陆地棉L-D1等位基因特异性分子标记的开发及应用

doi:10.11963/cs20210013

摘要/Abstract

摘要：

【目的】L-D₁基因调控陆地棉叶形。本研究设计特异分子标记精准鉴定L-D₁等位基因,为其在陆地棉冠层结构改良中的应用提供支撑。【方法】利用具有鲁棉研28号遗传背景的L-D₁等位基因近等基因系,分析不同等位基因组合对叶形的影响。根据4个等位基因的启动子和编码区的多态性设计特异分子标记,并对不同叶形中的等位基因进行检测。【结果】L-D₁基因从3叶期开始调控叶片叶裂的形成,4~8叶期叶裂不断加深,从9叶期开始基本稳定;L-D₁等位基因不同组合可形成相似叶形,仅从形态上难以准确辨别。克隆获得L-D₁位点4个等位基因起始密码子前约4 kb的启动子片段,发现24个SNPs（Single nucleotide polymorphism,单核苷酸多态性）、1个133 bp及1个14 bp的插入缺失。根据等位基因启动子及编码区的SNP和缺失插入开发了1个Indel分子标记InDel_8和2个衍生酶切扩增多态性序列标记dCAPS_192、dCAPS_519,分别为l₂、L₂^o、L₂^s的特异分子标记。【结论】根据陆地棉中控制叶形的L-D₁等位基因间的差异开发了3个特异性分子标记,可用来鉴定不同的L-D₁等位基因。

关键词: 冠层改良; 叶形; L-D₁位点; 特异分子标记; 陆地棉

Abstract:

[Objective] L-D₁ regulates leaf shape in upland cotton. This study develops specific molecular markers for L-D₁ alleles and provides a basis for the application of L-D₁ alleles in improving canopy structure. [Method] Morphological traits were investigated to analyze the effect of different combinations of L-D₁ alleles with the genetic background of Lumianyan 28 (LMY28) on leaf shape. Specific molecular markers based on polymorphism of alleles' promoters and CDS (Coding sequence) were used to check the combinations of L-D₁ alleles in different materials. [Result] L-D₁ alleles regulated the formation of leaf lobes from the 3-leaf stage. The lobes deepened from the 4-leaf stage to 8-leaf stage and stabilized from the 9-leaf stage. It was difficult to distinguish the combination of L-D₁ alleles from similar leaf shapes. A total of 24 SNPs (Single nucleotide polymorphism) and a 14 bp and a 133 bp insertion-deletion(InDel) were identified in the promoter of 4 alleles. Based on the SNPs and InDels in the promoter and coding sequences, three specific molecular markers, InDel_8, dCAPS_192, and dCAPS_519, were developed for l₂, L₂^o, and L₂^s, respectively. [Conclusion] Specific molecular markers were developed and could be used to identify L-D₁ alleles, which controlled leaf shape of cotton.

Key words: canopy improvement; leaf shape; L-D₁ locus; specific molecular marker; upland cotton

姜辉,郑锦秀,王永翠,张超,王秀丽,陈莹,高明伟,王家宝,柴启超,赵军胜. 陆地棉L-D₁等位基因特异性分子标记的开发及应用[J]. 棉花学报, 2021, 33(5): 412-421.

Jiang Hui,Zheng Jinxiu,Wang Yongcui,Zhang Chao,Wang Xiuli,Chen Ying,Gao Mingwei,Wang Jiabao,Chai Qichao,Zhao Junsheng. Development and application of specific molecular markers for L-D₁ alleles in upland cotton[J]. Cotton Science, 2021, 33(5): 412-421.

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多态性类型 Type of polymorphism	位置 Location/bp	4个等位基因的基因型 Genotype of four alleles
多态性类型 Type of polymorphism	位置 Location/bp	L₂^s	L₂^o	L₂^u	l₂
缺失 Deletion	-877~-745	-	-	+	+
	-1 412~-1 399	+	+	+	-
SNP	-166	T	T	T	C
	-192	T	G	T	T
	-250	A	A	A	G
	-436	C	C	C	A
	-519	A	G	G	G
	-689	T	T	T	G
	-968	A	A	A	T
	-991	A	A	A	G
	-1 468	T	T	T	C
	-1 785	G	G	G	T
	-1 977	A	A	A	G
	-2 485	C	G	C	C
	-2 610	C	C	C	T
	-2 638	A	A	A	G
	-2 661	A	A	A	C
	-2 736	A	A	A	G
	-2 740	T	T	T	C
	-2 819	A	A	A	T
	-2 917	A	A	A	G
	-3 018	A	A	A	C
	-3 284	T	T	T	A
	-3 330	A	A	A	C
	-3 527	T	T	T	C
	-3 814	A	A	A	G

引物名称 Primer name	引物序列 Primer sequences	扩增片段大小 Size of PCR product/bp	备注 Note
InDel_8_F2 InDel_8_R2	ACTCTCATACCTTTTACGCAGGT CCGCCGGAGATTTCCTTGTA	l₂：93;L₂^u,L₂^o,L₂^s：101	l₂特异分子标记 Specific marker for l₂
dCAPS _192_F3 dCAPS _192_R3	GTCCACCCTCTCTCCATCTCAATT CAATGGGGAGCAGACCAGAA	L₂^u,L₂^s,l₂：121;L₂^o：101	L₂^o特异分子标记 Specific marker for L₂^o
dCAPS _519_F4 dCAPS _519_R4	GATGTAGTGAATGAGTATTTATCCAT TGTACATTTACAATCCCTCATCA	L₂^u,l₂,L₂^o：183; L₂^s：210	L₂^s特异分子标记 Specific marker for L₂^s

陆地棉L-D₁等位基因特异性分子标记的开发及应用

Development and application of specific molecular markers for L-D₁ alleles in upland cotton

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