Verticillium is a small genus of ascomycete fungi containing 10 species. Several of these species are destructive phytopathogens that cause vascular diseases on diverse crops, including ornamentals, in different regions of the world. Traditional methods of differentiating the species based on morphological characteristics can lead to misidentification. Although rapid PCR detection assays are available, there remains a lack of universal and accurate methods for the identification of the 10 species. The aim of this research was to develop novel species-specific primers for the molecular detection of 10 Verticillium species. The primers were designed based on screening of species-specific genomic regions. Among the 10 primer sets tested, the detection limits ranged from approximately 0.1 to 1 ng of Verticillium DNA. The primer sets were further assessed on a collection of Verticillium strains from 10 species. Additionally, DNA extracted from two plant species or soil samples infested with eight different species was employed to further validate the utility of these primers. These species-specific primers facilitate the rapid identification of Verticillium species.

Molecular markers play a crucial role in marker-assisted breeding and varietal identification. However, the application of insertion/deletion markers (InDels) in grapevines has been limited by the low throughput and separability of gel electrophoresis. To developed effective InDel markers for grapevines, this study reports a novel, effective and high-throughput pipeline for InDel marker development and identification. After rigorous filtering, 11 polymorphic multi-allelic InDel markers were selected. These markers were then used to perform genetic identification of 123 elite grape cultivars using agarose gel electrophoresis and next-generation sequencing (NGS). The polymorphism rate of the InDel markers identified by gels was 37.92%, while the NGS-based results demonstrated a higher polymorphism rate of 61.12%. Finally, the NGS-based fingerprints successfully distinguished 122 grape varieties (99.19%), surpassing the gels, which could distinguish 116 grape varieties (94.31%). Specifically, we constructed phylogenetic trees based on the genotyping results from both gels and NGS. The population structure revealed by the NGS-based markers displayed three primary clusters, consisting of the patterns of the evolutionary divergence and geographical origin of the grapevines. Our work provides an efficient workflow for multi-allelic InDel marker development and practical tools for the genetic discrimination of grape cultivars.

The plant NAC (NAM, ATAF1/2, and CUC2) transcription factor family plays an important regulatory role in stress response. In this study, we analyzed the rice transcription factor OsNAC113 and elucidated its tissue-specific characteristics and stress response regulatory mechanisms. qRT-PCR results showed that under laboratory-simulated drought, high salt, temperature stress, and hormone treatments, such as abscisic acid (ABA) and gibberellic acid (GA₃), the expression level of OsNAC113 significantly changed, indicating that OsNAC113 responds to various stress conditions. Targeted creation of the rice (Oryza sativa L. spp. japonica) OsNAC113 (LOC_os08g10080.1) mutant based on the CRISPR-Cas9 genome editing strategy revealed its response to salt stress (200 mM). The growth status and survival rate of the mutant under high-salt stress were significantly higher than those of the wild type. Testing showed that the mutant exhibited increased relative water, chlorophyll, and soluble sugar contents under salt stress than the wild type. The malondialdehyde content in the mutant was lower, and the activities of superoxide dismutase, peroxidase, and catalase were higher than those in the wild type, indicating that the mutant with functional loss caused by knocking out OsNAC113 had a significantly enhanced tolerance to salt treatment. Using RNA-seq to detect genome-wide changes in OsNAC113 mutant materials under stress, KEGG annotation showed that knocking out OsNAC113 resulted in regulatory changes in “plant hormone signaling pathway” and “MAPK signaling pathway,” and GO and KEGG annotations showed significant changes in “amino acid transport and metabolism,” “carbohydrate transport and metabolism,” “lipid transport and metabolism,” and “replication, recombination, and repair.” OsNAC113 may be involved in the response to salt stress by regulating these signaling pathways. Using comparative metabolomic analysis, we further elucidated the function of OsNAC113 in physiological metabolic pathways. The knockout of OsNAC113 resulted in changes in various important metabolic pathways in plants, including flavonoid biosynthesis and ABC transporters. Therefore, it is suggested that OsNAC113 is involved in these metabolic processes and affects their regulation in high-salt environments. These results provide a theoretical foundation and reliable material for the molecular breeding of rice.