Chilling injury owing to low temperatures severely affects the growth and development of maize (Zea mays.L) seedlings during the early and late spring seasons. The existing maize germplasm is deficient in the resources required to improve maize’s ability to tolerate cold injury. Therefore, it is crucial to introduce and identify excellent gene/QTLs that confer cold tolerance to maize for sustainable crop production. Wild relatives of maize, such as Z. perennis and Tripsacum dactyloides, are strongly tolerant to cold and can be used to improve the cold tolerance of maize. In a previous study, a genetic bridge among maize that utilized Z. perennis and T. dactyloides was created and used to obtain a highly cold-tolerant maize introgression line (MIL)-IB030 by backcross breeding. In this study, two candidate genes that control relative electrical conductivity were located on MIL-IB030 by forward genetics combined with a weighted gene co-expression network analysis. The results of the phenotypic, genotypic, gene expression, and functional verification suggest that two candidate genes positively regulate cold tolerance in MIL-IB030 and could be used to improve the cold tolerance of cultivated maize. This study provides a workable route to introduce and mine excellent genes/QTLs to improve the cold tolerance of maize and also lays a theoretical and practical foundation to improve cultivated maize against low-temperature stress.

Helictotrichon virescens is a perennial herbaceous plant with a life expectancy of about 10 years. It has high cold and heat resistance and can successfully survive over winter in the habitats with a temperature range of −25 to 25°C. Therefore, this study aimed to identify the key genes regulating low-temperature stress responses in H. virescens and analyze cold tolerant at molecular level. This study used RNA sequencing (RNA-Seq) and weighted gene co-expression network analysis (WGCNA) to identify the hub genes associated with cold tolerance in H. virescens. RT-PCR was conducted, homologous genes were identified, and related bioinformatics were analyzed to verify the identified hub genes. Moreover, WGCNA analysis showed that only the brown module had the highest correlation with the active-oxygen scavenging enzymes [peroxide (POD), superoxide dismutase (SOD), and catalase (CAT)]. The expression levels of three hub genes in the brown module (Cluster-37118.47362, cluster-37118.47713, and cluster-37118.66740) were significantly higher under low-temperature stress than those under control conditions. Furthermore, gene ontology (GO) and KEGG annotations showed that the three hub genes were mainly enriched in the metabolism pathways of sphingolipids, selenocompounds, glyoxylate, and dicarboxylate, carotenoids biosynthesis, and other biological pathways. The results of this study also showed that the subcellular localization prediction results showed that the cold tolerance hub genes were all localized to the plasma membrane. By constructing a protein interaction network, it was found that the hub gene Cluster-37118.66740 interacted with Sb09g003460.1 and Sb04g020180.1 proteins in Sorghum bicolor. By constructing phylogenetic trees of the four species of H. virescens, Sorghum bicolo, Oryza sativa Japonica, and Arabidopsis thaliana, the results showed that, the hub gene Cluster 37118.66740 (of H. virescens) and Os03g0340500 (of Oryza sativa Japonica) belonged to the same ancestral branch and were in the same subfamily. Thus, this study provides methodology and guidance to identify the cold tolerance genes for other herbage and their cold tolerant molecular mechanisms at molecular level.

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Summary Low-temperature chilling injury in early spring or late spring severely restricts maize production. Therefore, improving its temperature tolerance is one of the important goals of maize breeding. The wild maize relatives such as Z. perennis and T. dactyloides have strong cold tolerance and thus can be used to improve the cold tolerance of cultivated maize. In the previous study, the allohexaploid MTP ( Zea . Z. perennis and T. dactyloides ) was created as a genetic bridge material and the introgressed cross of the backbone maize inbred line to obtain the MTP-maize introgression line with strong cold tolerance. In this study, F 2:3 populations was developed of the cold-tolerant maize introgression line IB030 and the cold-sensitive maize introgression line IB021. A high-density genetic linkage map was constructed using SSR genetic markers and performed QTL mapping for related traits at 2°C, RNA-seq was performed using cold-tolerant introgression line IB030 and recurrent parent B73 (cold-sensitive) after low temperature stress at seedling stage. The key genes controlling REC, SDW and SFW of maize introgression line seedlings were located by forward genetics combined with WGCNA analysis strategy. Key genes function verification results showed that, Zm00001d037590 REC and Zm00001d012321 REC have the highest expression levels in introgression line IB030, followed by the wild type B73, and finally the EMS mutant after low temperature stress, these results initially concluded that key genes positively regulate the cold tolerance of maize, and also laid a theoretical and practical foundation for improvement of cultivated maize against low-temperature stress.