一个高抗玉米南方锈病基因的QTL定位及遗传分析

doi:10.3864/j.issn.0578-1752.2019.12.002

摘要/Abstract

摘要：

【目的】通过对一个热带高抗玉米南方锈病材料S313与4个高感玉米南方锈病材料组配的8个F₂群体进行两年三季的遗传分析,在表型鉴定的基础上,利用其中1个F₂群体进行局部遗传图谱构建及抗性基因定位,并利用大群体及新开发的分子标记对抗性主效QTL进行精细定位,为进一步克隆该基因奠定基础。【方法】连续三季对8个F₂分离群体,分3个时期进行病原菌接种,玉米生长后期按1—9级标准等级记载各单株的抗南方锈病级数,进行抗性表型鉴定,最终确定抗、感分离比例。利用56K芯片筛选出2个亲本间多态标记,选择其中均匀分布的192个标记对S313×PHW52的F₂群体中各30个高抗、高感子代进行KASP分型。利用第10染色体短臂上19个SNP标记对整个F₂群体进行分型,构建局部遗传图谱;将遗传图谱与田间抗性表型鉴定结果相结合进行抗性QTL定位。开发初定位区间内10个SNP标记对次级群体进行标记分型,根据交换单株数量大小进一步缩小定位区间。根据玉米B73 Ref Gen_V4参考基因组信息,列出对应定位区间内的所有基因,利用基因的功能注释信息,确定可能与玉米抗南方锈病相关的候选基因。【结果】 8个F₂群体田间抗、感分离比均符合3﹕1的分离比例,说明热带自交系S313对玉米南方锈病的抗性是由1个效应比较大的主效QTL控制。利用56K芯片筛选出2个亲本间的多态标记16 426个。利用192个标记对各30个抗、感子代进行连锁分析,获得了1个抗性连锁标记Affx-90241059。利用19个SNP标记构建了总遗传距离为31.8 cM,标记间平均距离1.77 cM的局部遗传图谱。利用复合区间作图法把该主效QTL定位在标记Affx-91298359与标记Affx-91182449之间,区间大小约2 M。进一步利用F₂大群体及10个SNP标记把该区间缩小到474 K的范围内。玉米参考基因组B73的对应区间内共有63个基因,其中3个基因LOC103640657、LOC100191493、LOC103640673编码的蛋白质与植物抗病性有关,因此把这3个基因列为热带玉米种质S313高抗玉米南方锈病的抗性候选基因。【结论】 S313对玉米南方锈病的抗性是由1个主效QTL控制,并且S313的主效基因定位在第10染色体短臂约0.47 M的范围内。在该范围内有3个玉米南方锈病抗性候选基因。

关键词: 玉米, 南方锈病, 抗性, QTL定位

Abstract:

【Objective】This study aimed at constructing regional genetic map and positioning the resistant gene to Southern Corn Rust by using one of the 8 F₂ populations, deriving from crosses of the highly resistant tropical maize line S313 with 4 highly susceptible inbred lines to Southern Corn Rust, in 3 seasons of two years. In addition, a major QTL for Southern Corn Rust resistance was fine-mapped by applying new-developed molecular markers to a larger mapping population.【Method】Total 8 F₂ populations were planted in 3 consecutive seasons of 2016 and 2017, all of which received triple inoculation. Individual F₂ plants grown in the field were scored for resistance to southern rust using a nine-point relative scale at late growth stage. Finally, the segregation ratio of resistant to susceptible plants for each F₂ population was investigated by phenotypic identification. A 56K-SNP chip was used for screening polymorphisms between the two parents, among which 192 SNP markers were picked for genotyping the 30 high resistant and 30 susceptible plants in an F₂population of S313×PHW52. Nineteen newly developed SNP markers on the short arm of chromosome 10 were used for genotyping the whole F₂ population and constructing a regional genetic map. The resistant QTL mapping was proceeded by the combination of genetic map and phenotypic identification. The genotyping to second population with 10 newly developed SNP markers within the initial mapping region was performed, and the interval was delimited according to the number of exchanged plants. According to the information of maize B73 genome, all genes in corresponding interval were listed. By the gene functional annotation information, the candidate genes were determined.【Result】 By Chi-square test, the ratio of resistant plants to susceptible plants in these 8 F₂ populations was all statistically a 3:1 ratio, which indicated that the resistance to Southern Corn Rust of S313 was controlled by a main effect QTL. A total of 16426 polymorphic markers between the two parents were screened out from the 56K-SNP chip. A resistance-linked marker Affx-90241059 was identified by the linkage analysis of 30 resistant and 30 susceptible plants with 192 markers of uniform distribution across chromosomes. A regional genetic map with total distance of 31.8cM and average distance 1.77 cM was constructed by using 19 SNPs. By the composite interval mapping method, the main effect QTL was located within the interval between marker Affx-91298359 and marker Affx-91182449, which size was 2M approximately. Furtherly, the interval was delimited to the size of 474K by using a larger F₂population and 10 SNP markers. There are 63 genes in the 474k interval of B73 reference genome, among which, gene LOC103640657, LOC100191493 and LOC103640673 encode plant disease resistance-related proteins. Thus, these three genes were listed as candidate genes resistant to Southern Corn Rust of tropical maize inbred line S313. 【Conclusion】 By field phenotypic identification, the ratio of resistant plants to susceptible plants in all F₂ populations was determined as 3:1, which indicated that the resistance to Southern Corn Rust of S313 was under controlled by a main effect QTL. By the methods of population construction and molecular marker, the main effect QTL of S313 was located in the interval of about 0.47M size on the short arm of maize chromosome 10. There are 3 candidate genes related to Southern Corn Rust in this region.

Key words: maize, southern corn rust, disease resistance, QTL mapping

王兵伟, 覃嘉明, 时成俏, 郑加兴, 覃永嫒, 黄安霞. 一个高抗玉米南方锈病基因的QTL定位及遗传分析[J]. 中国农业科学, 2019, 52(12): 2033-2041.

WANG BingWei, QIN JiaMing, SHI ChengQiao, ZHENG JiaXing, QIN YongAi, HUANG AnXia. QTL Mapping and Genetic Analysis of a Gene with High Resistance to Southern Corn Rust[J]. Scientia Agricultura Sinica, 2019, 52(12): 2033-2041.

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年份 Year	群体 Population	总株数 Total plants	抗病株数 Resistant plants	感病株数 Susceptible plants	卡方值 χ² value	χ²测验 Chi-square test	实际抗﹕感值 Actual R﹕S value
2016秋 2016 Autumn	S313×ZD415	356	271	85	0.18	3﹕1	3.19
2016秋 2016 Autumn	ZD415×S313	284	209	75	0.23	3﹕1	2.79
2016秋 2016 Autumn	S313×PHW52	313	232	81	0.09	3﹕1	2.86
2016秋 2016 Autumn	PHW52×S313	260	189	71	0.62	3﹕1	2.66
2016秋 2016 Autumn	S313×368M	277	217	60	1.47	3﹕1	3.62
2016秋 2016 Autumn	368M×S313	300	222	78	0.11	3﹕1	2.85
2016秋 2016 Autumn	S313×TS647	288	214	74	0.04	3﹕1	2.89
2016秋 2016 Autumn	TS647×S313	385	285	100	0.15	3﹕1	2.85
2016秋 2016 Autumn	TS647×齐319	314	103	211	295.95	非3﹕1 Non 3﹕1	0.49
2016秋 2016 Autumn	S313×齐319	266	244	22	38.82	非3﹕1 Non 3﹕1	11.09
2017春 2017 Spring	S313×TS647	747	539	208	3.07	3﹕1	2.59
2017春 2017 Spring	S313×PHW52	275	201	74	0.44	3﹕1	2.72
2017春 2017 Spring	S313×ZD415	631	457	174	2.10	3﹕1	2.63
2017秋 2017 Spring	S313×PHW52	6705	4984	1721	1.56	3﹕1	2.90
2017秋 2017 Spring	S313×TS647	4206	3182	1024	0.92	3﹕1	3.11

F₂病级 F₂ disease grade	家系数 Family number	F_2:3抗病鉴定 F_2:3 resistant identification
1	20	6个家系全表现1—3级抗病,14个家系抗感分离 6 families exhibiting resistant of 1-3 grade,14 families exhibiting separation of resistance and susceptible
3	20	4个家系全表现1—3级抗病,16个家系抗感分离 4 families exhibiting resistant of 1-3 grade,16 families exhibiting separation of resistance and susceptible
5	20	全感5—9级 All exhibiting susceptible of 5-9 grade
7	20	全感7—9级 All exhibiting susceptible of 7-9 grade
9	20	全感7—9级 All exhibiting susceptible of 7-9 grade