QTL analysis of femaleness in monoecious spinach and fine mapping of a major QTL using an updated version of chromosome-scale pseudomolecules

Abstract

Although spinach is predominantly dioecious, monoecious plants with varying proportions of female and male flowers are also present. Recently, monoecious inbred lines with highly female and male conditions have been preferentially used as parents for F1-hybrids, rather than dioecious lines. Accordingly, identifying the loci for monoecism is an important issue for spinach breeding. We here used long-read sequencing and Hi-C technology to construct SOL_r2.0_pseudomolecule, a set of six pseudomolecules of spinach chromosomes (total length: 879.2 Mb; BUSCO complete 97.0%) that are longer and more genetically complete than our previous version of pseudomolecules (688.0 Mb; 81.5%). Three QTLs, qFem2.1, qFem3.1, and qFem6.1, responsible for monoecism were mapped to SOL_r2.0_pseudomolecule. qFem3.1 had the highest LOD score and corresponded to the M locus, which was previously identified as a determinant of monoecious expression, by genetic analysis of progeny from female and monoecious plants. The other QTLs were shown to modulate the ratio of female to male flowers in monoecious plants harboring a dominant allele of the M gene. Our findings will enable breeders to efficiently produce highly female- and male-monoecious parental lines for F1-hybrids by pyramiding the three QTLs. Through fine-mapping, we narrowed the candidate region for the M locus to a 19.5 kb interval containing three protein-coding genes and one long non-coding RNA gene. Among them, only RADIALIS-like-2a showed a higher expression in the reproductive organs, suggesting that it might play a role in reproductive organogenesis. However, there is no evidence that it is involved in the regulation of stamen and pistil initiation, which are directly related to the floral sex differentiation system in spinach. Given that auxin is involved in reproductive organ formation in many plant species, genes related to auxin transport/response, in addition to floral organ formation, were identified as candidates for regulators of floral sex-differentiation from qFem2.1 and qFem6.1.

Fig 1. Molecular linkage map of spinach based on the mapping population 03–009 x 03–336 F₂.

Black bars on the right side of the map represent QTLs (95% CI) for femaleness (proportion of female flowers per plant). The left and right values in parentheses represent LOD values and phenotypic variance explained (PVE) values of the QTLs.

Fig 2. Physical map of the M locus.

a. Schematic diagrams representing allelic bacterial artificial chromosome clones of the M locus from lines 03–009 and 03–336. Positions of the DNA markers are shown on the diagrams. b. Graphical genotypes and femaleness of progeny families from monoecious selections in S₅BC₂F₁ and BC₁F₄ populations produced by crosses between 03–009 and 03–336. Black boxes represent homozygous 03–336 segments, gray boxes indicate heterozygous 03–009/03–336 regions, and white boxes represent homozygous 03–009 segments. Indices of femaleness (expressed as the percentage of female flowers per plant) are shown to the right of the boxes. c. Schematic diagrams of protein-coding genes and a lncRNA locus predicted in the 19.5-kb M candidate region. Exons and coding regions are represented as gray and black boxes, respectively. Introns are depicted as horizontal gray lines. Gene directions are indicated by arrowheads. Vertical blue, red and green lines represent SNPs, small indels (≤ 7 bp) and structural variants (SV) found between lines 03–009 and 03–336. Diamonds (a–d) represent regions having significant sequence homology with the spinach PISTILLATA (PI) and APETALA3 (AP3) genes. seq1, the 97-bp region, has homology with the third intron of PI; seq2, the 343-bp region, has homology with the 5th intron of PI, and the upstream region of AP3; seq3, the 75-bp region, has homology with the 6th intron of AP3.

Fig 3. RT-qPCR analysis of SoRL2a.

a-e, RT-qPCR analysis of SoRL2a in male and female plants from the dioecious line 03–009, and monoecious plants from line NIL-M; f, RT-qPCR analysis of SoRL2a in leaves, and female and male flowers of monoecious plants. The amount of SoRL2a mRNA was normalized to that of Actin-7.

Fig 4. Violin plots showing the distribution of femaleness exhibited by MM plants selected from F₂ and F₃ progeny of the cross between 03–009 and 03–336.

Statistical significance was determined by Tukey-Kramer test (N.S. p ≥ 0.05; * p < 0.05; ** p < 0.01). A, homozygous for the 03–009 allele; H, heterozygous for the 03–009 and 03–336 alleles; B, homozygous for the 03–336 allele.