New DNA Markers Distinguish Desert Poplar Hybrids from Their Parent Species

Researchers have developed and validated a set of species-specific DNA markers that can reliably distinguish Populus euphratica from Populus pruinosa and identify putative hybrids between the two desert poplars, according to a study published in BMC Genomic Data. The work, led by Ya Li, Shanhe Zhang and colleagues with senior author Zhongshuai Gai, addresses a long-standing gap in the molecular toolkit available for desert riparian forest conservation in northwest China, where these two tree species form the dominant vegetation along fragile arid waterways.

Populus euphratica and Populus pruinosa both belong to section Turanga of the genus Populus and serve as core germplasm resources for desert riparian forests. The two species occupy overlapping ranges in arid northwest China, where natural interspecific gene flow occurs and morphologically intermediate individuals appear in contact zones. These intermediate trees combine traits of both parents: P. euphratica tolerates drought, salinity and wind erosion but grows slowly with often curved trunks, while P. pruinosa grows rapidly with straight stems but has weaker resistance to abiotic stress. Individuals carrying complementary traits from both species are of interest for stress-tolerant poplar breeding, which makes accurate hybrid identification an essential prerequisite for crossbreeding programs and germplasm purification.

The problem, as the authors describe it, is that morphological identification of hybrids is unreliable. Intermediate individuals collected from natural contact zones show highly overlapping phenotypic traits with both parental species under field conditions, making visual discrimination extremely difficult. Traditional approaches based on phenotypic observation and isozyme profiling are heavily affected by environmental conditions and offer low detection efficiency and accuracy. Conventional DNA markers carry their own technical defects: dominant markers such as RAPD and AFLP suffer poor amplification repeatability and cannot distinguish homozygous from heterozygous genotypes, while co-dominant SSR markers produce complex banding patterns and few loci show strict species-specific alleles between closely related poplar species.

Insertion/deletion (InDel) markers, by contrast, offer co-dominant inheritance, stable PCR amplification and intuitive fragment length polymorphisms, making them low-cost tools for identifying hybrids of closely related woody plants. However, publicly available and standardized genome-wide InDel marker resources specifically designed for Turanga desert poplars remained scarce. Most previously reported poplar InDel markers were developed from small numbers of resequenced individuals or transcriptome data, lacking population-level verification, and many showed intraspecies polymorphism when extended to natural wild populations. Previous phylogenomic studies had confirmed that Turanga species form an independent ancient evolutionary clade with genetic backgrounds distinct from other poplar groups, so markers developed for other sections could not simply be transferred.

To build the new resource, the team drew on large-scale whole-genome resequencing data from 439 P. euphratica and 146 P. pruinosa individuals. Clean paired-end reads were mapped to a chromosome-level reference genome assembly of P. euphratica using the Burrows-Wheeler Aligner, with SAMtools used to convert alignment files and remove PCR duplicates, and genome-wide SNP and InDel variants detected with GATK. Custom Python scripts then filtered candidate species-diagnostic InDel loci against unified thresholds: an intraspecies conserved allele frequency of at least 95 percent within each species, an interspecific fragment length difference exceeding 5 base pairs, and localization in non-repetitive genomic regions.

The screening identified 56,864 interspecific InDel loci with fragment length differences over 5 bp, of which 3,417 loci showed interspecific allele frequency differences greater than 0.9 and met the strict criteria for high-confidence candidate sites. Primer pairs were designed with Primer3 v2.3.7 using unified parameters: GC content between 20 and 46 percent, optimal annealing temperatures of 45 to 55 degrees Celsius, and predicted amplification fragments of 85 to 120 base pairs. All designed primer sequences were aligned against both species’ reference genomes using SeqHunter2 software, and primers achieving a genome matching rate of at least 95 percent with consistent single amplification targets in both species were defined as universal candidates. This pipeline yielded 11 universal InDel primer pairs with stable single-locus amplification.

Laboratory validation proceeded in two stages. In the first stage, genomic DNA from three randomly selected individuals of each species was amplified with all 11 candidate primer pairs and separated on 8 percent native polyacrylamide gels stained with silver nitrate. Only primers P1599-3 and P1599-4 produced clear single bands in both species with consistent and significant interspecific fragment length differences; the remaining nine primer pairs showed ambiguous bands or fragment gaps too small to serve as diagnostic markers. In the second stage, all 50 P. euphratica and 41 P. pruinosa natural individuals were genotyped with the two candidate primers. Both markers displayed complete intraspecies conservation, with zero intraspecific fragment length polymorphism and no non-specific amplification products detected across the 91 parental samples.

The banding patterns were unambiguous. For primer P1599-3, a stable 90 bp fragment appeared in every P. euphratica sample while a fixed 79 bp band appeared in all P. pruinosa individuals. For primer P1599-4, a 100 bp fragment was specific to P. euphratica and a 92 bp fragment was exclusive to P. pruinosa. Because InDel markers are co-dominant, a true interspecific hybrid should amplify both parental bands simultaneously at each locus. The researchers tested this expectation on 93 morphologically selected putative-hybrid individuals collected from natural contact zones across Xinjiang and adjacent Central Asian distribution zones, samples that had been characterized in a previously published morphological survey of Turanga poplars serving as independent pre-molecular screening evidence.

In an initial test of twelve P. euphratica, twelve P. pruinosa and twelve putative hybrid individuals sampled across Xinjiang, primer P1599-3 amplified only the 90 bp band in P. euphratica, only the 79 bp band in P. pruinosa, and both characteristic bands in all 12 putative hybrids. Primer P1599-4 behaved identically with its 100 bp and 92 bp products. Further testing of the remaining 81 candidates showed that all morphologically selected individuals generated dual species-specific diagnostic bands consistent with a heterozygous genotype combining alleles inherited from each parent. Within the 93 putative hybrids validated in the study, both primer pairs successfully discriminated the interspecific hybrids, and the authors note that the combined use of the two primers allows mutual verification, improving the reliability and stability of identification for their tested materials.

The authors frame the two verified co-dominant markers as reusable molecular resources for preliminary germplasm authentication and screening of putative hybrids within Populus sect. Turanga, with the full set of 11 candidate primers supplying alternative loci for follow-up population genetic analysis. They also present the workflow itself—large-population resequencing variation filtering, primer design, and multi-stage PAGE population validation—as a repeatable technical reference that can be transferred to other intercrossable woody species with weak interspecific genetic differentiation, supporting marker-assisted breeding and germplasm conservation of arid poplars. The study does include acknowledged limitations: the experimental samples came mainly from Xinjiang and Central Asia, so broader geographical sampling is needed to confirm universal applicability, and the two core diagnostic markers map only to chromosomes 04 and 06, an uneven genomic distribution that may restrict larger-scale genetic analysis. The authors state that future work will mine additional species-diagnostic InDel loci distributed evenly across all chromosomes. Supporting data, including resequencing reads deposited under NCBI BioProject accessions PRJNA1427129 and PRJNA865525, plus primer sequences, the genotype matrix for all 184 experimental individuals and original gel imaging files, are designated for permanent open-access release.

Subject of Research: Development and validation of species-specific InDel markers for identifying putative hybrids between two desert poplar species

Article Title: Population-resourced species-specific InDel markers for identifying putative interspecific hybrids between Populus euphratica and Populus pruinosa

Article References: Li, Y., Zhang, S., Ma, W., Long, X., Chen, Y., & Gai, Z. (2026). Population-resourced species-specific InDel markers for identifying putative interspecific hybrids between Populus euphratica and Populus pruinosa. BMC Genomic Data, 27(1), Article 78. https://doi.org/10.1186/s12863-026-01486-8

Image Credits: AI Generated

DOI: 10.1186/s12863-026-01486-8

Keywords: Populus euphratica, Populus pruinosa, InDel markers, interspecific hybrids, desert riparian forests, whole-genome resequencing, molecular markers, germplasm authentication, native-PAGE, Xinjiang, marker-assisted selection, poplar breeding