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

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.

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.

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.

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.