Population-resourced Species-Specific InDel Markers for Identifying Putative Interspecific Hybrids Between Populus Euphratica and Populus Pruinosa

Populus euphratica and Populus pruinosa represent core germplasm resources of desert riparian forests in northwest China. Accurate discrimination of putative interspecific hybrids between the two species is fundamental to population genetic research of desert poplars. Currently, publicly available and standardized genome-wide InDel marker resources specifically for desert poplars remain scarce. Traditional morphological identification exhibits low accuracy and reliability, and conventional markers (RAPD, AFLP, SSR) have limited repeatability and species specificity, failing to support standardized genotyping. Based on large-scale whole-genome resequencing data, this study developed and validated a set of highly specific and stable InDel markers to provide valuable genomic resources for subsequent genetic research on desert poplars. A total of 56,864 interspecific InDel loci with fragment length differences over 5 bp were screened from 439 P. euphratica and 146 P. pruinosa resequencing accessions, among which 3,417 loci had interspecific allele frequency differences > 0.9. After primer design and native polyacrylamide gel electrophoresis (native-PAGE) validation, 11 universal InDel primer pairs were obtained, and two robust diagnostic markers (P1599-3, P1599-4) were finally verified. Population validation involving 50 P. euphratica, 41 P. pruinosa and 93 morphologically selected putative-hybrid individuals revealed that this dual-marker set could reliably detect biparental banding patterns among tested candidates. The two verified co-dominant InDel markers provide reusable molecular resources for preliminary germplasm authentication and screening of putative hybrids within Populus sect. Turanga. The standardized screening and PAGE genotyping pipeline provided in this study can be transferred to other woody hybrid species, supporting marker-assisted breeding and germplasm conservation of arid poplars.