基于简化基因组的甜叶菊分子标记开发

doi:10.11924/j.issn.1000-6850.casb20190800564

摘要/Abstract

摘要：

本研究旨在筛选和开发具有多态性的甜叶菊基因组分子标记。选取4个甜叶菊品种为材料,利用简化基因组测序技术(RAD-seq)开发SSR和SNP标记。开发并验证了149对多态性丰富、重复性好、特异性高的简单重复序列(SSR)和4组功能性的SNP标记,开发的多态性基因组分子标记中包含了甜叶菊糖苷合成途径关键基因的新标记StKASPSNP-7。验证了基于KASP技术开发甜叶菊功能SNP标记的可行性,为甜叶菊遗传图谱构建、分子鉴定奠定了基础。本研究开发的SSR和SNP标记可应用于甜叶菊的遗传图谱分析、品种真实性的高效快速鉴定以及高糖苷含量新品种的开发。

关键词: 甜叶菊, 简化基因组测序(RAD-seq), 简单重复序列(SSR), 单核苷酸多态性(SNP), 竞争性等位基因特异性PCR(KASP)

Abstract:

The aim is to select and develop polymorphic molecular markers of Stevia rebaudiana genome. 4 Stevia rebaudiana varieties were used as materials, SSR markers and SNP markers were developed with RAD-seq. 149 pairs of SSR with rich polymorphism, high repeatability and specificity, and 4 functional SNP markers were developed and identified. These new genome molecule markers included new markers StKASPSNP-7 of key gene in Stevia rebaudiana glycoside synthesis pathway. The feasibility of developing SNP markers of Stevia rebaudiana based on KASP technology was verified, which laid a foundation for constructing genetic map and molecular identification of Stevia rebaudiana. SSR markers and KASP markers developed in the study could be used to analyze the genetic map of Stevia rebaudiana, identify its variety authenticity with high efficiency and speed, and develop new varieties with high glycoside content.

Key words: Stevia rebaudiana, RAD-seq, SSR, SNP, KASP

中图分类号:

S566.9

孙雨茜, 倪万潮, 杜平, 崔晓霞, 郭书巧, 蒋璐. 基于简化基因组的甜叶菊分子标记开发[J]. 中国农学通报, 2020, 36(27): 111-117.

Sun Yuxi, Ni Wanchao, Du Ping, Cui Xiaoxia, Guo Shuqiao, Jiang Lu. Development of Molecular Markers in Stevia rebaudiana Based on Reduced-Representation Genome Sequencing[J]. Chinese Agricultural Science Bulletin, 2020, 36(27): 111-117.

图/表 6

参考文献 24

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类别	数量	频率/%
检测序列	612904
检测序列碱基数/bp	223872168
检测到的SSR位点	16985
包含SSR的序列数	15710
含有1个以上SSR的序列数	1088	6.41
SSR复合物	886	5.22
单核苷酸	2667	15.07
二核苷酸	6564	38.65
三核苷酸	4736	27.88
四核苷酸	1833	10.79
五核苷酸	665	3.92

类别	数量	频率/%
检测序列	612904
检测序列碱基数/bp	223872168
检测到的SSR位点	16985
包含SSR的序列数	15710
含有1个以上SSR的序列数	1088	6.41
SSR复合物	886	5.22
单核苷酸	2667	15.07
二核苷酸	6564	38.65
三核苷酸	4736	27.88
四核苷酸	1833	10.79
五核苷酸	665	3.92

标记名称	重复单元	正向引物	反向引物	扩增片段/bp
StSSR20	(GA)10	ATTGGGTGTTGCCAAGAAAG	GGCTACCAGCCATTGAAGAG	157
StSSR21	(TTC)9	CCAGAGGTAGAGCTCTGTGC	GTTTTTGCGTCTGAATCGGT	134
StSSR23	(GCG)7	ATGCTTGGAGGCCAATGTAG	CTTTCTATCTCACAACCATGTCG	177
StSSR27	(TCA)7	CTGGTTCTTCTGCACCACTG	GTGGAGAATGCTGACCGTTT	229
StSSR33	(TTC)5	TTGTGAATCGACGAGAACCA	TTCGTTTGTCGGAACCCTAC	193
StSSR47	(ATC)5	GCACAATTTGCAGATGGTCTT	GCAGATGGGCTGCTAGACTT	246
StSSR51	(TCA)7	CAAAGCCTCATCATCCACCT	TGCTTTAGATGCTGGTGCTG	211
StSSR52	(AGA)7	TCGATTTTCCACTTTCACCC	CGTCTGCTACAGTCCCACAA	236
StSSR54	(TTA)7	ACAAGTCATATGCATCCGCA	ACAGCGCACGTTACCCTAGT	279
StSSR59	(TCA)6	ATGTGGTCTTCCTTTGGTGG	ATCACGCGGTAAATCCAGAC	240
StSSR60	(CCA)6	TCATGGCTTCCATTTCCTTC	TGTAGTTTTCCGGCAGGTTT	270
StSSR63	(AAT)6	ACACCCTTAAGGCATGCAAC	TCATACCCAATTTCAACCCA	198
StSSR66	(AAT)6	AGCTGAAGTGAAAATGGCGT	GGGCACAGACCCTTTGTTTA	235
StSSR67	(CAA)6	ACGCAACAACAATGGTCAAA	CAATTGTTGCTGTTGATGCC	246
StSSR69	(ATC)6	TGAAGTTGTGCTTGGATTGG	GTTCTTCAGTCGGGGAATCA	245
StSSR85	(CAC)6	CCTCCACAACATCCAAAACC	AGCCTTTGTAGTCTCGGCAA	259
StSSR88	(GGT)6	TGGCAACACCTGTACCTGAA	TGCATCTGTAGCAAGATCCG	188
StSSR100	(TCA)6	ATCGATCCATCTGCTTTTGC	CAACGTCATCTTGTCGGTCA	280
StSSR110	(AC)7	TTTCCCATGTCAACGAAGTG	GGAATCAAAGAGTGTGAAACCA	156
StSSR112	(AT)8	TCGGTTAAATTCGCACATGA	TGTAGTACACGGGTCAATAGGC	122
StSSR114	(TG)7	TGCCACCATGTGTTTCTGAT	CCGGTCATCCTCAAAACCTA	197
StSSR125	(ATA)6	TCAGAAGCCTGTAAGCAATTCA	ATTCAACTGCCAAAAATGGG	245
StSSR133	(AAC)5	CCCCCACTTGGGTACTCTTT	TTATGTGTGTCGGTGAGGGA	202
StSSR136	(ATA)5	ATGAAATCGAAACCCAGGTG	TAGGGGCACGTAATCCAAAA	145
StSSR139	(TAA)5	AACAATGAAAAGCTAGTATGGGTT	GGAACAATGCAGCAGTTTGA	130
StSSR141	(AAA)5	CAAAATGTAACAAAATCCCCCA	TCAAAGGGATTCATCTCCCA	140
StSSR143	(CAA)5	CGAAATGGATTCAATGGAGG	TGCTCGTTTTCACATTTCCA	165
StSSR155	(AG)16	ACAAACAACCTCTTTCCGGC	CACTTGTTCTCTTTCGGCAA	122
StSSR157	(CAT)9	GAAGCGTCGCATTAAAGGTC	CTTTCGGAGCAGCCTCTCTA	280
StSSR163	(CCA)8	GACCCATTTAACcCCGTTTT	GCAGTGATTGGTTCATTGGA	243
StSSR166	(TG)16	TTCGGTGTTAATTATGTGTGGA	CAATCCAGCTACTCCGATCC	269
StSSR169	(TC)14	TTCATATAAGTCTTTCACACACACTCT	GAAATATTCGGGGGTTCGTT	115
StSSR172	(TTG)9	TTCGACCCAAAGGTTACTCG	ATGGGCCAAACATCCAATAA	225
StSSR179	(AAC)9	TGACCCAAAACATCAACGAA	GTAAACTTCTCGGGATCGCA	127
StSSR181	(GAA)8	TTGGGAAACTTGATTTTGGG	TTGCACCATGTGATGTAAGGA	190
StSSR183	(CAC)8	ACAACCCACGATGAAGATCC	AATGGTGGTAAACGGTGGAC	280
StSSR191	(GAT)9	CTTTGCAATGGATGTCTCCC	CGTTTTGTTGTGTAACTTGCG	239
StSSR213	(ATC)8	TTCTTGGGGTTCTTTTGCTG	TGTTGATGATGGCAATGAAAA	225
StSSR215	(GAT)4	AAAGGTTCGCAACTTCGCTA	ACCCATTTGACCGTTTTTCA	260
StSSR219	(TTA)10	AAACATGGTTAGTCAGATATCACTCG	TGGTCCTTGTGTGTTAAGCG	203

标记名称	重复单元	正向引物	反向引物	扩增片段/bp
StSSR20	(GA)10	ATTGGGTGTTGCCAAGAAAG	GGCTACCAGCCATTGAAGAG	157
StSSR21	(TTC)9	CCAGAGGTAGAGCTCTGTGC	GTTTTTGCGTCTGAATCGGT	134
StSSR23	(GCG)7	ATGCTTGGAGGCCAATGTAG	CTTTCTATCTCACAACCATGTCG	177
StSSR27	(TCA)7	CTGGTTCTTCTGCACCACTG	GTGGAGAATGCTGACCGTTT	229
StSSR33	(TTC)5	TTGTGAATCGACGAGAACCA	TTCGTTTGTCGGAACCCTAC	193
StSSR47	(ATC)5	GCACAATTTGCAGATGGTCTT	GCAGATGGGCTGCTAGACTT	246
StSSR51	(TCA)7	CAAAGCCTCATCATCCACCT	TGCTTTAGATGCTGGTGCTG	211
StSSR52	(AGA)7	TCGATTTTCCACTTTCACCC	CGTCTGCTACAGTCCCACAA	236
StSSR54	(TTA)7	ACAAGTCATATGCATCCGCA	ACAGCGCACGTTACCCTAGT	279
StSSR59	(TCA)6	ATGTGGTCTTCCTTTGGTGG	ATCACGCGGTAAATCCAGAC	240
StSSR60	(CCA)6	TCATGGCTTCCATTTCCTTC	TGTAGTTTTCCGGCAGGTTT	270
StSSR63	(AAT)6	ACACCCTTAAGGCATGCAAC	TCATACCCAATTTCAACCCA	198
StSSR66	(AAT)6	AGCTGAAGTGAAAATGGCGT	GGGCACAGACCCTTTGTTTA	235
StSSR67	(CAA)6	ACGCAACAACAATGGTCAAA	CAATTGTTGCTGTTGATGCC	246
StSSR69	(ATC)6	TGAAGTTGTGCTTGGATTGG	GTTCTTCAGTCGGGGAATCA	245
StSSR85	(CAC)6	CCTCCACAACATCCAAAACC	AGCCTTTGTAGTCTCGGCAA	259
StSSR88	(GGT)6	TGGCAACACCTGTACCTGAA	TGCATCTGTAGCAAGATCCG	188
StSSR100	(TCA)6	ATCGATCCATCTGCTTTTGC	CAACGTCATCTTGTCGGTCA	280
StSSR110	(AC)7	TTTCCCATGTCAACGAAGTG	GGAATCAAAGAGTGTGAAACCA	156
StSSR112	(AT)8	TCGGTTAAATTCGCACATGA	TGTAGTACACGGGTCAATAGGC	122
StSSR114	(TG)7	TGCCACCATGTGTTTCTGAT	CCGGTCATCCTCAAAACCTA	197
StSSR125	(ATA)6	TCAGAAGCCTGTAAGCAATTCA	ATTCAACTGCCAAAAATGGG	245
StSSR133	(AAC)5	CCCCCACTTGGGTACTCTTT	TTATGTGTGTCGGTGAGGGA	202
StSSR136	(ATA)5	ATGAAATCGAAACCCAGGTG	TAGGGGCACGTAATCCAAAA	145
StSSR139	(TAA)5	AACAATGAAAAGCTAGTATGGGTT	GGAACAATGCAGCAGTTTGA	130
StSSR141	(AAA)5	CAAAATGTAACAAAATCCCCCA	TCAAAGGGATTCATCTCCCA	140
StSSR143	(CAA)5	CGAAATGGATTCAATGGAGG	TGCTCGTTTTCACATTTCCA	165
StSSR155	(AG)16	ACAAACAACCTCTTTCCGGC	CACTTGTTCTCTTTCGGCAA	122
StSSR157	(CAT)9	GAAGCGTCGCATTAAAGGTC	CTTTCGGAGCAGCCTCTCTA	280
StSSR163	(CCA)8	GACCCATTTAACcCCGTTTT	GCAGTGATTGGTTCATTGGA	243
StSSR166	(TG)16	TTCGGTGTTAATTATGTGTGGA	CAATCCAGCTACTCCGATCC	269
StSSR169	(TC)14	TTCATATAAGTCTTTCACACACACTCT	GAAATATTCGGGGGTTCGTT	115
StSSR172	(TTG)9	TTCGACCCAAAGGTTACTCG	ATGGGCCAAACATCCAATAA	225
StSSR179	(AAC)9	TGACCCAAAACATCAACGAA	GTAAACTTCTCGGGATCGCA	127
StSSR181	(GAA)8	TTGGGAAACTTGATTTTGGG	TTGCACCATGTGATGTAAGGA	190
StSSR183	(CAC)8	ACAACCCACGATGAAGATCC	AATGGTGGTAAACGGTGGAC	280
StSSR191	(GAT)9	CTTTGCAATGGATGTCTCCC	CGTTTTGTTGTGTAACTTGCG	239
StSSR213	(ATC)8	TTCTTGGGGTTCTTTTGCTG	TGTTGATGATGGCAATGAAAA	225
StSSR215	(GAT)4	AAAGGTTCGCAACTTCGCTA	ACCCATTTGACCGTTTTTCA	260
StSSR219	(TTA)10	AAACATGGTTAGTCAGATATCACTCG	TGGTCCTTGTGTGTTAAGCG	203

标记名称	SNP位点序列	注释	检测
StKASPSNP-1	AAAGTGAGAAACAAGGTGGTCATCGTGCATTCAAGCTTGTGTTGCCAACT[ /T]ATCGGCGGTTATGAAAGTTCCATGAAAACCATAAGGAACTCTCGTCGGCA	NCED3	+
StKASPSNP-2	ACGGTGGTGAGCCGTTGTTTCTTCCGAGCTCCGGCGACGAAGACGATGGT[C/T]ACATTTTAGCATTCGTTCATGATGAGGCTATATGGGAATCCGAACTTCAA	NCED3	+
StKASPSNP-3	TAAGCATTCGGGTCGGAAGAAGGACTCAAATCATCCATATTAATTATGTA[A/G]CGACTTTTGTAAACTAACCTTGACTTTTATTGTTTGTTGATTTGAGAATG	none	-
StKASPSNP-4	TAAGCCCGAGCCGCCGCCTCCGCTGTCTCGAACGTACCTAACCACACCCG[A\T]GCCGCTCGTTGCGGGTCCCGAATCTCCGCCGCCCATTTCCCCCACGGTC	ERF	-
StKASPSNP-5	GGCATTACGTGGCAACATGGCTTGTTTGAATTTTGCTGACTCTGTTTGGC[A\G]GTTGCCAGTACCAGTTACTAATGATGCTAAGGATATTCGTAGAGCGGCAG	ERF035	+
StKASPSNP-6	TGGTATATACTTGGCTAACATCGAGGGAAAAATACAATTATTAACTATAT[A\C]AAGCTCATTCTTATCTAGTGAAATGCTTGATTCAGAAACTCCATAATGAC	CBF1	-
StKASPSNP-7	CCTTCCAAACTATGAAGTAAAGTTGTGGGCACTTTGTGCAATACTTCCAT[T/C]GGTATCCTATTAGTGACAAAAAAAGAAACGAAAAGACCGTCATTATATGT	CPPS	+
StKASPSNP-8	TTTTTAAACATCATCTTCCCATCTTCAGCGTCTGGATTCGTTGCAGAGGC[C/G]TTTAATCGGAACTGAAAAAACATTGATCGACATGTTAGCAACTAAATTCC	DXS	+
StKASPSNP-9	ATCTTTAATGATTCTTGTTTAAGCAAGACTACATTATTGGCAAGGTAATC[T/C]CTTTTTTGATTAAGCTGATATGTATAAGATAAAGGCTAAACCAACATGTT	none	-
StKASPSNP-10	GTCATGAAAACAAACATCAATACATCATAAAGTCTGAAAGTGTACTTTGT[C/T]GTAAATTAAGCGTCATAAAAAGTGTAGAAATAAAATGAACAATAAAAACA	none	-
StKASPSNP-11	ATATTTATGTAYGAAAATCGTAAATTAGTATATCAATCACATGATACATT[A/C]ATTTACCAAAACAACATCATTGTATTATCTATTTGACTATTTGTAATCTT	none	+