基于SSR分子标记的核桃涩味遗传多样性分析

doi:10.7668/hbnxb.20193708

摘要/Abstract

摘要：

为研究核桃种质资源涩味的遗传多样性,以4个不同核桃涩味群体共118份种质资源为材料,利用SSR毛细管电泳荧光标记技术进行遗传多样性研究,并构建树状聚类图。结果表明:12对引物共检测到93个等位变异位点,变异范围为2~18,平均每对SSR引物可检测到7.75个等位位点;多态性信息量(PIC)为0.357 9~0.785 2,平均0.541 1。4个群体的多态位点数Np为91、多态位点百分率PPB为97.85%、观测等位基因数Na为1.978 5、有效等位基因数Ne为1.198 5、Shannon's信息指数I为0.209 7、Nei's多样性指数H为0.126 3、总遗传多样性指数Ht为0.126 7、群体内遗传多样性指数Hs为0.122 2,说明4个核桃群体的遗传多样性和变异度不高。群体间遗传分化系数Gst为0.035 5,说明群体内遗传变异占总变异的96.45%。其中稍涩群体多态性位点百分率最大,为72.04%,说明稍涩群体在4个群体中遗传多样性最丰富。UPGMA聚类分析表明,4个群体的遗传相似系数为0.989 8~0.997 1,群体的整体相似系数高,说明这4个群体的遗传距离很近。当相似系数为0.993 4时,可将4个群体分成2组。其中,第1组包括不涩、稍涩和较涩3个群体,剩余涩群体单独为一组。通过计算不同群体93个位点的基因频率,发现12对引物的41个位点可将不同涩味等级的资源区分开。利用SSR分子标记技术对4个核桃群体进行与涩味相关的遗传多样性分析,根据转录组测序结果筛选出12对引物用于SSR分析,共扩增出93个SSR位点,多态位点百分率为97.85%,说明引物的多态性高,适用于核桃的遗传多样性分析,为培育轻涩味的优良品种提供技术指导,可用于后续核桃涩味的遗传多样性及育种研究。

关键词: 核桃, SSR, 涩味, 遗传多样性, 毛细管电泳

Abstract:

To study the genetic diversity of astringency in walnut germplasm resources,118 germplasm resources from 4 different astringency walnut populations were used as materials,which was studied by using SSR capillary electrophoresis fluorescence labeling technology,and the dendrogram of 4 populations was constructed.The results showed that a total of 93 allelic variants were detected by 12 pairs of primers,ranging from 2 to 18,with an average of 7.75 alleles detected by each pair of SSR primers.Polymorphism information content (PIC) ranged from 0.357 9 to 0.785 2,with an average of 0.541 1.The number of polymorphic sites (Np) was 91,the percentage of polymorphic sites (PPB) was 97.85%,the number of observed alleles (Na) was 1.978 5,the effective number of alleles (Ne) was 1.198 5,Shannon's information index (I) was 0.209 7,Nei's diversity index (H) was 0.126 3,number of total genetic diversity index (Ht) was 0.126 7 and intra-population genetic diversity index (Hs) was 0.122 2,indicating that the genetic diversity and variation of the four walnut populations were not high.The coefficient of genetic differentiation among populations was 0.035 5,indicating that the genetic variation within populations was 96.45%.The percentage of polymorphic loci in the slightly astringent population was the largest,which was 72.04%,indicating that the slightly astringent population had the richest genetic diversity among the four populations.UPGMA cluster analysis showed that the genetic identity between four populations ranged from 0.989 8 to 0.997 1,indicating that the genetic distance among four populations was close.The four populations can be divided into two groups at the coefficient of 0.993 4.The first group included unastringent,slightly astringent and more astringent populations,with the astringent populations as a separate group.By calculating the gene frequency of 93 locis in different populations,41 locis of 12 pairs of SSR primers were found to be able to distinguish the resources with different levels of astringency.SSR capillary electrophoresis fluorescence labeling technology was used to analyze the genetic diversity related to astringency in 4 walnut populations.According to the transcriptomic sequencing results,12 pairs of primers were selected for SSR analysis.A total of 93 SSR locis were amplified,and the percentage of polymorphic sites was 97.85%,indicating that the primers had high polymorphism and were suitable for genetic diversity analysis of walnut,which can be used for subsequent studies on genetic diversity and breeding of astringency in walnut.

Key words: Walnut, SSR, Astringency, Genetic diversity, Capillary electrophoresis

刘警, 李扬, 刘金利, 张馨方, 白仲奎, 于秋香. 基于SSR分子标记的核桃涩味遗传多样性分析[J]. 华北农学报, 2024, 39(S1): 23-30. doi: 10.7668/hbnxb.20193708.

LIU Jing, LI Yang, LIU Jinli, ZHANG Xinfang, BAI Zhongkui, YU Qiuxiang. Genetic Diversity Analysis about Astringency of Walnut by SSR Molecular Marker[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 23-30. doi: 10.7668/hbnxb.20193708.

http://www.hbnxb.net/CN/Y2024/V39/IS1/23

图/表 7

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涩味等级 Astringency grade	品种来源 Origin	资源 Resource
不涩	新疆	2-21、2-27、4-3
Unastringent	河北	魁香、台营2、旮旯铺、3-17、5-39、D1、D4
	辽宁	礼品2号、辽宁10号、寒丰
	山东	花生1号、丰香、A19
	河南	中核2号
	山西	晋龙1号
	日本	43号心形
稍涩	新疆	新光、扎木台3号、新早丰、1-41、2-7、2-17、2-19、2-35、3-30、4-22、4-26、6-3、6-5、6-21、
Slightly astringent		6-29、6-33
	河北	绿岭、早硕、龙珠、硕香、昌黎2、昌黎3、昌黎4、昌黎5、台营3、台营4、SJL、Y1、1-32、1-38、2-15、
		2-16、2-40、3-10、3-12、3-22、3-26、3-34、3-39、5-28、5-30、5-35、5-36、5-37、5-42、6-1
	辽宁	辽宁5号、辽宁6号、辽宁7号、礼品1号、辽瑞丰
	山东	华山5号
	朝鲜	安边2号
	日本	清香
较涩	新疆	1-43、2-37、2-39、3-1、4-5、4-15、6-25、6-43
More astringent	河北	西岭、台营1、1-34、2-8、2-12、2-14、2-20、2-32、2-34、3-5、3-7、3-20、3-21、3-29、3-32、3-33、
		4-10、5-9、5-12、5-34
	辽宁	辽宁1号、辽宁4号、10901优系
	山东	B76、鲁果6号、岱香
	北京	美香
	河南	绿波
涩	新疆	2-25、2-29、4-29、6-7、纸皮3号
Astringent	河北	2-22、2-42
	山西	金薄丰
	河南	中核3号

涩味等级 Astringency grade	品种来源 Origin	资源 Resource
不涩	新疆	2-21、2-27、4-3
Unastringent	河北	魁香、台营2、旮旯铺、3-17、5-39、D1、D4
	辽宁	礼品2号、辽宁10号、寒丰
	山东	花生1号、丰香、A19
	河南	中核2号
	山西	晋龙1号
	日本	43号心形
稍涩	新疆	新光、扎木台3号、新早丰、1-41、2-7、2-17、2-19、2-35、3-30、4-22、4-26、6-3、6-5、6-21、
Slightly astringent		6-29、6-33
	河北	绿岭、早硕、龙珠、硕香、昌黎2、昌黎3、昌黎4、昌黎5、台营3、台营4、SJL、Y1、1-32、1-38、2-15、
		2-16、2-40、3-10、3-12、3-22、3-26、3-34、3-39、5-28、5-30、5-35、5-36、5-37、5-42、6-1
	辽宁	辽宁5号、辽宁6号、辽宁7号、礼品1号、辽瑞丰
	山东	华山5号
	朝鲜	安边2号
	日本	清香
较涩	新疆	1-43、2-37、2-39、3-1、4-5、4-15、6-25、6-43
More astringent	河北	西岭、台营1、1-34、2-8、2-12、2-14、2-20、2-32、2-34、3-5、3-7、3-20、3-21、3-29、3-32、3-33、
		4-10、5-9、5-12、5-34
	辽宁	辽宁1号、辽宁4号、10901优系
	山东	B76、鲁果6号、岱香
	北京	美香
	河南	绿波
涩	新疆	2-25、2-29、4-29、6-7、纸皮3号
Astringent	河北	2-22、2-42
	山西	金薄丰
	河南	中核3号

引物名称 Primers	正向引物(5'—3') Forward primers(5'—3')	反向引物(5'—3') Reverse primers(5'—3')	扩增物大小/bp The size of fragment	等位基因数 No. of alleles
PAL-1	F:CAGCAGCGACTGGGTCATAA	R:AGCATGGGACTTTTCCACCTATA	322~327	4
ANR-6	F:CCACCTAAGAAGCCACCGTT	R:GGCTCTCATACTTTTGCGATGC	101~141	11
UFGT-6	F:TTGGCTGGACGCGATAATGA	R:ATCTTCGACGGTTGGGCAAT	237~278	11
4CL	F:TCGAGACGTCCAGATCCAGT	R:CGAGGATGGGTCGCTTTCTT	131~156	4
DFR-2	F:GCACCGTTCGTTTATACCGC	R:GGTGGCCACCGAGAAATGTA	147~189	18
C4H	F:TCATTCGTGCCTATGTCGATACT	R:GCCTTTGTGCTCGACAGTTT	260~294	5
F3'H	F:GCCAAAGCCAAAGAAGAGGG	R:GCTTCAACAACCTTGCCATCA	385~465	8
UFGT-1	F:TCCCTCGATACATGTTGGCA	R:GCACATCTCACCACTCCTCC	288~299	8
CHS	F:GAGAAGTTCAAGCGCATGTGT	R:CCTTGGTGGCTGCTTCTTTG	484~518	6
ANR-3	F:TTGGTGGACGCTTGGTGATT	R:AGCCTCCACTCCGATCTCTT	222~258	11
ANR-5	F:CAGTAGAGTCTGCGTCACGG	R:ACTTTGGATGTGTCGCCTGT	282~311	5
FHT-4	F:CGAGTTGACACCCTCCACTC	R:ACGAAGCTTTTCCTCGGGAG	546~548	2

引物名称 Primers	正向引物(5'—3') Forward primers(5'—3')	反向引物(5'—3') Reverse primers(5'—3')	扩增物大小/bp The size of fragment	等位基因数 No. of alleles
PAL-1	F:CAGCAGCGACTGGGTCATAA	R:AGCATGGGACTTTTCCACCTATA	322~327	4
ANR-6	F:CCACCTAAGAAGCCACCGTT	R:GGCTCTCATACTTTTGCGATGC	101~141	11
UFGT-6	F:TTGGCTGGACGCGATAATGA	R:ATCTTCGACGGTTGGGCAAT	237~278	11
4CL	F:TCGAGACGTCCAGATCCAGT	R:CGAGGATGGGTCGCTTTCTT	131~156	4
DFR-2	F:GCACCGTTCGTTTATACCGC	R:GGTGGCCACCGAGAAATGTA	147~189	18
C4H	F:TCATTCGTGCCTATGTCGATACT	R:GCCTTTGTGCTCGACAGTTT	260~294	5
F3'H	F:GCCAAAGCCAAAGAAGAGGG	R:GCTTCAACAACCTTGCCATCA	385~465	8
UFGT-1	F:TCCCTCGATACATGTTGGCA	R:GCACATCTCACCACTCCTCC	288~299	8
CHS	F:GAGAAGTTCAAGCGCATGTGT	R:CCTTGGTGGCTGCTTCTTTG	484~518	6
ANR-3	F:TTGGTGGACGCTTGGTGATT	R:AGCCTCCACTCCGATCTCTT	222~258	11
ANR-5	F:CAGTAGAGTCTGCGTCACGG	R:ACTTTGGATGTGTCGCCTGT	282~311	5
FHT-4	F:CGAGTTGACACCCTCCACTC	R:ACGAAGCTTTTCCTCGGGAG	546~548	2

群体名称 Population	份数 Number	多态位点数 Np	多态位点百分率/% PPB	观测等位基因数 Na	有效等位基因数 Ne	Nei's多样性指数 H	Shannon's 信息指数 I
不涩Unastringent	19	55	59.14	1.591 4	1.196 2	0.123 8	0.200 6
稍涩Slightly astringent	54	67	72.04	1.720 4	1.195 1	0.123 1	0.201 5
较涩More astringent	36	63	67.74	1.677 4	1.201 4	0.126 5	0.205 7
涩Astringent	9	41	44.09	1.440 9	1.183 1	0.115 5	0.182 9
总计Total	118	91	97.85	1.978 5	1.198 5	0.126 3	0.209 7

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