Table_2_RNA-seq analysis revealed considerable genetic diversity and enabled the development of specific KASP markers for Psathyrostachys huashanica.xls

Psathyrostachys huashanica, which grows exclusively in Huashan, China, is an important wild relative of common wheat that has many desirable traits relevant for wheat breeding. However, the poorly characterized interspecific phylogeny and genomic variations and the relative lack of species-specific molecular markers have limited the utility of P. huashanica as a genetic resource for enhancing wheat germplasm. In this study, we sequenced the P. huashanica transcriptome, resulting in 50,337,570 clean reads that were assembled into 65,617 unigenes, of which 38,428 (58.56%) matched at least one sequence in public databases. The phylogenetic analysis of P. huashanica, Triticeae species, and Poaceae species was conducted using 68 putative orthologous gene clusters. The data revealed the distant evolutionary relationship between P. huashanica and common wheat as well as the substantial diversity between the P. huashanica genome and the wheat D genome. By comparing the transcriptomes of P. huashanica and Chinese Spring, 750,759 candidate SNPs between P. huashanica Ns genes and their common wheat orthologs were identified. Among the 90 SNPs in the exon regions with different functional annotations, 58 (64.4%) were validated as Ns genome-specific SNPs in the common wheat background by KASP genotyping assays. Marker validation analyses indicated that six specific markers can discriminate between P. huashanica and the other wheat-related species. In addition, five markers are unique to P. huashanica, P. juncea, and Leymus species, which carry the Ns genome. The Ns genome-specific markers in a wheat background were also validated regarding their specificity and stability for detecting P. huashanica chromosomes in four wheat–P. huashanica addition lines. Four and eight SNP markers were detected in wheat–P. huashanica 2Ns and 7Ns addition lines, respectively, and one marker was specific to both wheat–P. huashanica 3Ns, 4Ns, and 7Ns addition lines. These markers developed using transcriptome data may be used to elucidate the genetic relationships among Psathyrostachys, Leymus, and other closely-related species. They may also facilitate precise introgressions and the high-throughput monitoring of P. huashanica exogenous chromosomes or segments in future crop breeding programs.

华山独有之植物 Psathyrostachys huashanica，为中国特有，乃小麦之重要野生近缘种，具备诸多对小麦育种有益的优良性状。然而，其物种间系统发育关系的描述尚不明确，基因组变异情况亦未得到充分阐释，加之物种特异性分子标记的相对匮乏，限制了 Psathyrostachys huashanica 作为遗传资源在小麦种质改良中的应用。本研究对 Psathyrostachys huashanica 转录组进行测序，获得 50,337,570 条清洁读段，组装成 65,617 个非冗余基因，其中 38,428 个（占 58.56%）至少与公共数据库中的一个序列相匹配。采用 68 个假定的直系同源基因簇对 Psathyrostachys huashanica、Triticeae 属物种及 Poaceae 科物种进行了系统发育分析。数据揭示了 Psathyrostachys huashanica 与普通小麦之间遥远的进化关系，以及 Psathyrostachys huashanica 基因组与小麦 D 基因组之间的显著多样性。通过比较 Psathyrostachys huashanica 与 Chinese Spring 的转录组，识别了 Psathyrostachys huashanica Ns 基因及其在普通小麦中的同源基因之间 750,759 个候选单核苷酸多态性位点。在 90 个具有不同功能注释的外显子区域单核苷酸多态性位点中，经 KASP 基因分型验证，有 58 个（占 64.4%）被确认为普通小麦背景下的 Ns 基因组特异性单核苷酸多态性位点。标记验证分析表明，六种特定标记能够区分 Psathyrostachys huashanica 与其他小麦相关物种。此外，五种标记仅存在于 Psathyrostachys huashanica、Psathyrostachys juncea 及 Leymus 属物种中，这些标记携带 Ns 基因组。在小麦背景中，Ns 基因组特异性标记在检测四个小麦-Psathyrostachys huashanica 补加系中的 Psathyrostachys huashanica 染色体方面的特异性和稳定性也得到了验证。在小麦-Psathyrostachys huashanica 2Ns 和 7Ns 补加系中，分别检测到四个和八个单核苷酸多态性标记，有一个标记对小麦-Psathyrostachys huashanica 3Ns、4Ns 和 7Ns 补加系均具有特异性。这些基于转录组数据开发的标记可能被用于阐明 Psathyrostachys、Leymus 及其他密切相关的物种之间的遗传关系，并有助于精确导入和未来作物育种计划中对 Psathyrostachys huashanica 外源染色体或片段的高通量监测。