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

Abstract

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

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.

Figures/Tables 8

Table 1

Fig. 1

Table 2

Fig. 2

Table 3

Fig. 3

Fig. 4

Table 4

Screened 16 pairs of primers with significant polymorphism"

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

Table 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

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

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