Tissue-specific transcriptomic analysis reveals the molecular mechanisms responsive to cold stress in Poa crymophila, and development of EST-SSR markers linked to cold tolerance candidate genes.

Abstract

Background: Poa crymophila is a perennial, cold-tolerant, native grass species, widely distributed in the Qinghai-Tibet Plateau. However, the tissue-specific regulatory mechanisms and key regulatory genes underlying its cold tolerance remain poorly characterized. Therefore, in this study, based on the screening and evaluation of cold tolerance of four Poa species, the cold tolerance mechanism of P. crymophila's roots, stems, and leaves and its cold tolerance candidate genes were investigated through physiological and transcriptomic analyses.

Results: Results of the present study suggested that the cold tolerance of the four Poa species was in the following order: P. crymophila > P. botryoides > P. pratensis var. anceps > P. pratensis. Cold stress significantly changed the physiological characteristics of roots, stems, and leaves of P. crymophila in this study. In addition, the transcriptome results showed that 4434, 8793, and 14,942 differentially expressed genes (DEGs) were identified in roots, stems, and leaves, respectively; however, 464 DEGs were commonly identified in these three tissues. KEGG enrichment analysis showed that these DEGs were mainly enriched in the phenylpropanoid biosynthesis pathway (roots), photosynthesis pathway (stems and leaves), circadian rhythm-plant pathway (stems and leaves), starch and sucrose metabolism pathway (roots, stems, and leaves), and galactose metabolism pathway (roots, stems, and leaves). A total of 392 candidate genes involved in Ca²⁺ signaling, ROS scavenging system, hormones, circadian clock, photosynthesis, and transcription factors (TFs) were identified in P. crymophila. Weighted gene co-expression network analysis (WGCNA) identified nine hub genes that may be involved in P. crymophila cold response. A total of 200 candidate gene-based EST-SSRs were developed and characterized. Twenty-nine polymorphic EST-SSRs primers were finally used to study genetic diversity of 40 individuals from four Poa species with different cold tolerance characteristics. UPGMA cluster and STRUCTURE analysis showed that the 40 Poa individuals were clustered into three major groups, individual plant with similar cold tolerance tended to group together. Notably, markers P37 (PcGA2ox3) and P148 (PcERF013) could distinguish P. crymophila from P. pratensis var. anceps, P. pratensis, and P. botryoides.

Conclusions: This study provides new insights into the molecular mechanisms underlying the cold tolerance of P. crymophila, and also lays a foundation for molecular marker-assisted selection for cold tolerance improvement in Poa species.