基于转录组数据的芦笋EST-SSR标记的开发及通用性分析

doi:10.3864/j.issn.0578-1752.2023.22.011

Abstract

Abstract:

【Objective】The aim of this study was to clarify the distribution law of SSR loci across the transcriptome of Asparagus officinalis, to develop highly informative EST-SSR markers and to analyze their transferability, so as to provide the tools for phylogenetic analysis, functional gene mining and molecular marker-assisted breeding of asparagus plants.【Method】Based on the RNA-seq data of 15 asparagus roots obtained from the previous stage by our research group, MISA software was used to retrieve SSR loci, and Primer 3.0 software was employed to design primers in batches. Then, the ineffective primers were eliminated by performing batch e-PCR with TB-tools software and one-to-one e-PCR with the Primer-blast programme. The information of EST-SSR markers (such as gene id, physical location, and potential function) was obtained by comparison with the genome of asparagus officinalis. The DNA of 9 A. officinalis varieties, 7 A. setaceus varieties, 5 A. cochinchinensis varieties, and 3 A. umbellatus varieties were used as templates, and 50 pairs of randomly synthesized primers were used as primers to detect the effectiveness, polymorphism and transferability of the primers developed.【Result】A total of 36 590 simple sequence repeats (SSRs) loci distributed in 30 229 unigenes with a frequency of 4.78% and an average distance of 9.17 kb were identified based on data from 15 root transcriptomes of A. officinalis. The SSRs were unevenly distributed in the 10 chromosomes, with the highest number in chromosome 7 (4 642) and the highest density in chromosome 3 (37.86 SSRs/Mb). The SRRs were distributed from di- to hexa-, with tri- (46.92%) and AG/CT (16.58%) as the most abundant repeat type and predominant repeat motif, respectively. A total of 19 695 pairs of EST-SSR primers were successfully designed, and 15 147 pairs ineffective primers were eliminated by e-PCR. Among them, 3 085 pairs ineffective primers didn’t produce any amplification products, 10 102 pairs produced severely inconsistent amplification products in terms of fragment size, 1 289 pairs had unknown physical positions in the genome, 402 pairs gave other amplification products of similar size to the target fragments, and 269 pairs generated amplification products without SSRs. Based on 2 517 EST-SSR markers located in the gene region developed in this study, the chromosome density distribution map was constructed, with a total coverage length of 1 125.51 Mb and an average distance of 447.16 kb. The potential functions of these markers were involved in many aspects, such as yield, quality, stress resistance, and so on. All 50 pairs of randomly synthesized primers could amplify target bands clearly, of which 36 pairs were polymorphic, and the average polymorphic information content was 0.330. These markers could be used in three other species of Asparagus: the transferability to A. cochinchinensis, A. setaceus, and A. umbellatus were 100%, 92%, and 88%, respectively. Cluster analysis based on the EST-SSR alleles grouped the 24 accessions into four clusters that corresponded to the species of A. officinalis, A. setaceus, A. cochinchinensis, and A. umbellatus.【Conclusion】In this study, 2 517 highly informative EST-SSR markers of asparagus were successfully developed, and the effective amplification rate was 100%. The total coverage length of the physical map was 1 125.51 Mb, and the average distance was 447.16 kb, which could be used for phylogenetic analysis of asparagus and related species. Moreover, it provided a reference for the development of EST-SSR markers in other species.

Key words: transcriptome, asparagus, EST-SSR, chromosome density distribution map, functional annotation

YI ZeHui, ZHAO Jing, MAO LiPing. Development and Transferability of EST-SSR Markers Based on Transcriptome Data from Asparagus officinalis[J].Scientia Agricultura Sinica, 2023, 56(22): 4490-4505.

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URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2023.22.011

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Figures/Tables 11

Table 1

Fig. 1

Table 2

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Table 3

Information of 50 pairs of randomly synthesized EST-SSR primers developed from Asparagus officinalis"

名称 Name	重复基序 Repeat motif	开放阅读框 ORF	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')	退火温度 Tm (℃)	产物大小 Product size (bp)	染色体Chr	起始位置 Start position (bp)	预测功能 Putative function	基因<BOLD>ID</BOLD> Gene<BOLD> ID</BOLD>	等位基因数Na	多态性信息含量PIC
SSR20	(CCG)₅	CDS	TTATCGAGGAACAGCAGCCT	TTCGTCCTCCTCTACTCCGA	59.9	143	1	2228458	未知蛋白 LOC109845910 Uncharacterized protein LOC109845910	109845910	1	0.210
SSR44	(TC)₁₂	5'-UTR	GCGAGTAAGACTACCGCGAG	GATTTTGGGGAGAATTCGGT	60.1	156	1	6946254	未知蛋白 DDB_G0294196 Putative uncharacterized protein DDB_G0294196	109829946	2	0.494
SSR170	(AGA)₅	CDS	TATGATGAGAACCGAAGGGC	CATCCGTGCCAAACTATCCT	60.0	204	1	58943187	SCL蛋白28 Scarecrow-like protein 28	109834799	1	0.000
SSR189	(CAG)₇	CDS	CCCCAGAACCCTAAACCCTA	GAAGTAGTAGTCGGCGCTGG	60.0	238	1	79487997	可能的蛋白质精氨酸N-甲基转移酶1 Probable protein arginine N-methyltransferase 1	109826536	1	0.000
SSR249	(TGG)₅	CDS	CCTCTGGTCGTGGTGAGATT	TCAGGTTTTCCTGCACACAG	59.9	264	1	112933201	锌指蛋白8 Zinc finger CCHC domain-containing protein 8-like	109829023	2	0.444
SSR354	(CTC)₅	5'-UTR	TGACTGGTTGTTAGGGCCTC	CCTCCGGATAGATAGCACCA	60.1	224	2	2364488	未知蛋白 LOC109829884 Uncharacterized protein LOC109829884	109829884	3	0.593
SSR414	(ATA)₆	CDS	TCAAGCCTATTGTTGAATCTGC	GCTTAAGCAAGGGGGTCTTC	60.0	258	2	15586523	自噬相关蛋白13B Autophagy-related protein 13b-like	109830378	2	0.198
SSR486	(TTG)5	3'-UTR	CATGCACGCACACAAATGTA	TGAAACCAATGGTAAAGGCA	59.0	136	2	76807149	胆碱/乙醇胺磷酸转移酶2 Choline/ethanolamine-phospho- transferase 2-like	109831674	2	0.494
SSR491	(ATC)5	5'-UTR	GGGAAAATGAGAACTCGCAA	CCAGTTTTCTGCGGATCATT	60.1	250	2	77458426	DNA介导的RNA聚合酶II和IV亚基5A DNA-directed RNA polymerases II and IV subunit 5A	109831692	2	0.494
SSR518	(GAA)₅	CDS	GCCCTCACTCTCTCCATCAG	GGGGTGATGTCGTAGACGTT	59.9	214	2	83538318	核出口调节因子NEMF Nuclear export mediator factor NEMF	109831911	2	0.198
SSR572	(AAG)₇	Intron	GCACGCATTCGTTAAAGTTG	TTTGATTCAATTGTCCTTCGG	59.9	157	3	9251743	核孔复合蛋白NUP1 Nuclear pore complex protein NUP1-like	109832884	2	0.543
SSR595	(AAG)₅	CDS	TGGTTCTATTTTTCGGCTGG	CTTGCGACAAAACTCGATGA	60.0	171	3	14367170	八氢番茄红素合成酶2，叶绿体 Phytoene synthase 2, chloroplastic-like	109833104	2	0.198
SSR725	(GA)₈	5'-UTR	ATCGCTCCTTTCCCCTTTT	CTCCTCTGTCGCATCAATCA	59.9	138	3	41477413	MARD1蛋白 Protein MARD1-like	109835001	2	0.345
SSR782	(ATA)₅	CDS	TGCCCATGTCTGTTAAAGGAA	AGACCGCTAAGGTGGTGATG	60.0	188	3	73982482	未知蛋白 LOC109834334 Uncharacterized protein LOC109834334	109834334	2	0.198
SSR806	(TCT)₁₂	CDS	TTTGACCTTGGCAGACACAC	TGGCTTTGGAGGTCTCAAGT	59.8	141	3	102124175	叶绿素酶2，叶绿体 Chlorophyllase-2, chloroplastic	109835385	3	0.643
SSR857	(TC)₂₉	Intron	TGCCAATGCCTTTAAGGTTC	ACACGGGAGATGGAACAGAG	60.1	185	4	5629132	未知蛋白 LOC109836451 Uncharacterized protein LOC109836451	109836451	3	0.185
SSR883	(AAT)₅	Intron	CGAGTACTTCTCTTGCGCCT	TCTCCAACTACAACCCCTGG	60.0	156	4	15373511	脯氨酸转运蛋白2 Proline transporter 2-like	109836720	2	0.346
SSR952	(TC)₈	CDS	CCTAGTGAGGCAGGATGTCA	AGGGGATCGAGATGAATGTG	60.0	192	4	44331895	双向糖转运蛋白SWEET13 Bidirectional sugar transporter SWEET13-like	109837341	2	0.444
SSR991	(AGA)₇	3'-UTR	TGTGGCGTGCACTGTTAGAT	TTGGGGCTCCATAACATAGC	59.9	235	4	65334575	未知蛋白 LOC109837601 Uncharacterized protein LOC109837601	109837601	3	0.593
SSR1087	(T)₁₆	Intron	ACCTTCAACGGAAGGGTCTT	ACTGAGGAAGTGTGACCGCT	59.9	121	4	137703636	原叶绿素还原酶 Protochlorophyllide reductase-like	109839366	1	0.000
SSR1131	(ATG)₅	Intron	TCTACGCAAACCATCCAATG	GCAGTCTGATAGGGGCAAAG	59.8	150	5	1864366	T复合体蛋白1亚基β T-complex protein 1 subunit beta	109841430	3	0.568
SSR1177	(TG)₁₀	Intron	TACAGACCCCTGTTTTTCGC	CCCTCAGCTTAACGTGCATT	60.3	234	5	14904240	ABC转运蛋白家族I成员6 ABC transporter I family member 6	109843163	1	0.000
SSR1224	(AAT)₈	Intron	ATAGCCTCGTCGCTTGAGAA	TCCAAGGGGAATATACGCAA	60.3	208	5	69242913	四肽α-吡喃酮还原酶1 Tetraketide alpha-pyrone reductase 1	109843750	4	0.691
SSR1324	(GCT)₅	CDS	ATTAGACACAAACCCCTGCG	AGAAGAGGGGAAGCAAAAGC	60.0	162	5	114882457	葡糖苷酶2亚基位β Glucosidase 2 subunit beta	109840787	1	0.000
SSR1386	(TTC)₁₀	CDS	CCTATCGCGTCCTATCCAAA	CGTGTAACGCGATTATGGTG	60.0	198	5	129216684	可能的丝氨酸/苏氨酸受体蛋白激酶At5g57670 Probable receptor-like serine/threonine-protein kinase At5g57670	109841319	3	0.185
SSR1412	(AACCCC)₄	CDS	CAACACCAAGCCCGTTATCT	TGGTGGAGAGAAGAGGAGGA	59.9	207	6	1910664	肽基-脯氨酰顺反异构酶FKBP17-1 Peptidyl-prolyl cis-trans isomerase FKBP17-1	109845585	1	0.000
SSR1463	(TCT)₅	CDS	TCATGGGCAATCCTCTTGTT	AGACTCCATGTCGGATGAGG	60.1	204	6	8996684	细胞分裂素羟化酶 Cytokinin hydroxylase-like	109845014	2	0.346
SSR1494	(TAA)₈	Intron	GGAAAGGAGGGGTAAAATGG	TTGTCCAAAATGTCATGCGT	60.0	270	6	18667772	丝氨酸/苏氨酸蛋白磷酸酶PP2A-1催化亚基 Serine/threonine-protein phosphatase PP2A-1 catalytic subunit-like	109845040	1	0.000
SSR1519	(GA)₁₂	5'-UTR	TGTTTCCTCTCCTTCTTTCACA	TTTTTCCTCGCCATCTTCAC	60.2	170	6	34974597	双元件响应调节器ORR24 Two-component response regulator ORR24-like	109845367	2	0.198
SSR1555	(CCT)₅	CDS	ACTCTTACGATGACACCGGC	ATCGGAGCTGAGGTTGTTGT	59.7	134	6	76122250	含NAC结构域的蛋白21/22 NAC domain-containing protein 21/22-like	109845418	3	0.494
SSR1715	(GAA)₆	CDS	AGCCGCAAGTTTCTAGGGTT	TGTATGTTCTTGAGGCTGCG	60.0	145	7	58010025	富含谷氨酸WD重复序列的蛋白1 Glutamate-rich WD repeat-containing protein 1	109849288	3	0.642
SSR1779	(TCT)₅	CDS	GACGTGCTTGGTGCTCTCTT	AACTGCTGCAATGATGCAAG	60.0	207	7	88705397	NEP1相互作用蛋白1 NEP1-interacting protein 1-like	109850734	3	0.593
SSR1802	(TG)₈	Intron	TGTCTGCTAAGACCTCTGCG	CAAAGTGGGGGCTTGAATAA	59.9	218	7	117477415	未知蛋白LOC109848868 Uncharacterized protein LOC109848868	109848868	2	0.198
SSR1819	(AAT)₅	Intron	TGTTTGAGCTTCGGGTTCTT	CGTTCCTGATGATTTTCGCT	60.2	224	7	129709783	非共生血红蛋白1 Non-symbiotic hemoglobin 1-like	109849734	2	0.444
SSR1902	(GAAAA)₄	Intron	TGTGTGACCAGAATGATACCAA	CAGCAAGATGTAATCGGGGT	60.0	170	7	149292304	角蛋白，II型细胞骨架1 Keratin, type II cytoskeletal 1-like	109847973	3	0.630
SSR1952	(GAT)₈	CDS	ACTCTTCAGATCAGGCCGAA	TCTCTCTGTCACCTCTGCGA	59.9	173	8	3856499	预测的未知蛋白YGR160 Putative uncharacterized protein YGR160	109819557	1	0.000
SSR2056	(CTT)₅	CDS	ACTTGACTTCCACATTGGGC	GATTCTGAGGCTGAGGCAAG	60.1	169	8	35842857	未知蛋白 LOC109819575 Uncharacterized protein LOC109819575	109819575	2	0.580
SSR2105	(CTC)₆	CDS	AGAGAACCCTCAGACGCAAA	CCACTCCATGTCCTCGATCT	60.1	174	8	88072165	双苯甲酰胺生物合成蛋白3 Diphthamide biosynthesis protein 3-like	109822197	1	0.000
SSR2113	(CT)₈	CDS	CTCAAATGGCCGTCTACCTC	GATGGATGGTGAATGGAACC	60.0	162	8	97036927	核转录因子Y亚基C-4-like Nuclear transcription factor Y subunit C-4-like	109822868	2	0.198
SSR2158	(AATA)₇	Intron	CCTGCTTGCTCACCTGAGAT	TCGCGACTGACAGAGATCAG	60.3	272	8	122287668	β-D-木糖苷酶1 Beta-D-xylosidase 1-like	109819692	2	0.198
SSR2219	(TA)₁₀	CDS	GTTAGCCTCCGGTCAATCAA	CGTAGCGCTCACCTGACATA	60.0	158	9	613886	SP蛋白 Protein SELF-PRUNING-like	109824684	1	0.000
SSR2259	(CAA)₅	CDS	CGATTTCTCATCACAGGGGT	ACACTCCACGCTTTCTCGTT	59.9	194	9	7569754	B-box锌指蛋白22 B-box zinc finger protein 22-like	109823141	2	0.198
SSR2297	(GAG)₇	CDS	AAAGCCAACACCACCAAGAG	AGATGGCTCCCAGTCAGTGT	59.7	150	9	40584132	氯通道蛋白CLC-a Chloride channel protein CLC-a-like	109823966	2	0.346
SSR2322	(CCG)₅	CDS	TTGGAGCTCACATCCACAGA	CTCTTCTCCCCCAGATGCTA	59.4	176	9	65579962	预测的酪蛋白激酶II亚基β-4 Putative casein kinase II subunit beta-4	109823813	3	0.494
SSR2325	(CCT)₅	CDS	CAAAGCTACCCTTCCCCTCT	TGGGGGAGACGGTGTAGTAG	60.0	194	9	66180515	lysM结构域受体样激酶3 LysM domain receptor-like kinase 3	109823957	1	0.000
SSR2365	(ATT)₆	5'-UTR	AATGCAGCCAAAGTTCATCC	CCGGGTCGGGTAACTCTATT	60.2	208	10	2729728	磷酸甘露糖长醇利用缺陷型基因1的同源物2 Mannose-P-dolichol utilization defect 1 protein homolog 2-like	109825639	1	0.000
SSR2398	(GAT)₅	CDS	TTGGGGTCTCAACAGAATCC	GGAAGGGCCTAATCTCCAAG	60.0	276	10	6915115	未知蛋白LOC109825889 Uncharacterized protein LOC109825889	109825889	2	0.444
SSR2463	(TTA)₅	CDS	AGTGAACGAAAGATGGCTGC	GAGCAGAGAGCGAGACCTGT	60.0	146	10	58522448	硒蛋白F Selenoprotein F	109826215	1	0.000
SSR2492	(GA)₁₁	Intron	CCCTGGAGACAAAAAGCAGA	CTTGGCTTTATCCTGCAAGC	59.8	140	10	70357333	细胞分裂素脱氢酶3 Cytokinin dehydrogenase 3-like	109826192	2	0.346
SSR2517	(A)₂₃	5'-UTR	GGAGTTAAAATTAGGCTCTGCG	TTTCCCCGCCTAAATTACCT	59.8	165	10	73126476	mRNA前体剪接因子SLU7 Pre-mRNA-splicing factor SLU7	109825241	3	0.494

Table 3

Fig. 8

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编号 No.	名称 Name	物种 Species	来源 Origin	编号 No.	名称 Name	物种 Species	来源 Origin
1	冠军 Guanjun	芦笋 Asparagus officinalis	中国潍坊 Weifang, China	13	广东天门冬3 GD-tmd3	天门冬 Asparagus setaceus	中国广东 Guangdong, China
2	翡翠明珠 Feicuimingzhu		中国潍坊 Weifang, China	14	江苏天门冬1 JS-tmd1		中国江苏 Jiangsu, China
3	京紫芦2号 Jiangzilunsun No.2		中国北京 Beijing, China	15	江苏天门冬2 JS-tmd2		中国江苏 Jiangsu, China
4	井岗111 Jinggang111		中国江西 Jiangxi, China	16	浙江天门冬1 ZJ-tmd1		中国浙江 Zhejiang, China
5	紫色激情 Purple passion		美国 USA	17	福建文竹1 FJ-wz1	文竹 Asparagus cochinchinensis	中国福建 Fujian, China
6	格兰德 Grande		美国 USA	18	河南文竹1 HN-wz1		中国河南 Henan, China
7	阿特拉斯 Atlas		美国 USA	19	山东文竹1 SD-wz1		中国山东 Shandong, China
8	王子 Prince		日本 Japan	20	山东文竹2 SD-wz2		中国山东 Shandong, China
9	荷兰1号 Holland tongfu No.1		荷兰 Holland	21	浙江文竹1 ZJ-wz1		中国浙江 Zhejiang, China
10	福建天门冬1 FJ-tmd1	天门冬 Asparagus setaceus	中国福建 Fujian, China	22	福建蓬莱松1 FJ-pls1	蓬莱松 Asparagus umbellatus	中国福建 Fujian, China
11	广东天门冬1 GD-tmd1		中国广东 Guangdong, China	23	广东蓬莱松1 GD-pls1		中国广东 Guangdong, China
12	广东天门冬2 GD-tmd2		中国广东 Guangdong, China	24	上海蓬莱松1 SH-pls1		中国上海 Shanghai, China

重复基序类型 Repeat motif type	重复次数 Repeat times									总计 Total	平均距离 Average distance (kb)	百分率 Percentage (%)
重复基序类型 Repeat motif type	4	5	6	7	8	9	10	10-20	>20	总计 Total	平均距离 Average distance (kb)	百分率 Percentage (%)
单核苷酸 Mono-	0	0	0	0	0	0	0	6220	739	6959	48.20	19.02
二核苷酸 Di-	0	0	3291	1834	1260	856	572	1762	370	9945	33.73	27.18
三核苷酸 Tri-	0	9086	3854	2050	1288	201	218	443	28	17168	19.54	46.92
四核苷酸 Tetra-	0	548	216	32	25	7	10	18	0	856	391.86	2.34
五核苷酸 Penta-	597	134	15	8	4	4	0	6	0	768	436.76	2.10
六核苷酸 Hexa-	765	69	25	16	7	7	0	5	0	894	375.20	2.44
总计 Total	1362	9837	7401	3940	2584	1075	800	8454	1137	36590	9.17	100
平均距离 Average distance (kb)	246.28	34.10	45.32	85.13	129.81	312.03	419.29	39.68	295.01	9.17
百分率 Percentage (%)	3.72	26.88	20.23	10.77	7.06	2.94	2.19	23.10	3.11	100

Development and Transferability of EST-SSR Markers Based on Transcriptome Data from Asparagus officinalis

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