Wild germplasm resources are crucial for gene mining and molecular breeding because of their special trait performance. Haplotype-resolved genome is an ideal solution for fully understanding the biology of subgenomes in highly heterozygous species. Here, we surveyed the genome of a wild walnut tree from Gongliu County, Xinjiang, China, and generated a haplotype-resolved reference genome of 562.99 Mb (contig N50 = 34.10 Mb) for one haplotype (hap1) and 561.07 Mb (contig N50 = 33.91 Mb) for another haplotype (hap2) using PacBio high-fidelity (HiFi) reads and Hi-C technology. Approximately 527.20 Mb (93.64%) of hap1 and 526.40 Mb (93.82%) of hap2 were assigned to 16 pseudochromosomes. A total of 41039 and 39744 protein-coding gene models were predicted for hap1 and hap2, respectively. Moreover, 123 structural variations (SVs) were identified between the two haplotype genomes. Allele-specific expression genes (ASEGs) that respond to cold stress were ultimately identified. These datasets can be used to study subgenome evolution, for functional elite gene mining and to discover the transcriptional basis of specific traits related to environmental adaptation in wild walnut.

Flower bud development is a defining feature of walnut, which contributes to the kernel yield, yield stability, fruit quality and commodity value. However, little is known about the mechanism of the flower bud development in walnut. Here, the stages of walnut female flower bud development were divided into five period (P01-05) by using histological observation. They were further studied through PacBio Iso-Seq and RNA-seq analysis. Accordingly, we obtained 52,875 full-length transcripts, where 4,579 were new transcripts, 3,065 were novel genes, 1,437 were consensus lncRNAs and 20,813 were alternatively spliced isoforms. These transcripts greatly improved the current genome annotation and enhanced our understanding of the walnut transcriptome. Next, RNA sequencing of female flower buds at five periods revealed that circadian rhythm-plant was commonly enriched along with the flower bud developmental gradient. A total of 14 differentially expressed genes (DEGs) were identified, and six of them were confirmed by real-time quantitative analysis. Additionally, six and two differentially expressed clock genes were detected to be regulated by AS events and lncRNAs, respectively. All these detected plant circadian genes form a complex interconnected network to regulate the flower bud development. Thus, investigation of key genes associated with the circadian clock could clarify the process of flower bud development in walnut.

Cold damage poses a significant challenge to the cultivation of soft-seeded pomegranate varieties, hindering the growth of the pomegranate industry. The genetic basis of cold tolerance in pomegranates has remained elusive, largely due to the lack of high-quality genome assemblies for cold-tolerant varieties and comprehensive population-scale genomic studies. In this study, we addressed these challenges by assembling a high-quality chromosome-level reference genome for 'Sanbai', a pomegranate variety renowned for its freezing resistance, achieving an impressive contig N50 of 15.93 Mb. This robust assembly, enhanced by long-read sequencing of 38 pomegranate accessions, facilitated the identification of 14 239 polymorphic structural variants, revealing their critical roles in genomic diversity and population differentiation related to cold tolerance. Of particular significance was the discovery of a ~ 5.4-Mb inversion on chromosome 1, which emerged as an important factor affecting cold tolerance in pomegranate. Moreover, through the integration of bulked segregant analysis, differential selection analysis, and genetic transformation techniques, we identified and validated the interaction between the PgNAC12 transcription factor and PgCBF1, disclosing their pivotal roles in response to cold stress. These findings mark a significant advancement in pomegranate genomics, offering novel insights into the genetic mechanisms of cold tolerance and providing valuable resources for the genetic improvement of soft-seeded pomegranate varieties.

SPL transcription factors affect plant growth and development, including blooming and photoperiod control. The investigation began with transcriptome data screening of 28 JrSPL genes in walnut (Junglans regia L.) ‘Wen185’. These genes were discovered on all chromosomes except 6 and 15. Phylogenetic study divides the 28 JrSPL genes into five groupings. The biggest cluster, cluster IV, has 12 JrSPL genes. The expression of JrSPL genes in different tissues was investigated by qRT-PCR. JrSPL02 gene expression was greater in walnut female and male flower tissues than other genes. Subcellular localization has shown the JrSPL02 gene resides in the nucleus. Jre-miR156 may target JrSPL02’s 3′-UTR region, according to miRNA sequencing, RACE, and BiFC studies. Arabidopsis plants expressing the JrSPL02 gene flowered 3 days faster than the wild type, according to phenotypic observation. Transgenic lines had more stem branches and siliques than the control group but fewer rosette leaves. In summary, this study functionally analyses the metamorphosis of the miR156-SPL module during the blooming stage and the underlying mechanisms that govern early fruiting in early-fruiting walnuts in Xinjiang.

Walnuts are among the globally significant woody food and oil tree species. At high latitudes, they frequently experience late-frost damage, inducing low-temperature stress, which significantly affects walnut seedlings. The aim of this study was to investigate the physiological and biochemical alterations in walnut seedlings under low-temperature (LT) stress along with its underlying molecular mechanisms. Physiological indices were determined, and the transcriptome and miRNA were sequenced by sampling leaves (0 h, 6 h, 12 h, 24 h, and 48 h) of two-month-old live seedlings of walnuts treated with a low temperature of 4 °C. The results indicated that LT stress induced an increase in electrical conductivity and malondialdehyde content while simultaneously causing a reduction in Fv/Fm. From the transcriptome comparison between the control and treated groups, a total of 12,566 differentially expressed genes (DEGs) were identified, consisting of 6829 up-regulated and 5737 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs were primarily enriched in polysaccharide metabolic processes, responses to abscisic acid and phenylpropanoid biosynthesis pathways. Furthermore, the miRNA database identified 1052 miRNAs in response to low-temperature stress in walnuts; these miRNAs were found to target 7043 predicted genes. Through the integration and analysis of transcriptome and miRNA data, 244 differential DEGs were identified. Following GO and KEGG enrichment analyses of the differential target genes, we identified that these genes primarily regulate pathways involved in starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, and glycerophospholipid biosynthesis, as well as phenylalanine, tyrosine, and tryptophan biosynthesis, in walnut leaves under LT stress. Additionally, we conducted an in-depth analysis of the associations between differentially expressed genes (DEGs) and differentially expressed microRNAs (DEMs) within the starch and sucrose metabolism pathway. Real-time fluorescent quantitative PCR (qRT-PCR) validation of the expression patterns of a subset of differential genes confirmed the accuracy of the transcriptome data. This study unveils the potential molecular mechanisms underlying walnut’s response to low-temperature stress, providing valuable genetic resources for future research on the cold tolerance mechanisms of walnut in response to late-frost damage.

Elucidating the genetic architecture of key quality traits and understanding population differentiation in walnut (Juglans regia) are critical for advancing molecular breeding and enhancing environmental adaptability. In this study, we performed whole-genome resequencing of 282 representative walnut accessions encompassing wild populations, ancient landraces, and modern cultivars. Walnuts in Xinjiang exhibit clear genetic differentiation and can be categorized into two main groups: wild walnuts from Yili and ancient landraces from Aksu, Kashi, and Hotan. The Yili region in northern Xinjiang, China, represents an independent center of walnut genetic diversity. In contrast, the ancient landraces and cultivated walnuts from other regions of Xinjiang are closely related to wild walnuts from the Middle East, suggesting a shared origin. Analysis of deleterious mutations uncovered contrasting accumulation patterns between Wild-Yili_China and Ancient Landrace-Xinjiang_China groups, likely shaped by demographic history and local environmental adaptation. Genome-wide association studies (GWAS) identified key loci and candidate genes, including JruL2x, JrCRK26 and JrRHF2A, that are associated with important quality traits. Furthermore, we functionally validated JrCYP98A2, which encodes a ferulate 5-hydroxylase, as a regulator of shell thickness. Together, these findings provide insights into walnut domestication and the genetic basis of quality trait variation, offering valuable genomic resources for future molecular breeding and genetic improvement of walnut.