Cultivated spinach (Spinacia oleracea L.) is one of the most widely cultivated types of leafy vegetable in the world, and it has a high nutritional value. Spinach is also an ideal plant for investigating the mechanism of sex determination because it is a dioecious species with separate male and female plants. Some reports on the sex labeling and localization of spinach in the study of molecular markers have surfaced. However, there have only been two reports completed on the genetic map of spinach. The lack of rich and reliable molecular markers and the shortage of high-density linkage maps are important constraints in spinach research work. In this study, a high-density genetic map of spinach based on the Specific-locus Amplified Fragment Sequencing (SLAF-seq) technique was constructed; the sex-determining gene was also finely mapped. Through bio-information analysis, 50.75 Gb of data in total was obtained, including 207.58 million paired-end reads. Finally, 145,456 high-quality SLAF markers were obtained, with 27,800 polymorphic markers and 4080 SLAF markers were finally mapped onto the genetic map after linkage analysis. The map spanned 1,125.97 cM with an average distance of 0.31 cM between the adjacent marker loci. It was divided into 6 linkage groups corresponding to the number of spinach chromosomes. Besides, the combination of Bulked Segregation Analysis (BSA) with SLAF-seq technology(super-BSA) was employed to generate the linkage markers with the sex-determining gene. Combined with the high-density genetic map of spinach, the sex-determining gene X/Y was located at the position of the linkage group (LG) 4 (66.98 cM–69.72 cM and 75.48 cM–92.96 cM), which may be the ideal region for the sex-determining gene. A high-density genetic map of spinach based on the SLAF-seq technique was constructed with a backcross (BC1) population (which is the highest density genetic map of spinach reported at present). At the same time, the sex-determining gene X/Y was mapped to LG4 with super-BSA. This map will offer a suitable basis for further study of spinach, such as gene mapping, map-based cloning of Specific genes, quantitative trait locus (QTL) mapping and marker-assisted selection (MAS). It will also provide an efficient reference for studies on the mechanism of sex determination in other dioecious plants.

Sex chromosomes have evolved independently in many different plant lineages. Here, we describe reference genomes for spinach (Spinacia oleracea) X and Y haplotypes by sequencing homozygous XX females and YY males. The long arm of the 185 Mb chromosome 4 carries a 13 Mb X-linked region (XLR) and 24.1 Mb Y-linked region (YLR), of which 10 Mb is Y-specific. We describe evidence that this reflects insertions of autosomal sequences creating a “Y duplication region” or “YDR” whose presence probably directly reduces genetic recombination in the immediately flanking regions, although both the X and Y Sex-Linked Regions are within a large pericentromeric region of chromosome 4 that recombines rarely in meiosis of both sexes. Sequence divergence estimates using synonymous sites indicate that YDR genes started diverging from their likely autosomal progenitors about 3 MYA, around the time when the flanking YLR stopped recombining with the XLR. These flanking regions have a higher density of repetitive sequences in the YY than the XX assembly and include slightly more pseudogenes compared with the XLR, and the YLR has lost about 11% of the ancestral genes, suggesting some degeneration. Insertion of a male-determining factor would have caused Y-linkage across the entire pericentromeric region, creating physically small, highly recombining, terminal pseudo-autosomal regions. These findings provide a broader understanding of the origin of sex chromosomes in spinach.

Long terminal repeat (LTR)-retrotransposons (LTR-RTs) comprise a major portion of many plant genomes and may exert a profound impact on genome structure, function, and evolution. Although many studies have focused on these elements in an individual species, their dynamics on a family level remains elusive. Here, we investigated the abundance, evolutionary dynamics, and impact on associated genes of LTR-RTs in 16 species in an economically important plant family, Cucurbitaceae. Results showed that full-length LTR-RT numbers and LTR-RT content varied greatly among different species, and they were highly correlated with genome size. Most of the full-length LTR-RTs were amplified after the speciation event, reflecting the ongoing rapid evolution of these genomes. LTR-RTs highly contributed to genome size variation via species-specific distinct proliferations. The Angela and Tekay lineages with a greater evolutionary age were amplified in Trichosanthes anguina, whereas a recent activity burst of Reina and another ancient round of Tekay activity burst were examined in Sechium edule. In addition, Tekay and Retand lineages belonging to the Gypsy superfamily underwent a recent burst in Gynostemma pentaphyllum. Detailed investigation of genes with intronic and promoter LTR-RT insertion showed diverse functions, but the term of metabolism was enriched in most species. Further gene expression analysis in G.pentaphyllum revealed that the LTR-RTs within introns suppress the corresponding gene expression, whereas the LTR-RTs within promoters exert a complex influence on the downstream gene expression, with the main function of promoting gene expression. This study provides novel insights into the organization, evolution, and function of LTR-RTs in Cucurbitaceae genomes.

Spinacia is a genus of important leafy vegetable crops worldwide and includes cultivated Spinacia oleracea and two wild progenitors, Spinacia turkestanica and Spinacia tetrandra. However, the chloroplast genomes of the two wild progenitors remain unpublished, limiting our knowledge of chloroplast genome evolution among these three Spinacia species. Here, we reported the complete chloroplast genomes of S. oleracea, S. turkestanica, and S. tetrandra obtained via Illumina sequencing. The three chloroplast genomes exhibited a typical quadripartite structure and were 150,739, 150,747, and 150,680 bp in size, respectively. Only three variants were identified between S. oleracea and S. turkestanica, whereas 690 variants were obtained between S. oleracea and S. tetrandra, strongly demonstrating the close relationship between S. turkestanica and S. oleracea. This was further supported by phylogenetic analysis. We reported a comprehensive variant dataset including 503 SNPs and 83 Indels using 85 Spinacia accessions containing 61 S. oleracea, 16 S. turkestanica, and eight S. tetrandra accessions. Thirteen S. oleracea accessions were derived through introgression from S. turkestanica that acts as the maternal parent. Together, these results provide a valuable resource for spinach breeding programs and improve our understanding of the phylogenetic relationships within Amaranthaceae.

The members of the myeloblastosis (MYB) family of transcription factors (TFs) participate in a variety of biological regulatory processes in plants, such as circadian rhythm, metabolism, and flower development. However, the characterization of MYB genes across the genomes of spinach Spinacia oleracea L. has not been reported. Here, we identified 140 MYB genes in spinach and described their characteristics using bioinformatics approaches. Among the MYB genes, 54 were 1R-MYB, 80 were 2R-MYB, 5 were 3R-MYB, and 1 was 4R-MYB. Almost all MYB genes were located in the 0–30 Mb region of autosomes; however, the 20 MYB genes were enriched at both ends of the sex chromosome (chromosome 4). Based on phylogeny, conserved motifs, and the structure of genes, 2R-MYB exhibited higher conservation relative to 1R-MYB genes. Tandem duplication and collinearity of spinach MYB genes drive their evolution, enabling the functional diversification of spinach genes. Subcellular localization prediction indicated that spinach MYB genes were mainly located in the nucleus. Cis-acting element analysis confirmed that MYB genes were involved in various processes of spinach growth and development, such as circadian rhythm, cell differentiation, and reproduction through hormone synthesis. Furthermore, through the transcriptome data analysis of male and female flower organs at five different periods, ten candidate genes showed biased expression in spinach males, suggesting that these genes might be related to the development of spinach anthers. Collectively, this study provides useful information for further investigating the function of MYB TFs and novel insights into the regulation of sex determination in spinach.