Development of Molecular Markers in Stevia rebaudiana Based on Reduced-Representation Genome Sequencing

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

Abstract

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

CLC Number:

S566.9

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.

Figures/Tables 6

References 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	+