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.

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.

Spinach (Spinacia oleracea L.) is commonly considered a dioecious plant with heterogametic (XY) and homogametic (XX) sex chromosomes. The characteristic is also utilized for the production of spinach hybrid seeds. However, the molecular mechanisms of sex determination in spinach are still unclear because of a lack of genomic and transcriptomic information. In this study, RNA sequencing (RNA-seq) was performed in male and female inflorescences to provide insight into the molecular basis of sex determination in spinach. Comparative transcriptome analyses showed that 2278 differentially expressed genes (DEGs) were identified between male and female inflorescences. A high correlation between the RNA-Seq and qRT-PCR validation for DEGs was observed. Among these, 182 DEGs were annotated to transcription factors including the MYB family protein, bHLH family, and MADS family, suggesting these factors might play a vital role in sex determination. Moreover, 26 DEGs related to flower development, including nine ABCE class genes, were detected. Expression analyses of hormone pathways showed that brassinosteroids may be key hormones related to sex determination in spinach. Overall, this study provides a large amount of DEGs related to sexual expression and lays a foundation for unraveling the regulatory mechanism of sex determination in spinach.

Spinach downy mildew, caused by Peronospora effusa (=P. farinosa f. sp. spinaciae), is the most economically important disease of spinach worldwide including China (1). Although the pathogen has been formally reported in China (4, 5), there are no reports of any races or race diversity in this country even though 15 races of the pathogen have been reported worldwide (2). In recent years, foreign seed companies have introduced hybrid spinach seed varieties which account for approximately 75% of the spinach grown in China. Reports of resistance and DM-1 marker PCR amplification (3) confirmed that many foreign spinach hybrids planted in China contain the RPF1 gene confirming providing resistance to P. effusa races 1-7, 9, 11, 13 and 15. To evaluate which races of the downy mildew pathogen could be infecting spinach in China, five field isolates of the pathogen were recovered from symptomatic spinach in China in 2014 and 2015. Three isolates ( BJ-pfs1, BJ-pfs2, and BJ-pfs3) from cv. Shubo No.1 in Haidian and Shunyi Districts, Beijing, were collected in November 2014; an isolate (SD-pfs1) from cv. HuangjiaSuteng in Liaocheng, Shandong Province，was collected in March 2015; and an isolate (SX-pfs1) from cv Shubo No.1 in Xinzhou, Shanxi Province，was collected in May 2015. The cultivar Shubo No.1 has no known reported resistance and HuangjiaSuteng is reported to be resistant to races 1 to 7, 9 and 11. The isolates were returned to a laboratory and examined morphologically. In addition, the isolates were inoculated directly onto a set of international spinach differential lines, or onto the susceptible cv. Shubo No.1 to increase inoculum, prior to inoculation of the differentials, following previously published procedures with this pathogen (2). The sporangia and sporangiophores were consistent with the morphological description of P. effusa, the only downy mildew pathogen known to infect spinach (1). Each isolate was tested in a minimum of three independent inoculation tests. The three isolates collected from cv. Shubo No.1 from Beijing typed out as race 9 whereas the isolate from Shubo No.1 from Shanxi typed out as race 5. The isolate from cv. HuangjiaSuteng from Shandong typed out as race 8. The differentials Viroflay, Resistoflay, and Clermont were susceptible to all five isolates; Campania was susceptible to all isolates except SX-pfs1; Boeing was resistant to all isolates except SD-pfs1; Lion, Lazio, Whale, and Pigeon were resistant to all five isolates. The races identified indicate that many spinach hybrids currently grown in China are susceptible to the downy mildew pathogen. Continued examination of race diversity of the downy mildew pathogen in China will assist in deploying cultivars with the most effective resistance. References: (1) J. C. Correll et al. Eur. J. Plant Path. 129:193, 2011. (2) C. Feng et al. Plant Dis. 98:145, 2014. (3) B. M. Irish et al. Phytopathology 98:894, 2008. (4) Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking. (5) S.C. Teng. 1996. Fungi of China. Mycotaxon Ltd. Ithaca, NY.

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.

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.