Sexual reproduction is the primary means of reproduction for the vast majority of macroscopic organisms, including almost all animals and plants. Sex chromosomes are predicted to play a central role in sexual dimorphism. Sex determination in spinach is controlled by a pair of sex chromosomes. However, the mechanisms of sex determination in spinach remain poorly understand. Here, we assembled the genomes of both a female (XX) and a male (YY) individual of spinach, and the genome sizes were 978 Mb with 28,320 predicted genes and 926 Mb with 26,537 predicted genes, respectively. Based on reported sex-linked markers, chromosomes 4 of the female and male genome were defined as the X and Y chromosomes, and a 10 Mb male-specific region of the Y chromosome (MSY) from approximately 95– 105 Mb, was identified that contains abundant transposable elements (92.32%). Importantly, a large-scale inversion of about 13 Mb in length was detected on the X chromosome, corresponding to ~9 Mb and ~4 Mb on the Y chromosome, which were located on both sides of the MSY with two distinct evolutionary strata. Almost all sex-linked/Y-specific markers were enriched on the inversions/MSY, suggesting that the flanked inversions might result in recombination suppression between the X and Y chromosomes to maintain the MSY. Forty-nine genes within the MSY had functional homologs elsewhere in the autosomal region, suggesting movement of genes onto the MSY. The X and Y chromosomes of spinach provide a valuable resource for investigating spinach sex chromosomes evolution from wild to cultivated spinach and also provide a broader understanding of the sex determination model in the Amaranthaceae family.

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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 However, the chloroplast genomes of the two wild progenitors remain unpublished, limiting our knowledge of chloroplast genome evolution among these three Spinacia species.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 sex-linked region (SLR) plays an important role in determining the sex of a plant. The SLR of the Y chromosome, composed of a 14.1-Mb inversion and a 10-Mb Y-duplication region (YDR), was deciphered in Spinacia oleracea previously. However, our understanding of the SLR in its wild relatives, S. turkestanica and S. tetrandra, remains limited. In this study, we used 63 resequencing data from the three Spinacia species to infer the evolution of the SLR among the Spinacia species. In the SLR, all the cultivated spinach and S. turkestanica accessions were clustered into two distinct categories with both sexes, while the S. tetrandra accessions of both sexes were grouped. This suggests that S. oleracea shared a similar SLR with S. turkestanica, but not with S. tetrandra, which was further confirmed based on the population structure and principal component analysis. Furthermore, we identified 3910 fully sex-linked SNPs in S. oleracea and 92.82% of them were available in S. turkestanica, while none of the SNPs were adopted in S. tetrandra. Genome coverage in males and females supported the hypothesis that the YDR increasingly expanded during its evolution. Otherwise, we identified 13 sex-linked transposable element insertion polymorphisms within the inversion in both S. oleracea and S. turkestanica, demonstrating that the transposable element insertions might have occurred before the recombination suppression event of the inversion. The SLR was conserved compared with the pseudoautosomal region given that the genetic hitchhiking process occurred in the SLR during its evolution. Our findings will significantly advance our understanding of the characteristics and evolution of the SLR in Spinacia species.

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.

Background Spinach (Spinacia oleracea L.) is an important leafy vegetable crop, and leaf-related traits including leaf length, leaf width, and petiole length, are important commercial traits. However, the underlying genes remain unclear. The objective of the study was to conduct QTL mapping of leaf-related traits in spinach. Results A BC1 population was used to construct the linkage map and for QTL mapping of leaf length, leaf width, petiole length, and the ratio of leaf length to width in 2015 and 2019. Two genetic linkage maps were constructed by specific locus amplified fragment sequencing (SLAF-seq), and kompetitive allele specific PCR (KASP) technology, respectively using BC1 population in 2015. Based on the results of 2015, the specific linkage groups (LG) detected QTLs were generated using BC1 population in 2019. A total of 13 QTLs were detected for leaf-related traits, only five QTLs being repeatedly detected in multiple years or linkage maps. Interestingly, the major QTLs of leaf length, petiole length, and the ratio of leaf length to width were highly associated with the same SNP markers (KM3102838, KM1360385 and KM2191098). A major QTL of leaf width was mapped on chromosome 1 from 41.470−42.045 Mb. And 44 genes were identified within the region. Based on the GO analysis, these genes were significantly enriched on ribonuclease, lyase activity, phosphodiester bond hydrolysis process, and cell wall component, thus it might change cell size to determine leaves shape. Conclusions Five QTLs for leaf-related traits were repeatedly detected at least two years or linkage maps. The major QTLs of leaf length, petiole length, and the ratio of leaf length to width were mapped on the same loci. And three genes (Spo10792, Spo21018, and Spo21019) were identified as important candidate genes for leaf width.

Downy mildew is a major threat to the economic value of spinach. The most effective approach to managing spinach downy mildew is breeding cultivars with resistance genes. The resistance allele RPF2 is effective against races 1–10 and 15 of Peronospora farinosa f. sp. Spinaciae (P. effusa) and is widely used as a resistance gene. However, the gene and the linked marker of RPF2 remain unclear, which limit its utilization. Herein, we located the RPF2 gene in a 0.61 Mb region using a BC1 population derived from Sp39 (rr) and Sp62 (RR) cultivars via kompetitive allele specific PCR (KASP) markers. Within this region, only one R gene, Spo12821, was identified based on annotation information. The amino acid sequence analysis showed that there were large differences in the length of the LRR domain between the parents. Additionally, a molecular marker, RPF2-IN12821, was developed based on the sequence variation in the Spo12821, and the evaluation in the BC1 population produced a 100% match with resistance/susceptibility. The finding of the study could be valuable for improving our understanding of the genetic basis of resistance against the downy mildew pathogen and breeding resistance lines in the future.