小麦成株期抗叶锈病基因<i>Lr12</i>精细定位

doi:10.7668/hbnxb.20194881

摘要/Abstract

摘要：

成株期抗叶锈病基因Lr12在生产上具有良好抗性,为Lr12进行精细定位并开发可靠的分子标记,以感病材料Thatcher和含Lr12抗性基因的近等基因系RL6011为亲本杂交产生F₁,进一步自交产生F₂单株和F_2∶3家系。在田间利用5种强毒力叶锈菌混合小种(PHTT、THKS、THTT、PHTS和PHKS)接种F₂单株和F_2∶3家系进行成株抗叶锈性鉴定和抗性遗传分析。随后利用16K液相芯片对F₂的10个抗病单株和10个感病单株进行基因分型,获得与Lr12紧密连锁的SNP位点,确定抗病基因所在的染色体物理区间,开发SSR分子标记并构建遗传连锁图谱。结果表明,对RL6011(Lr12)/Thatcher的3 494个F₂单株进行抗叶锈性鉴定,抗病单株与感病单株之间的分离比符合3∶1( $χ 3 ∶ 1 2$ =0.14;P=0.71)。对685个F_2∶3家系进行抗叶锈性鉴定,抗病单株、抗病杂合单株与感病单株之间的分离比符合1∶2∶1( $χ 1 ∶ 2 ∶ 1 2$ = 2.01;P=0.37),表明Lr12为显性基因且群体分离符合单基因遗传规律。通过遗传连锁图谱分析成株期抗叶锈病基因Lr12基因位于SSR分子标记YK12817和YK12928之间,遗传区间为0.38 cM,对应中国春参考基因组(IWGSC.Ref.V1.0)中4BL染色体579.44~581.53 Mb物理范围内共2.09 Mb的物理区间。上述结果为预测候选基因提供了确定的依据。

关键词: 小麦, 叶锈病, 基因定位, 连锁标记, Lr12

Abstract:

The adult plant resistance gene Lr12 exhibits excellent resistance in production systems.To fine map and develop reliable molecular markers for Lr12,a cross was made between the susceptible variety Thatcher and the resistant near-isogenic line RL6011 containing the Lr12 gene.The F₁ generation resulting from this cross was self-pollinated to generate F₂ individual plants and F_2∶3 families.Field evaluations were conducted using a mixture of five highly virulent leaf rust pathotypes (PHTT, THKS, THTT, PHTS, and PHKS) to inoculate F₂ individual plants and F_2∶3 families for adult plant resistance assessment and genetic analysis of resistance.Subsequently,genotyping was performed using a 16K liquid chip on 10 resistant and 10 susceptible individuals from the F₂ generation to identify SNP markers closely linked to Lr12.This enabled the determination of the chromosomal physical interval containing the resistance gene,the development of SSR molecular markers,and the construction of a genetic linkage map.The results indicate that the segregation ratio of resistance to leaf rust in 3 494 F₂ individuals derived from the RL6011(Lr12)/Thatcher cross was consistent with a 3∶1 ratio ( $χ 3 ∶ 1 2$ =0.14;P=0.71). In the assessment of 685 F_2∶3 families, the segregation ratio among resistant individuals, resistant heterozygous individuals, and susceptible individuals conformed to a 1∶2∶1 ratio ( $χ 1 ∶ 2 ∶ 1 2$ =2.01;P=0.37), suggesting that Lr12 is a dominant gene and the population segregation follows Mendelian single-gene inheritance patterns. Genetic linkage map analysis localized the adult plant leaf rust resistance gene Lr12 between SSR molecular markers YK12817 and YK12928,within a genetic interval of 0.38 cM.This corresponds to a physical interval of 2.09 Mb within the physical range of 579.44 Mb to 581.53 Mb on chromosome 4BL of the Chinese Spring reference genome(IWGSC.Ref.V1.0).These findings provide a solid basis for predicting candidate genes.

Key words: Wheat, Leaf rust, Gene mapping, Linkage marker, Lr12

刘志杰, 王新海, 高璞, 董瑞, 李帅杰, 张培培, 刘大群, 李在峰. 小麦成株期抗叶锈病基因Lr12精细定位[J]. 华北农学报, 2024, 39(5): 179-185. doi: 10.7668/hbnxb.20194881.

LIU Zhijie, WANG Xinhai, GAO Pu, DONG Rui, LI Shuaijie, ZHANG Peipei, LIU Daqun, LI Zaifeng. Fine Mapping of Wheat Adult-plant Resistance Gene Lr12 to Leaf Rust[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 179-185. doi: 10.7668/hbnxb.20194881.

http://www.hbnxb.net/CN/Y2024/V39/I5/179

图/表 4

表1 小麦叶锈病侵染型鉴定分级标准

Tab.1 The standard for infection types of wheat leaf rust

侵染型 Infection types	症状 Symptoms
0	无可见的侵染点或孢子堆
;	有褪绿斑或坏死,但是无孢子堆
1	有坏死或者有褪绿斑但孢子堆很小
2	有坏死或者有褪绿斑且孢子堆中等偏小
X	不同大小的孢子堆随机分布
3	叶片有或无褪绿斑,孢子堆中等大小
4	无褪绿斑或者坏死,孢子堆大

表2 RL6011(Lr12)/ Thatcher的F₂及F_2∶3群体的抗叶锈性遗传分析

Tab.2 Genetic analysis of leaf rust resistance in the F₂ and F_2∶3 populations of RL6011(Lr12)/Thatcher

材料名称 Materials	总株数 Total plant numbers	抗病株数 Resistant plant numbers	抗感分离 Heterozygous	感病株数 Susceptible plant numbers	卡方测验 Chi-square tests
Thatcher	6			6
RL6011 (Lr12)	6	6
F₂	3 494	2 630		864	$χ 3 ∶ 1 2$ =0.14;P=0.71
F_2∶3	685	169	329	187	$χ 1 ∶ 2 ∶ 1 2$ =2.01;P=0.37

表2 RL6011(Lr12)/ Thatcher的F₂及F_2∶3群体的抗叶锈性遗传分析

Tab.2 Genetic analysis of leaf rust resistance in the F₂ and F_2∶3 populations of RL6011(Lr12)/Thatcher

材料名称 Materials	总株数 Total plant numbers	抗病株数 Resistant plant numbers	抗感分离 Heterozygous	感病株数 Susceptible plant numbers	卡方测验 Chi-square tests
Thatcher	6			6
RL6011 (Lr12)	6	6
F₂	3 494	2 630		864	$χ 3 ∶ 1 2$ =0.14;P=0.71
F_2∶3	685	169	329	187	$χ 1 ∶ 2 ∶ 1 2$ =2.01;P=0.37

图1 抗叶锈基因Lr12与SSR标记在4BL染色体上的遗传连锁图谱 A.13个标记(紫色标记为新设计标记)检测RL6011(Lr12)/Thatcher的685个 F_2:3家系构建的遗传连锁图谱;B.6个紧密连锁标记检测RL6011(Lr12)/Thatcher的3 494个F₂单株的精细遗传连锁图谱;C.部分重组系中的标记检测带型:A.抗病亲本带型;B.感病亲本带型;H.杂合带型;Res.抗病表型;Sus.感病表型;Het.抗感分离。

Fig.1 Genetic linkage map of leaf rust resistance gene Lr12 and SSR markers on the 4BL chromosome A.The genetic linkage map of Lr12 and 13 SSR markers(The purple color is marked as the indicator for the new design)based on 685 F_2:3 lines from RL6011(Lr12)/Thatcher;B.The fine genetic linkage map of Lr12 and 6 SSR markers based on 3 494 F₂ plants from RL6011(Lr12)/Thatcher;C.The molecular marker bands in selected recombinant lines:A.The resistant parental genotype;B.The susceptible parental genotype;H.The heterozygous genotype;Res.Resistant;Sus.Susceptible;Het.Heterozygous.

表3 多态性SSR标记的引物序列

Tab.3 Primer sequences for the amplification of polymorphic SSR markers

标记名称 Marker IDs	正向引物序列(5'-3') Forward primer sequence(5'-3')	反向引物序列(5'-3') Reverse primer sequence(5'-3')
Xgwm495	GAGAGCCTCGCGAAATATAGG	TGCTTCTGGTGTTCCTTCG
Xwmc657	CGGGCTGCGGGGGTAT	CGGTTGGGTCATTTGTCTCA
SSR802	CTTGGCAAGGTCTCGGATGT	TGCCCATCCTCATTCCACAC
SSR921	CAGCATGCGTTGTGTGGAAA	GCAAAAGACACGGGGTTGTC
Xgwm149	CATTGTTTTCTGCCTCTAGCC	CTAGCATCGAACCTGAACAAG
ZBSF4B332	GCTTCTGCGTTGATGGTGTT	ATCACGTTGCCAAGAAAGCC
Xgwm251	CAACTGGTTGCTACACAAGCA	GGGATGTCTGTTCCATCTTAG
YK12817	ACCAGTTCTTCCTCTTCCTC	CTGACTGCGTTGTTAGCTC
YK12928	TATGTGACATGCCTCAACTG	CATGCACGGAAGTTGTTAC
YK14228	GGCTAGAGAGCCGATAAGAG	CTTAGTGCCCTCACCTCTC
ZBSF4B68	ATAATTTGGTCCCATAGGATG	TGGGTGCACAAATAATGTAGT
YK15718	TGCCAGATGTTCCTGACATT	TGTTGCTTCCTTCGCTTCTT
ZBSF4B138	TTAGATTTGGCACTACCATGA	GCAATAAGCTGCATTTAAGAC

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小麦成株期抗叶锈病基因Lr12精细定位

Fine Mapping of Wheat Adult-plant Resistance Gene Lr12 to Leaf Rust

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Fine Mapping of Wheat Adult-plant Resistance Gene Lr12 to Leaf Rust

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