To clarify the basic characteristics of single-copy nuclear gene sequences in Littledalea racemosa and obtain effective single-copy nuclear gene molecular markers， the single-copy homologous genes were identified using OrthorFinder software based on third-generation full-length transcriptome data of L. racemosa in this study. And then through PCR amplification and population testing experiments， 10 pairs of single-copy nuclear gene molecular marker primers for L. racemosa were successfully developed. The results showed that a total of 48 219 CDS （coding sequence） regions were detected in the L. racemosa transcriptome， with an average of 1.57 coding regions per transcript. A total of 301 single-copy nuclear gene sequences were obtained based on the amino acid sequences of transcriptome CDS and finally， 10 pairs of single-copy nuclear gene primers were obtained， which demonstrated effective amplification performance and exhibited intraspecific variation. A total of 64 individuals of L. racemosa were successfully amplified through all 10 pairs of single-copy nuclear genes primers. The gene sequences exhibited extensive variation in polymorphism. The number of segregating sites （S） ranged from 13 to 42， with an average of 24.4. The number of haplotypes （h） ranged from 16 to 32， with an average of 23.7. The average number of nucleotide differences （k） ranged from 2.275 to 16.497， with an average of 7.686. The haplotype diversity （H_d） ranged from 0.776 to 0.896， with an average of 0.841. The nucleotide diversity （π） ranged from 3.05×10^-3 to 15.61×10^-3， with an average of 10.50×10^-3， and the Watterson’s estimator （θw） ranged from 3.27×10^-3 to 9.92×10^-3， with an average of 6.21×10^-3. The genetic differentiation results showed that the genetic variation of 15 populations of L. racemosa mainly existed within populations. Phylogenetic trees constructed using the 10 single-copy nuclear genes demonstrated congruent relationships among five Poaceae species. The 10 pairs of L. racemosa single-copy nuclear gene primers developed in this study， providing abundant candidate markers for future research on phylogeny， and resource conservation and utilization for Littledalea species.

Littledalea racemosa  /  Transcriptome  /  Single-copy nuclear genes  /  Phylogeny