Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L. in responses to waterlogging stress

doi:10.3969/j.issn.1004-1524.20230443

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (3): 544-558.DOI: 10.3969/j.issn.1004-1524.20230443

• Horticultural Science • Previous Articles Next Articles

Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L. in responses to waterlogging stress

LIU Huichun(), XU Wenting, ZHOU Jianghua, ZHANG Jiaqiang, SHI Xiaohua, ZHU Kaiyuan()

Zhejiang Institute of Landscaping Plants and Flowers, Hangzhou 311251, China

Received:2023-04-10 Online:2024-03-25 Published:2024-04-09

Abstract

Abstract:

Waterlogging stress is one of the most severe abiotic stress that limits cultivational expansion, fast growth, and high yield of P.suffruticosa. In order to elucidate the mechanism of P.suffruticosa during waterlogging stress, transcriptomic analysis was performed and SSR markers were developed in this study. Six cDNA libraries constructed from mRNAs of waterlogging-treated seedlings and control were sequenced by using the Illumina sequencing platform. Totally, 73 925 unigenes were obtained by denovo assembly, creating an initial reference transcriptome dataset of P. suffruticosa. Among them, 780 were identified as response genes in early stage of waterlogging including 155 up-regulated genes and 625 down-regulated genes. Functional analysis showed that modifications in the expression of genes involved in signal regulation by transcription factors, DNA replication, ribosome and pyrimidine metabolism might play important roles in responses to waterlogging stress in P. suffruticosa. Based on these high throughput transcriptome sequencing data, 5 204 SSR markers were excavated. Among these markers, dinucleotide repeats were 58.13%, trinucleotide repeats were 27.11%, and tetranucleotide repeats were 7.24%. 110 pairs of SSR marker primers were found in the unigenes of functional annotation related to the target genes of waterlogging stress traits of peony, and 45 pairs of primers were screened, of which 12 pairs could amplify clear target bands, and the polymorphic SSR marker primers accounted for 26.67% of the total primers. This study provides the first large-scale transcriptome annotation of P. suffruticosa which may beneficially elucidate the waterlogging response mechanisms. Besides, it provides a valuable genomic resource and a scientific basis for molecular marker assisted selection breeding in waterlogging-tolerant species of P. suffruticosa.

Key words: Paeonia suffruticosa, waterlogging stress, transciptome, RNA-seq, SSR

CLC Number:

S685.11

LIU Huichun, XU Wenting, ZHOU Jianghua, ZHANG Jiaqiang, SHI Xiaohua, ZHU Kaiyuan. Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L. in responses to waterlogging stress[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 544-558.

Figures/Tables 16

References 39

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功能组 Functional groups	富集P值 Enriched P value	基因数量 Gene number
生物进程Biological processes
核小体组织Nucleosome organization	5.73×10^-57	49
蛋白质-DNA复合物亚单位组织Protein-DNA complex subunit organization	1.48×10^-56	49
染色体组织Chromosome organization	3.60×10^-50	55
蛋白质复合物亚单位组织Protein complex subunit organization	2.69×10^-49	54
染色质组织Chromatin organization	4.54×10^-48	51
大分子复合物亚单位组织Macromolecular complex subunit organization	1.94×10^-44	56
细胞器组织Organelle organization	5.84×10^-44	60
单元元件组织Cellular component organization	1.77×10^-41	68
细胞成分组织或生物发生Cellular component organization or biogenesis	2.53×10^-39	68
二价金属离子输送Divalent metal ion transport	1.20×10^-9	13
分子功能Molecular functions
蛋白质二聚化活性Protein dimerization activity	8.09×10^-51	49
蛋白质结合Protein binding	2.16×10^-36	63
核酸结合Nucleic acid binding	1.09×10^-32	58
杂环化合物结合Heterocyclic compound binding	7.96×10^-14	83
结合Binding	3.10×10^-13	112
有机环状化合物结合Organic cyclic compound binding	4.95×10^-13	83
微管蛋白结合Tubulin binding	2.54×10^-4	6
DNA聚合酶活性DNA polymerase activity	3.59×10^-4	4
运动活性Motor activity	8.52×10^-4	5
细胞骨架蛋白结合Cytoskeletal protein binding	1.45×10^-3	6
细胞成分Cellular components
无膜细胞器Non-membrane-bounded organelle	3.68×10^-35	69
细胞内无膜细胞器Intracellular non-membrane-bounded organelle	3.68×10^-35	69
膜封闭官腔Membrane-enclosed lumen	8.35×10^-20	35
核腔Nuclear lumen	8.35×10^-20	35
细胞器腔Organelle lumen	8.35×10^-20	35
细胞器内腔Intracellular organelle lumen	8.35×10^-20	35
核部件Nuclear part	9.44×10^-19	35
核Nucleus	2.22×10^-18	35
细胞内细胞器Intracellular organelle	5.98×10^-14	95
细胞器Organelle	6.90×10^-14	95

功能组 Functional groups	富集P值 Enriched P value	基因数量 Gene number
生物进程Biological processes
核小体组织Nucleosome organization	5.73×10^-57	49
蛋白质-DNA复合物亚单位组织Protein-DNA complex subunit organization	1.48×10^-56	49
染色体组织Chromosome organization	3.60×10^-50	55
蛋白质复合物亚单位组织Protein complex subunit organization	2.69×10^-49	54
染色质组织Chromatin organization	4.54×10^-48	51
大分子复合物亚单位组织Macromolecular complex subunit organization	1.94×10^-44	56
细胞器组织Organelle organization	5.84×10^-44	60
单元元件组织Cellular component organization	1.77×10^-41	68
细胞成分组织或生物发生Cellular component organization or biogenesis	2.53×10^-39	68
二价金属离子输送Divalent metal ion transport	1.20×10^-9	13
分子功能Molecular functions
蛋白质二聚化活性Protein dimerization activity	8.09×10^-51	49
蛋白质结合Protein binding	2.16×10^-36	63
核酸结合Nucleic acid binding	1.09×10^-32	58
杂环化合物结合Heterocyclic compound binding	7.96×10^-14	83
结合Binding	3.10×10^-13	112
有机环状化合物结合Organic cyclic compound binding	4.95×10^-13	83
微管蛋白结合Tubulin binding	2.54×10^-4	6
DNA聚合酶活性DNA polymerase activity	3.59×10^-4	4
运动活性Motor activity	8.52×10^-4	5
细胞骨架蛋白结合Cytoskeletal protein binding	1.45×10^-3	6
细胞成分Cellular components
无膜细胞器Non-membrane-bounded organelle	3.68×10^-35	69
细胞内无膜细胞器Intracellular non-membrane-bounded organelle	3.68×10^-35	69
膜封闭官腔Membrane-enclosed lumen	8.35×10^-20	35
核腔Nuclear lumen	8.35×10^-20	35
细胞器腔Organelle lumen	8.35×10^-20	35
细胞器内腔Intracellular organelle lumen	8.35×10^-20	35
核部件Nuclear part	9.44×10^-19	35
核Nucleus	2.22×10^-18	35
细胞内细胞器Intracellular organelle	5.98×10^-14	95
细胞器Organelle	6.90×10^-14	95

KEGG途径 KEGG pathways	富集P值 Enriched P value	基因数量 Gene number
DNA复制DNA replication	1.04×10^-15	84
核糖体Ribosome	3.74×10^-6	604
嘧啶代谢Pyrimidine metabolism	5.20×10^-4	230
吞噬体Phagosome	1.48×10^-3	112
基底切除修复Base excision repair	3.31×10^-3	75
内质网中的蛋白质加工Protein processing in endoplasmic reticulum	4.59×10^-3	363
嘌呤代谢Purine metabolism	5.29×10^-3	264
不匹配修复Mismatch repair	6.52×10^-3	61
半胱氨酸和蛋氨酸代谢Cysteine and methionine metabolism	1.68×10^-2	136
同源重组Homologous recombination	1.69×10^-2	77
核苷酸切除修复Nucleotide excision repair	3.09×10^-2	90

KEGG途径 KEGG pathways	富集P值 Enriched P value	基因数量 Gene number
DNA复制DNA replication	1.04×10^-15	84
核糖体Ribosome	3.74×10^-6	604
嘧啶代谢Pyrimidine metabolism	5.20×10^-4	230
吞噬体Phagosome	1.48×10^-3	112
基底切除修复Base excision repair	3.31×10^-3	75
内质网中的蛋白质加工Protein processing in endoplasmic reticulum	4.59×10^-3	363
嘌呤代谢Purine metabolism	5.29×10^-3	264
不匹配修复Mismatch repair	6.52×10^-3	61
半胱氨酸和蛋氨酸代谢Cysteine and methionine metabolism	1.68×10^-2	136
同源重组Homologous recombination	1.69×10^-2	77
核苷酸切除修复Nucleotide excision repair	3.09×10^-2	90

重复类型 Repeat type	重复数量Repeat number												总数 Total	占比 Percentage/%
重复类型 Repeat type	4	5	6	7	8	9	10	11	12	13	14	≥15	总数 Total	占比 Percentage/%
二核苷酸Dinucleotide			1 084	656	513	350	241	110	17	1	9	44	3 025	58.13
三核苷酸Trinucleotide		901	292	149	38	12	9	1	1	2	3	3	1 411	27.11
四核苷酸Tetranucleotide	296	61	11	6	1		1			1			377	7.24
五核苷酸Pentanucleotide	104	21	3										128	2.46
六核苷酸Hexanucleotide	176	61	8	10	4	1	2		1				263	5.05
合计Total													5 204	100

Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L. in responses to waterlogging stress

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重复基序类型 Repeat motif type	主要重复基序 Main repeat motif	重复次数 Repeat times	SSR重复的百分比 Percentage of SSR repetition/%	SSR重复总体占比 Percentage of total SSR repetition/%
二核苷酸Dinucleotide	AG/CT	2153	71.2	41.4
	AT/AT	513	17.0	9.9
	AC/CT	352	11.6	6.8
三核苷酸Trinucleotide	AAG/CCT	362	25.7	7.0
	ACC/GGT	277	19.6	5.3
	ATC/ATG	196	13.9	3.8
	AAT/ATT	154	10.9	3.0
	AGC/CTG	133	9.4	2.6
	AAC/GTT	109	7.7	2.1
	AGG/CCT	101	7.2	1.9
四核苷酸Tetranucleotide	AAAT/ATTT	171	45.4	3.3
		38	10.1	0.7
		35	9.3	0.7

引物名称 Primers name	引物序列 Primer sequence (5'-3')	重复基序 Repeat motif	Tm/℃	片段长度 Fragment length/bp
Ps01017	F:CTTTCGAGAACCGCATTTTC R:ATCGAAACACGACCCACTTC	(TGA)5	59.9	223
Ps01382	F:TGGGAGAGCATTATCAAGGG R:GAAAACCTAATCCCCAATGC	(GA)8	60.0	122
Ps02163	F:GAGCCGCGGTGATATATGTT R:CCGCGAAATTTCTTCGATAA	(GA)9	60.1	228
Ps04601	F:TCCACGAACCAAACACTGAA R:TCTTTGTGATCTGGTGCTGC	(TTTA)4	60.1	251
Ps06615	F:AAGAAGGAAGGCGTACACGA R:CACTCCCAACCAAATAACCAA	(AG)10	59.8	174
Ps08889	F:GAGAGAAGCTCCGTTGTTGG R:GAATTACAACACCCCATGCC	(AGA)6	60.0	260
Ps11111	F:AGAATCCTCCCCTTCTTCCA R:CACCAGAGGTGAAGGTGGTT	(TC)6	60.0	186
Ps11416	F:ATGATTATGCTCCAGCAGCC R:ACCCCCATCTCTAGGAGGAA	(TGA)5	60.2	103
Ps11993	F:AGGCCGAGCTCCACATGTAT R:TGCGGAGGATGAGTTCTTCT	(CT)7	61.9	257
Ps14499	F:GCAGGGCAGGACTCTCTATG R:AAGCCCAGTCGAGCAGATTA	(TA)9	59.9	167
Ps17872	F:TACCTGTCTCGAACTCCCCA R:CTATGAGGGACCAGAGAGCG	(CT)10	60.6	200
Ps18852	F:CCAGAGCCACATGCCTTATT R:ATGTCATCTGAGGTTTCGGC	(CCGTGT)4	60.0	269
Ps25388	F:CCGTGTAGATCTGGTTCGGT R:TCACCGCTGAATCAGAACAG	(TA)7	59.9	168
Ps28246	F:ACCTCCTTCTTTAGCGCTCC R:CCGCCAATTTCAGAGAGAGA	(TC)8	59.9	245
Ps29032	F:TCCCACATTCTTGTGCAGAG R:GTTTGGGGAAATTGGGAACT	(TA)6	60.0	259
Ps29541	F:GTGAGAGGATGAAACGGGAA R:ACAACAAATCCCTCACTCGC	(TC)8	60.1	272
Ps29681	F:CGAAATGGTGAGACACGAAG R:AATCTCAAACGGCTCATTGG	(GA)6	60.0	198
Ps30272	F:CCGGGACTATTTTCTAGCCC R:CAAAACTCCATCAGCAGCAG	(GAC)6	59.9	259
Ps31923	F:CCATAACAGCGGTGAGGATT R:TATGCGAGCCATCTCCTTTT	(AG)7	59.9	183
Ps33766	F:CTCGGGTGTTGGAGTTCATT R:TGTCGCCCTGTCTCTCTTTT	(GA)8	59.9	132
Ps34118-1	F:TCTTGCCATTTCCTTGAAGC R:AAGGTGGCACGAGCATTAAC	(CT)8	60.3	207
Ps34118-2	F:ATGGCCGTAGCATAGTGTCC R:CACAAGAGGCCAACACGTAA	(AT)7	59.9	233
Ps34343	F:GGGTTGGGAGGGATTAAGAA R:CACAATTCGGACACAGCATC	(TGG)5	60.1	165
Ps34446	F:TCAGGGTTTTAGCTCGAGGA R:CTCCTTTGCCCATTGTCATC	(AG)6	59.9	142
Ps35645-1	F:CACAAAGCAACCACCCTTTT R:GCATCTGCATCTGGCTCATA	(ATGTAG)4	60.0	179
Ps35645-2	F:GCAGAGTCGATCCTGGCTAC R:GGGTACGTCTGCAACCTTGT	(TC)6	60.0	238
Ps44241	F:GCCATCGAAACTTTTGAACC R:TTCCGGAAAAATGAAAGACG	(CT)9	60.0	148
Ps45222	F:TTAGGATGAAAATCGGCTGG R:TCTTTTTAAGACCCAGACTCTTTGA	(GA)10	60.0	197
Ps45971	F:AAAACGTGTGGCCTCAAAAC R:GCGACGAATCAGGAGAGAAC	(AGA)5	60.0	204
Ps46734	F:CCATACCGTGCATATGACCA R:CAGCGCATCAATACCTCAAA	(TC)6	60.2	142
Ps47595	F:TCGCCTTGTATACCTTTCCG R:AGTGCAGTCCTTCTCGAATCT	(TC)7tt(TC)7	60.0	147
Ps48286	F:CTCTCCTCTTCCAGACACGC R:GCAGAAAGAACCAAACCCAA	(TG)6	60.1	177
Ps49879	F:ATAGGCAACGCCATGTTTTC R:CTCCCCTCTCTTTGGCTCTT	(GA)8	59.9	110
Ps51526	F:GCGAGGGGATAAAACTAGGG R:GTGCTTTCCTGAAACCCAAC	(TA)6	59.9	176
Ps54582	F:GAGAGAGATAGAGGGCGCAA R:TCACCTCCGATTCGTAAACC	(TCT)5	59.9	230
Ps54679	F:TGTACTTGGGCATCACCAGA R:TTAAGGGCAGCTCGGACTAA	(TC)8	60.1	257
Ps55401	F:AGCCTTAATCGGTCGAACCT R:TTGTGCCAGACACCAAGAAG	(GAT)6	60.0	272
Ps55802	F:CCCAAGTTTTTCCCTTCTCA R:GGTGGACTTTGACACGGACT	(TTA)5	60.0	144
Ps57846	F:TCCATACGCATTCTTTTCCC R:GGAGCCCAAGCGTAACAATA	(AGC)6	59.9	276
Ps57972	F:ATTGTTTCCGGGACCACTTC R:GTTTGGTAGGGGTTTTGGCT	(ACC)5	61.1	223
Ps58678	F:AAGGCCGGTAAGAAGCTACC R:TTCCCTAGAGGTGATGGTGG	(AGA)5	59.9	249
Ps58726	F:TCGAGTACCTTGAGGAGCGT R:CAGATGGGCTTCTGCTTGTT	(AGA)5	60.3	262
Ps60834	F:TCCCTCTCTTTTCTCACTGCAT R:GGTGCATGACTGGGAGGTAT	(TG)6	60.3	152
Ps64449	F:GTTGCCCCTCTCCTTCTCTT R:CTCTCTCCTTGCCGCTCTTA	(AT)6	59.8	168
Ps67490	F:GAACAAAAGCCTTGCCTCTG R:TACGATAGGCCACAACCACA	(CT)10	59.9	262

引物 Primers	观测等位基因数 Observed number of alleles (Na)	有效等位基因数 Effective number of alleles (Ne)	Nei’s基因多样性 Nei’s gene diversity index (H)	Shannon信息指数 Shannon’s information index (I)	多态性位点 Polymorphic sites	多态性百分比 Polymorphic percentage/%
Ps02163	2.000 0	1.371 1	0.244 2	0.396 1	14	100
P04601	1.833 3	1.487 2	0.285 6	0.428 2	10	83.33
Ps17872	1.857 1	1.405 1	0.259 9	0.404 3	6	85.71
Ps18852	2.000 0	1.413 7	0.264 5	0.422 7	5	100
Ps28246	1.777 8	1.294 4	0.192 8	0.310 2	7	77.78
Ps29032	1.333 3	1.266 7	0.148 1	0.212 2	1	33.33
Ps30272	1.833 3	1.265 1	0.177 9	0.295 1	5	83.33
Ps34118-1	1.833 3	1.351 7	0.217 0	0.343 4	5	83.33
Ps34118-2	1.500 0	1.060 4	0.053 9	0.109 6	1	50.00
Ps35645	2.000 0	1.692 6	0.397 2	0.581 8	5	100
Ps54679	2.000 0	1.479 4	0.297 7	0.462 5	10	100
Ps55401	2.000 0	1.573 6	0.332 2	0.497 4	3	100
Mean	1.830 7	1.388 4	0.256 1	0.372 0	6	83.00

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