的抗杨树叶锈病SNP位点开发

戴美丽,方乐成,尹佟明,李小平^*

作者信息 +

Devleopment of SNP molecular markers against the foliar rust of poplar using SLAF-seq technique

DAI Meili, FANG Lecheng, YIN Tongming, LI Xiaoping^*

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文章历史 +

摘要

【目的】探索美洲黑杨抗叶锈病的分子机制,为杨树抗病分子育种提供理论参考。【方法】选取抗锈病差异显著的美洲黑杨‘2-2'与‘2-38'为亲本,通过控制授粉杂交获得80 个F1代个体。用特异性位点扩增(specific-locus amplified fragmenl sequencinq, SLAF-seq)简化基因组技术对所获F1代进行深度测序,以美洲黑杨第3代组装基因组为参照。通过序列比对筛选特异长度的DNA片段,构建SLAF-seq文库,获得特异性SNP位点。经美洲黑杨叶锈病的病原菌经形态学与分子工具鉴定,确定为落叶松-杨栅锈菌(Melampsora larici-populina Kleb.)。通过叶盘法控制条件接种并对被锈菌侵染后的杨树抗性进行评估; 应用Kruskal-Wallis方法对接种结果进行检验。【结果】通过接种,获得了孢子堆数量和大小等表型数据; 基于SNP标记的连锁分析,一共有8 723 个SNP连锁至遗传图谱上; 共识别出19 个连锁群,总遗传距离为3 610.67 cM; 通过Kruskal-Wallis检验可得到37 个紧密连锁位点(P<0.005),标记分别位于I、IV、VI、XI、XV和XIX号连锁群上。【结论】利用SLAF-seq技术开发出了37 个与杨树叶锈病抗病性状紧密关联的SNP分子标记,其中Marker42056位点连锁程度最高。

Abstract

【Objective】Explore the molecular mechanism of resistance to rust in Populus deltoides and provide the theoretical basis for the poplar molecular breeding.【Method】 In this study, eighty F1 progenies were selected for genetic analysis using P. detloides parents of ‘2-2'(female parent)and ‘2-38'(male parent)controlled pollination hybridization which possess substantial differences in resistance to rust. The F₁ progeny were sequenced by SLAF-seq, using the third generation genome of P. deltoides as reference. Therefore, specific DNA fragments were extracted to construct the SLAF-seq library. Meanwhile, a great amount of specific SNP sites were identified. Melampsora larici-populina(Kleb.)that caused foliar rust of poplar were identified by morphylogy and molecular tools.And then the rust infection levels were evaluated under the control condition through leaf disk method. The data were analyzed with Kruskal-Wallis test.【Result】Leaf disks were inoculated with the rust and hold for at least eight days. Phenotypes of uredinial numbers and uredinial size were analyzed with the help of stereoscopy. Totally, 8 723 SNP tags are linked to genetic map and 19 linkage groups with a total genetic distance of 3 610.67 cM were identified through linkage analysis of SNP markers. Thirty-seven closely linked loci(P <0.005)are revealed and locates in linkage groups I, IV, VI, XI, XV and XIX respectively with Kruskal-Wallis test.【Conclusion】Thirty-seven loci were developed by SLAF-seq and found to be tightly linked with the rust resistance in P. deltoides and one of the loci, Marker42056, is the nearest to resistance site genetically.

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戴美丽,方乐成,尹佟明,李小平. 基于SLAF-seq技术的抗杨树叶锈病SNP位点开发[J]. 南京林业大学学报（自然科学版）. 2019, 43(02): 73-78 https://doi.org/10.3969/j.issn.1000-2006.201806020

DAI Meili, FANG Lecheng, YIN Tongming, LI Xiaoping. Devleopment of SNP molecular markers against the foliar rust of poplar using SLAF-seq technique[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2019, 43(02): 73-78 https://doi.org/10.3969/j.issn.1000-2006.201806020

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