蔗茅全基因组SSR标记特征分析与开发

doi:10.3864/j.issn.0578-1752.2025.05.003

中国农业科学 ›› 2025, Vol. 58 ›› Issue (5): 851-863.doi: 10.3864/j.issn.0578-1752.2025.05.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

蔗茅全基因组SSR标记特征分析与开发

罗正英¹(), 胡鑫²^,³(), 吴转娣²^,³, 钱禛锋¹, 田春艳²^,³, 刘新龙²^,³(), 李富生¹()

¹ 云南农业大学农学与生物技术学院/云南省作物生产与智慧农业重点实验室，昆明 650201
² 云南省农业科学院甘蔗研究所/云南甘蔗遗传改良重点实验室/农业农村部甘蔗生物学与遗传育种重点实验室（云南），云南开远 661699
³ 云南省农业科学院热带作物生物育种全国重点实验室，昆明 650205

收稿日期:2024-08-04 接受日期:2024-09-27 出版日期:2025-03-07 发布日期:2025-03-07
通信作者:
刘新龙，E-mail：lxlgood868@163.com。
李富生，E-mail：lfs810@sina.com
联系方式: 罗正英，E-mail：zhengyluo@163.com。胡鑫，E-mail：sugarhuxin@163.com。罗正英和胡鑫为同等贡献作者。
基金资助:
云南省农业科学院科研项目(2024KYZX-03); 云南省种子种业联合实验室项目(202205AR070001-09)

Genome-Wide Survey and Development of Novel SSR Markers in Erianthus fulvus

LUO ZhengYing¹(), HU Xin²^,³(), WU ZhuanDi²^,³, QIAN ZhenFeng¹, TIAN ChunYan²^,³, LIU XinLong²^,³(), LI FuSheng¹()

¹ College of Agronomy and Biotechnology, Yunnan Agricultural University/The Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province, Kunming 650201
² Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences/Yunnan Key Laboratory of Sugarcane Genetic Improvement/Key Laboratory of Sugarcane Biology and Genetic Breeding (Yunnan), Ministry of Agriculture and Rural Affairs, Kaiyuan 661699, Yunnan
³ National Key Laboratory for Biological Breeding of Tropical Crops, Yunnan Academy of Agricultural Sciences, Kunming 650205

Received:2024-08-04 Accepted:2024-09-27 Published:2025-03-07 Online:2025-03-07

摘要/Abstract

摘要：

【目的】蔗茅（Erianthus fulvus）是甘蔗重要的野生资源，可用于改良品种的抗逆性和产量。系统鉴定和开发蔗茅基因组中的简单重复序列（simple sequence repeat，SSR）位点，筛选可利用的多态性SSR标记，解析蔗茅资源的遗传多样性特征，开发重要性状关联分子标记，对于利用蔗茅基因资源改良品种性状具有重要意义。【方法】采用生物信息学软件TBtools中的SSRminer模块，对二倍体蔗茅全基因组序列进行系统性SSR位点挖掘，并对所得数据进行统计分析，揭示其在基因组中的分布模式及规律。利用Batch Target Region Primer Design功能批量设计SSR引物，并通过Primer check工具评估引物的特异性。在6个蔗茅种质中，通过对随机合成的50对SSR引物和14对来自甘蔗的SSR引物进行扩增效率和多态性比较分析，验证引物多态性。【结果】共鉴定出152 707个蔗茅SSR位点，平均检测频率为5.64 kb/个，大部分分布在基因间区。SSR类型分布以单碱基、二碱基、三碱基为主。二核苷酸SSR类型中的基序重复次数变化最大，而五核苷酸的基序重复次数变异最低。共检测出883种不同的SSR基序重复类型，其中，A/T和AT/TA是最为丰富。共设计144 692对SSR引物，其中85 025对显示高特异性。这些特异性引物在基因组上呈现出两端密集、中间稀疏的分布特点。扩增试验显示，50对随机合成的SSR引物中，有42对在蔗茅中扩增出稳定清晰的条带，其中32对显示出多态性，多态率为64.0%。与甘蔗SSR引物相比，蔗茅SSR引物表现出更高的扩增效率和更好的多态性水平。经过筛选，从32对有效的蔗茅SSR引物中确定了16对多态性好且扩增条带清晰的引物。这16对引物共扩增出72条条带，多态性信息量（polymorphism information content，PIC）范围为0.63—0.83，平均PIC值为0.74，表明它们在蔗茅种质资源的多态性分析和分子标记研究中具有有效性和实用性。【结论】揭示了蔗茅基因组中高丰度和多样性的SSR分布特征。获得16对扩增效率高、多态性良好的SSR引物。

关键词: 蔗茅, 基因组, 简单序列重复, 特异扩增, 多态性分析

Abstract:

【Objective】 Erianthus fulvus, serving as a crucial wild resource for sugarcane, is capable of enhancing the stress tolerance and yield of varieties. In order to utilize E. fulvus for sugarcane breeding, it is important to systematically identify and develop simple sequence repeat (SSR) loci in the E. fulvus genome, screen for polymorphic SSR markers, analyse the genetic diversity characteristics of E. fulvus resources and then develop SSR markers associated with important traits. 【Method】Using the SSRminer module in the software TBtools, a comprehensive exploration of SSR loci was conducted on the diploid E. fulvus whole genome sequence. The obtained data were statistically analyzed to reveal their distribution patterns and regularities within the genome. The Batch Target Region Primer Design function was employed for batch designing SSR primers, and the specificity of the primers was evaluated using the Primer check tool. To comparethe SSR polymorphism betweenE. fulvus and sugarcane, amplification experiments were performed on 50 pairs of randomly synthesized SSR primers and 14 pairs of SSR primers sourced from sugarcane across 6 E. fulvus germplasms. 【Result】A total of 152 707 SSR loci, which were distributed on E. fulvus genome with an average density of 5.64 kb/locus, were identified. The majority were located in intergenic regions. In terms of SSR type distribution, mononucleotide, dinucleotide, and trinucleotide had the highest density. Dinucleotide SSR types exhibited the greatest variation in motif repeat numbers, while pentanucleotide motif repeat variations were the least. Across the entire genome, 883 distinct SSR motif repeat types were identified, with A/T and AT/TA being the most abundant. A total of 144 692 pairs of SSR primers, of which 85 025 pairs exhibited high specificity, were designed. These specific primers displayed a distribution characteristic of dense ends and sparse middles on the genome. Amplification experiments showed that 42 out of the 50 randomly synthesized SSR primer pairs yielded stable and clear bands in E. fulvus, with 32 exhibiting polymorphisms, yielding a polymorphism rate of 64.0%. In contrast to the 14 sugarcane SSR primers, the E. fulvus SSR primers demonstrated superior amplification efficacy and greater polymorphism. After screening, 16 pairs of SSR primers with good polymorphism and clear amplification bands were determined from the 32 effective SSR primer pairs. These 16 pairs of primers amplified a total of 72 bands, with polymorphism information content (PIC) ranging from 0.63 to 0.83, and an average PIC value of 0.74, indicating their effectiveness and practicality in polymorphism analysis and molecular marker research of E. fulvus germplasm resources. 【Conclusion】This study comprehensively identified SSR loci in the E. fulvus genome, revealing the high abundance and diversity of SSR distribution features. Sixteen pairs of highly specific and polymorphic SSR primers were successfully screened.

Key words: Erianthus fulvus, genome, simple sequence repeat, specific amplification, polymorphic analysis

罗正英, 胡鑫, 吴转娣, 钱禛锋, 田春艳, 刘新龙, 李富生. 蔗茅全基因组SSR标记特征分析与开发[J]. 中国农业科学, 2025, 58(5): 851-863.

LUO ZhengYing, HU Xin, WU ZhuanDi, QIAN ZhenFeng, TIAN ChunYan, LIU XinLong, LI FuSheng. Genome-Wide Survey and Development of Novel SSR Markers in Erianthus fulvus[J]. Scientia Agricultura Sinica, 2025, 58(5): 851-863.

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表4

筛选出的蔗茅SSR引物及多态性信息"

序号 No.	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	退火温度 Tm (℃)	预期产物大小Product size (bp)	总条带数 Totol bands	多态性条带数 Polymorphic bands	多态性比例 Percentage of polymorphic bands (%)	多态性信息 PIC
C2	Erufi.01G0016800_1231_1267_1	F: GCGTGGGTCATACAATCAGC	55	260	4	4	100	0.69
C2	Erufi.01G0016800_1231_1267_1	R: TCACCGTGGTCCAGTAAAACA	55	260	4	4	100	0.69
C3	Erufi.01G0040420_4442_4492_1	F: GTGGCGGACCCAGGATTT	55	275	3	3	100	0.63
C3	Erufi.01G0040420_4442_4492_1	R: TGCAACAAATCATCACGGCT	55	275	3	3	100	0.63
C5	Erufi.01G0048750_226_258_1	F: CGCATCGAGCCATGGGAT	55	245	3	3	100	0.63
C5	Erufi.01G0048750_226_258_1	R: TCCTCGTACCCGATCCCG	55	245	3	3	100	0.63
C7	Erufi.02G0005560_1571_1631_1	F: AGCCTACCCCAACTTGCT	55	252	4	4	100	0.9
C7	Erufi.02G0005560_1571_1631_1	R: GGCCTGATTTTGGGTCTGGA	55	252	4	4	100	0.9
C9	Erufi.02G0022700_1697_1727_1	F: GGAGGGCATCACATCATTGC	55	224	6	6	100	0.80
C9	Erufi.02G0022700_1697_1727_1	R: CCTGGGCTCTTTCGGTGG	55	224	6	6	100	0.80
C11	Erufi.02G0041280_2595_2621_1	F: GGCCAGGATGATCCAGCC	55	245	7	7	100	0.81
C11	Erufi.02G0041280_2595_2621_1	R: TCCAGCATATGACGCCCAC	55	245	7	7	100	0.81
C12	Erufi.03G0008120_4062_4088_1	F: AAGGAAGCGAACGAGGCC	55	254	4	4	100	0.66
C12	Erufi.03G0008120_4062_4088_1	R: TGGAAGTGGTCGTCGCAA	55	254	4	4	100	0.66
C14	Erufi.03G0018800_963_997_1	F: GGAATGCCCTGGTTGGATT	55	221	3	3	100	0.72
C14	Erufi.03G0018800_963_997_1	R: TCACGTTGACAGTGATCAAGT	55	221	3	3	100	0.72
C18	Erufi.04G0000530_5476_5544_1	F: AGCCCCGGTAGTCACACT	55	235	4	4	100	0.80
C18	Erufi.04G0000530_5476_5544_1	R: GCATCAGCCAAACTGCCA	55	235	4	4	100	0.80
C28	Erufi.06G0007490_1977_2023_1	F: CCAAGGCCATCGCACTGA	55	221	6	6	100	0.82
C28	Erufi.06G0007490_1977_2023_1	R: CGAAGCTCTCCACCACGG	55	221	6	6	100	0.82
C30	Erufi.06G0016290_955_987_1	F: AATTTAGCAGCCGCCCCA	55	246	3	3	100	0.73
C30	Erufi.06G0016290_955_987_1	R: ACCGTGGTACGATCCCCA	55	246	3	3	100	0.73
C31	Erufi.06G0025250_3167_3207_1	F: ACCTGCAGATGAGAAGTCTCT	55	262	3	3	100	0.72
C31	Erufi.06G0025250_3167_3207_1	R: ACAGCGCACACAACAGGA	55	262	3	3	100	0.72
C34	Erufi.07G0007060_723_755_1	F: GCCGCTGTGATCTCGTCA	55	190	7	7	100	0.77
C34	Erufi.07G0007060_723_755_1	R: ACTGCCCTGACCTCCCAA	55	190	7	7	100	0.77
C39	Erufi.08G0009320_21599_21623_1	F: TGTGTTGCAGGAAACAAGTCA	55	277	5	5	100	0.74
C39	Erufi.08G0009320_21599_21623_1	R: ACCATGTTCCAGACAGTCAGT	55	277	5	5	100	0.74
C40	Erufi.08G0016710_156_210_1	F: GAGAAGGGGAGCGATGGC	55	260	6	6	100	0.77
C40	Erufi.08G0016710_156_210_1	R: CGCACCTGTACCGGATCC	55	260	6	6	100	0.77
C49	Erufi.10G0023270_6715_6749_1	F: TGGGGTTCACTGGTGCAT	55	263	4	2	50	0.63
C49	Erufi.10G0023270_6715_6749_1	R: GCACAATGCACCTGGTAACA	55	263	4	2	50	0.63
B1	mSSCIR1	F: CTTGTGGATTGGATTGGAT	55	130	5	5	100	0.73
B1	mSSCIR1	R: AGGAAATGGATTGCTCAGG	55	130	5	5	100	0.73
B2	mSSCIR26	F: AAAATCAGACAAACAGCAT	55	115	3	2	66.7	0.29
B2	mSSCIR26	R: AGAAGAAGCAGATACAGGT	55	115	3	2	66.7	0.29
B3	mSSCIR53	F: TGGTCTACTGAAGTTCGTG	52	197	1	1	100	0
B3	mSSCIR53	R: TGCTTCTAAGTCAACCAAA	52	197	1	1	100	0
B4	mSSCIR66	F: AGGTGATTTAGCAGCATA	55	120	1	1	100	0
B4	mSSCIR66	R: CACAAATAAACCCAATGA	55	120	1	1	100	0
B5	SMC21SA	F: CGTGAGCTTGGGTAGCTG	50	120	4	4	100	0.7
B5	SMC21SA	R: AAACATTCCCCATTGCTATC	50	120	4	4	100	0.7
B6	SMC119CG	F: TTCATCTCTAGCCTACCCCAA	55	106	0	0	/	/
B6	SMC119CG	R: AGCAGCCATTTACCCAGGA	55	106	0	0	/	/
B7	SMC226	F: GAGGCTCAGAAGCTGGCAT	50	136	1	1	100	0
B7	SMC226	R: ACCCTCTATTTCCGAGTTGGT	50	136	1	1	100	0
B8	SMC336BS	F: ATTCTAGTGCCAATCCATCTCA	50	190	6	6	100	0.76
B8	SMC336BS	R: CATGCCAACTTCCAAACAGAC	50	190	6	6	100	0.76
B9	SMC477	F: CCAACAACGAATTGTGCATGT	55	168	0	0	/	/
B9	SMC477	R: CCTGGTTGGCTACCTGTCTTCA	55	168	0	0	/	/
B10	SMC720BS	F: CGCACCGACGCACGTCT	55	100	1	0	0	0
B10	SMC720BS	R: GCCAATGGAACGGGTCTA	55	100	1	0	0	0
B11	SMC863	F: CGGTCGCTGTTGCATTGTAG	55	296	0	0	/	/
B11	SMC863	R: TGGATCACTCAATCTCACTTCG	55	296	0	0	/	/
B12	SMC1490CL	F: AGCGATGGGTGCTGACAT	55	140	0	0	/	/
B12	SMC1490CL	R: CAGGTTGCGTCTTCCAGCT	55	140	0	0	/	/
B13	So10H(CT)19	F: GACCGTGTTACTGCATGGTG	55	107	1	0	0	0
B13	So10H(CT)19	R: CAAGAAGCGGGAATTTGTCT	55	107	1	0	0	0
B14	SoChr01B(CA)7	F: CTTCCACTCTCCCCTCCTCT	55	163	4	3	75	0.74
B14	SoChr01B(CA)7	R: AGGGTGGTGCTACAACTTGG	55	163	4	3	75	0.74

表4

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编号 No.	种质名称 Name	株高 Plant height (cm)	有效茎 <BOLD>P</BOLD>roductive stem	茎径 Stalk diameter (cm)	采集地 Origin
1	贵州2020-41 Guizhou 2020-41	32	55	0.41	贵州省兴仁市 Xingren, Guizhou
2	贵州2020-83 Guizhou 2020-83	30	59	0.35	贵州省普安县 Puan, Guizhou
3	贵州2012-171 Guizhou 2012-171	52	95	0.55	贵州省平坝区 Pingba, Guizhou
4	四川2013-14 Sichuan 2013-14	30	110	0.34	四川省盐源县 Yanyuan, Sichuan
5	四川2013-18 Sichuan 2013-18	26	84	0.35	四川省得荣县 Derong, Sichuan
6	云南2009-39 Yunnan 2009-39	41	129	0.43	云南省香格里拉市 Shangrila, Yunnan

染色体 Chr．	单核苷酸 Mononucleotide	二核苷酸 Dinucleotide	三核苷酸 Trinucleotide	四核苷酸 Tetranucleotide	五核苷酸 Pentanucleotide	六核苷酸 Hexanucleotide	合计 Total	染色体长度 Chromosome length (Mb)	频率（kb/个） Frequency
Chr.01	12102	5225	3879	275	138	35	21654	117.958	5.447
Chr.02	10355	4625	3722	287	100	33	19122	101.827	5.325
Chr.03	9666	4304	3069	238	83	34	17394	94.001	5.404
Chr.04	8400	3764	2899	214	71	23	15371	85.168	5.541
Chr.05	8163	4540	3503	293	105	27	16631	101.333	6.093
Chr.06	6641	3215	2338	164	77	28	12463	77.063	6.183
Chr.07	6547	3386	2454	280	69	21	12757	72.906	5.715
Chr.08	5755	3169	2187	147	73	19	11350	62.755	5.529
Chr.09	6949	3199	2385	151	81	21	12786	73.208	5.726
Chr.10	7186	3247	2437	214	72	23	13179	72.480	5.500
合计Total	81764	38674	28873	2263	869	264	152707	858.700	5.646
比例Ratio (%)	53.54	25.33	18.91	1.48	0.17	0.57	100

SSR重复单元 SSR repeat unit	重复基序类型数 No. of repeat motif types	重复基序类型 Repeat motif types	重复基序数量 No. of repeat motifs	占本类型比例 Self scale (%)	总体比例 Overall scale (%)
单核苷酸 Mononucleotide	4	A/T C/G	72070 9694	88.14 11.85	47.19 6.35
二核苷酸 Dinucleotide	12	AT/TA AG/CT GA/TC 其他Others	15555 7625 6039 9545	40.22 19.72 15.62 24.68	10.19 4.99 3.95 6.25
三核苷酸 Trinucleotide	60	AAG/CTT GGC/GCC CGC/GCG GAG/CTC 其他Others	1939 1811 1791 1622 21710	6.72 6.27 6.20 5.62 75.19	1.27 1.19 1.17 1.06 14.22
四核苷酸 Tetranucleotide	213	TTAA/AATA AAAT/ATTT TATT/TTTA 其他Others	171 146 121 1825	7.56 6.45 5.35 80.65	0.11 0.10 0.08 1.20
五核苷酸 Pentanucleotide	179	AGGGA/TCCCT Others	14 250	5.30 94.70	0.01 0.16
六核苷酸 Hexanucleotide	415	TATAGA/TCTATA ATCTAT/ATAGAT 其他Others	41 40 788	4.72 4.60 90.68	0.03 0.03 0.52
合计Total	883		152707		100

蔗茅全基因组SSR标记特征分析与开发

Genome-Wide Survey and Development of Novel SSR Markers in Erianthus fulvus

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