Analysis of SSR Loci and Development of Molecular Markers in Isatis indigotica Genome

doi:10.11924/j.issn.1000-6850.casb2025-0244

Abstract

Abstract:

In response to the lack of effective molecular markers for the identification of Isatis indigotica germplasm and the difficulty of distinguishing cultivated types within the species using existing methods, this study aimed to develop simple sequence repeat (SSR) molecular markers suitable for germplasm identification and genetic diversity analysis in I. indigotica. Based on whole-genome data of I. indigotica, SSR loci were screened using MISA software, and primers were designed with Primer3.0. Using three cultivated types of I. indigotica (comprising 18 individual plants) as experimental materials, the amplification stability, polymorphism, effectiveness, and variety identification ability of the primers were validated. Genetic diversity parameters were also calculated. The results showed that: (1) a total of 139,852 SSR loci were detected in the I. indigotica genome. The most abundant were mononucleotide SSR loci, accounting for approximately 72.51% of the total. The most frequent SSR motif, A/T, also exhibited the highest total number of repeats among all nucleotide SSR motifs, with a count of 100,205, accounting for 71.65% of all repeated motifs. (2) Eight pairs of primers that can stably amplify genes and have rich polymorphisms were screened out. Among them, primer SSRSL02 could be used to distinguish the I. indigotica germplasms tested in this study. (3) The eight primer pairs exhibited the average expected heterozygosity (He) of 0.65, the average Shannon's information index (I) of 1.34, and the average polymorphic information content (PIC) of 0.614, indicating a relatively high level of genetic diversity within the tested I. indigotica samples. In summary, the eight SSR molecular markers developed in this study can be used for the identification of I. indigotica germplasm resources, genetic diversity analysis, and variety breeding. In the future, the sample size of I. indigotica germplasm could be expanded, and DNA fingerprinting could be constructed by integrating phenotypic data and active ingredient content, thereby providing more comprehensive technical support for the precise evaluation of I. indigotica germplasm resources.

Key words: Isatis indigotica, SSR, molecular markers, polymorphism, genetic diversity, germplasm identification

SUN Tianqi, ZHOU Lei, XU Lingui, SUN Xiaobo, CHEN Yong, SUI Chun. Analysis of SSR Loci and Development of Molecular Markers in Isatis indigotica Genome[J]. Chinese Agricultural Science Bulletin, 2026, 42(2): 50-56.

Figures/Tables 5

References 29

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重复次数	SSR基序						总数	百分比/%
重复次数	单核苷酸	二核苷酸	三核苷酸	四核苷酸	五核苷酸	六核苷酸	总数	百分比/%
5	-	-	5092	257	77	103	5529	3.95
6	-	9243	1775	68	16	40	11142	7.97
7	-	5523	732	24	6	15	6300	4.50
8	-	3987	367	9	6	11	4380	3.13
9	-	2853	222	3	1	3	3082	2.20
10	36401	1843	138	3	2	2	38389	27.45
11	19862	1262	70	1	-	5	21200	15.16
12	12700	897	43	2	-	0	13642	9.75
>12	32528	3529	123	0	1	7	36188	25.87

重复次数	SSR基序						总数	百分比/%
重复次数	单核苷酸	二核苷酸	三核苷酸	四核苷酸	五核苷酸	六核苷酸	总数	百分比/%
5	-	-	5092	257	77	103	5529	3.95
6	-	9243	1775	68	16	40	11142	7.97
7	-	5523	732	24	6	15	6300	4.50
8	-	3987	367	9	6	11	4380	3.13
9	-	2853	222	3	1	3	3082	2.20
10	36401	1843	138	3	2	2	38389	27.45
11	19862	1262	70	1	-	5	21200	15.16
12	12700	897	43	2	-	0	13642	9.75
>12	32528	3529	123	0	1	7	36188	25.87

SSR基序	SSR数目	基序频率/%
A/T	100,205	71.65
C/G	1,286	0.92
AC/GT	1,972	1.41
AG/CT	11,355	8.12
AT/AT	15,803	11.30
AAC/GTT	709	0.52
AAG/CTT	4,484	3.21
AAT/ATT	963	0.69
ACC/GGT	384	0.27
AGC/CTG	207	0.15
AGG/CCT	541	0.39
ATC/ATG	880	0.63
CCG/CGG	194	0.14
AAAT/ATTT	145	0.10

SSR基序	SSR数目	基序频率/%
A/T	100,205	71.65
C/G	1,286	0.92
AC/GT	1,972	1.41
AG/CT	11,355	8.12
AT/AT	15,803	11.30
AAC/GTT	709	0.52
AAG/CTT	4,484	3.21
AAT/ATT	963	0.69
ACC/GGT	384	0.27
AGC/CTG	207	0.15
AGG/CCT	541	0.39
ATC/ATG	880	0.63
CCG/CGG	194	0.14
AAAT/ATTT	145	0.10

引物	预期产物长度	引物序列
SSRSL01	223	F: TCGTCTGCCCTTATGCCTCTCA
SSRSL01	223	R: AGTCGCTGAGATGGGAGGTTCT
SSRSL02	186	F: TGTTGCCTCCACGTCATGATAA
SSRSL02	186	R: ACCAGTCGTACATGCGCCTAAG
SSRSL03	176	F: TCGTTCGGTTATGACGGCTCTT
SSRSL03	176	R: CGTAAGGTCCAATGGCGAATAT
SSRSL04	206	F: TCCGCACGAGAGAATGGCTAAT
SSRSL04	206	R: TCCTGACCGTCCATTCGAATTC
SSRSL05	192	F: ACTCTCAGGGCAGCGACAGAAA
SSRSL05	192	R: TCTCCCACCACCACCACAAATA
SSRSL06	253	F: CAAACCACCACCGGACCACTAT
SSRSL06	253	R: GCCTCTCCATCCTCGTCGTATT
SSRSL07	212	F: TGGAGCAAGAAGAGAGGTTAGG
SSRSL07	212	R: TTTGAAGCTCTGCAGGGAAAGT
SSRSL08	191	F: ACACACACAATTATCCGCATCT
SSRSL08	191	R: AGTGACCGACGATGGAGAATAA