Microsatellites or simple sequence repeats (SSRs) are distributed across both prokaryotic and eukaryotic genomes and have been widely used for genetic studies and molecular marker-assisted breeding in crops. Though an ordered draft sequence of hexaploid bread wheat have been announced, the researches about systemic analysis of SSRs for wheat still have not been reported so far. In the present study, we identified 364,347 SSRs from among 10,603,760 sequences of the Chinese spring wheat (CSW) genome, which were present at a density of 36.68 SSR/Mb. In total, we detected 488 types of motifs ranging from di- to hexanucleotides, among which dinucleotide repeats dominated, accounting for approximately 42.52% of the genome. The density of tri- to hexanucleotide repeats was 24.97%, 4.62%, 3.25% and 24.65%, respectively. AG/CT, AAG/CTT, AGAT/ATCT, AAAAG/CTTTT and AAAATT/AATTTT were the most frequent repeats among di- to hexanucleotide repeats. Among the 21 chromosomes of CSW, the density of repeats was highest on chromosome 2D and lowest on chromosome 3A. The proportions of di-, tri-, tetra-, penta- and hexanucleotide repeats on each chromosome, and even on the whole genome, were almost identical. In addition, 295,267 SSR markers were successfully developed from the 21 chromosomes of CSW, which cover the entire genome at a density of 29.73 per Mb. All of the SSR markers were validated by reverse electronic-Polymerase Chain Reaction (re-PCR); 70,564 (23.9%) were found to be monomorphic and 224,703 (76.1%) were found to be polymorphic. A total of 45 monomorphic markers were selected randomly for validation purposes; 24 (53.3%) amplified one locus, 8 (17.8%) amplified multiple identical loci, and 13 (28.9%) did not amplify any fragments from the genomic DNA of CSW. Then a dendrogram was generated based on the 24 monomorphic SSR markers among 20 wheat cultivars and three species of its diploid ancestors showing that monomorphic SSR markers represented a promising source to increase the number of genetic markers available for the wheat genome. The results of this study will be useful for investigating the genetic diversity and evolution among wheat and related species. At the same time, the results will facilitate comparative genomic studies and marker-assisted breeding (MAS) in plants.

Low-temperature stress significantly limits wheat growth and productivity. Poly-γ-glutamic acid (γ-PGA) is an environmentally friendly green molecular material that plays an important role in plant growth and regulation; however, its protective mechanisms against cold stress in wheat remain poorly understood. In this study, the effect of γ-PGA on both chilling (4 °C) and freezing (−18 °C) resistance in wheat seedlings and its underlying mechanisms were comparatively studied. The results showed that the γ-PGA-treated seedlings exhibited a 128.81% higher survival rate after freezing stress and maintained significantly greater biomass accumulation under both stress conditions (62.44% and 26.56% higher dry weight under chilling and freezing stress, respectively). A physiological analysis revealed that γ-PGA enhanced osmoprotectant (proline and soluble sugars) accumulation and activated key antioxidant enzymes (SOD, POD, and APX). Then, an RNA-seq analysis identified 11,401 and 7721 differentially expressed genes under chilling and freezing stress, respectively, with 3598 common genes constituting a core cold-response network. KEGG and GO analyses demonstrated significant enrichment in pathways related to carbon metabolism, glutathione metabolism, phenylpropanoid–flavonoid biosynthesis, fatty acid metabolism, and cell wall organization. Notably, γ-PGA strongly upregulated key genes in phenylpropanoid–flavonoid metabolism (TraesCS2B02G615000 and TraesCS2B02G624400), glutathione metabolism (TraesCS1B02G127900), and lipid metabolism (TraesCS1B02G018700). These results provide comprehensive molecular insights into γ-PGA-mediated cold tolerance and support its potential application in sustainable wheat production under low-temperature stress conditions.