Abstract
BackgroundThis study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.ResultsUsing bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.ConclusionsA set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.
Highlights
This study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars
We successfully developed a set of single-copy excellent SSR primers that combines high efficiency and stability, easy-to-read peak shapes, multiple allelic variations, high polymorphism information content (PIC) values, and uniform chromosome distribution
Analyzing the number of alleles of each SSR primer sequence in 48 cotton germplasm lines, we found that the more alleles, the stronger ability of the primers to identify different varieties
Summary
This study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials. Its fiber is a renewable textile raw material, and its seeds can be used for the production and processing of cottonseed oil. It is difficult to meet the needs of identification, standardization, and large-scale detection of cotton varieties currently planted in China
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