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MS-DNA(Microsatellite DNA) is widely used in gene mapping, clinical medicine, paternity testing, anthropology, population genetics, and species identification. In this study, we examined the variation and distribution of MS-DNA in the mitochondrial genome (mtDNA) and chloroplast genome (cpDNA) of cotton to develop more valuable MS-DNA molecular markers. Based on bioinformatics techniques, we selected full sequences of mtDNA and cpDNA from eight cotton species and utilized Perl language and open-source code (MISA) to create a tool for identifying and localizing MSDNA sequences for analysis. The results revealed that the lengths of mtDNA among the eight cotton species ranged from 644,311bp to 687,482bp, while their cpDNA lengths were between 160,072bp and 160,317bp, indicating that the length of mtDNA was approximately four times larger than that of cpDNA. The frequency of MS-DNA was in cpDNA higher than mtDNA. Regarding distribution characteristics, MS-DNA in the cotton cytoplasmic genome were mainly located in intergenic regions, with AT content exceeding CG content. Compared to the chloroplast genome, the mitochondrial genome contained more repetitive sequence regions, resulting in higher richness of MS-DNA. In terms of types, the microsatellite categories showed a higher prevalence of P4 type repeats over P3 type, with P5 and P6 repeats exhibiting specificity. Additionally, a total of 31 types (61 instances) of compound MS-DNA were detected across the eight cotton mtDNAs, while 18 types (32 instances) were found in cpDNAs. These loci can serve as candidate sites, providing assistance for the subsequent development of specific molecular markers.
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