During cross breeding, the import frequency of alleles into gametes often varies in the progeny population or a specific gene combination causes sterility, resulting in a segregation ratio that is inconsistent with Mendelian inheritance (i.e., segregation distortion). Segregation distortions have been detected in a wide range of taxa (Burt and Trivers, 2006) and are increasingly recognized as a potentially powerful evolutionary force (Taylor and Ingvarsson, 2003). Segregation distortion is thought to have diverse causes, some of which involve gametophytic lethality, embryonic lethality, or cross-incompatibility (Lashermes et al., 2001; Candela et al., 2011) or are related to (selfish) genetic elements that can enhance their own transmission (Dawkins, 1976; Werren et al., 1988). The selfish genetic factors include Cytoplasmic male sterility (Lewis, 1941), B chromosome (Ostergren, 1945), X chromosome drive (Gershenson, 1928; Sturtevant and Dobzhansky, 1936), Y chromosome drive (Hickey and Craig, 1966; Sweeny and Barr, 1978; Wood and Newton, 1991), t haplotype (Dobrovolskaia-Zavadskaia and Kobozieff, 1927), and transposable factors (McClintock, 1950).

Fig (Ficus carica L.) is a dioecious or gynodioecious species, and because only female plant fruits are edible, sex is an important target trait during breeding (Stover et al., 2007; Flaishman et al., 2008). The common fig has the following two sexual types: caprifig type (males) with both male and female flowers and fig type (females) with only female flowers. The progeny resulting from a normal fig type (G/G) × caprifig type (G/A) cross should theoretically be 50% fig and 50% caprifig (fig:caprifig = 1:1) (Storey, 1975).

Mori et al. (2017) identified FcRAN1 and determined that it is highly correlated with the fig sex phenotype on the basis of several analyses, including a genome-wide association study. Additionally, because of a single nucleotide polymorphism (SNP), its alleles are heterozygous (G/A) in males (caprifig type) and homozygous (G/G) in females (fig type) (Storey, 1975). Moreover, FcRAN1 is the only gene in the fig genome that carries a polymorphism that perfectly corresponds to the sex phenotype (Mori et al., 2017). In our current fig breeding program, we genotyped the FcRAN1 alleles of some breeding populations to identify sex phenotypes. We observed that individuals with male type alleles are produced at a high rate only in a specific cross combination (M119-226 × H238-107), suggesting that the FcRAN1 locus is affected by segregation distortion. Clarifying the cause of this distortion may lead to improved sex control and selection during fig breeding, but it may also provide insights into sex ratio transitions and sex chromosome evolution in fig.

In the present study, we examined the segregation distortion in fig breeding populations and performed preliminary analyses to explore the responsible genetic factors. More precisely, we investigated the distribution of SNPs specific to each pollen and seed parent generating segregation distortion in a whole-chromosome pseudomolecule and considered the biological and evolutionary background related to the occurrence of the segregation distortion.

Crossing experiments involving 10 cross combinations of seven seed parents (M119-226, V180-13, Masui Dauphine, H180-2, HIG5, Toyomitsuhime, and Burjassotte Greece) and six pollen parents (H238-107, M238-1, VD238-83, VS238-53, NG60, and CH-13) generated 1,232 seedlings. The progeny population sizes ranged from 12 to 370 (Table 1). Three cross combinations involving three seed parents (Violette de Dauphine, Violette de Solies, and Royal Vineyard) and two pollen parents (M106-238 and VC-180) from a previous study (Mori et al., 2017) were also analyzed. In 12 of the 13 cross combinations, the segregation ratio of the male (G/A) and female (G/G) genotypes of FcRAN1 was close to the theoretical segregation ratio of 1:1, ranging from 3:9 to 192:176 (G/A:G/G; χ²-test: p = 0.014–1.00). In contrast, for the cross combination (C100) involving the seed parent M119-226 and the pollen parent H238-107, the segregation ratio was 178:7 (G/A:G/G; χ²-test: p < 2.2e-16) (Table 1 and Figure 1). An examination of pollen conditions using an optical microscope revealed that more than 99% of the pollen grains were clearly stained and had a normal appearance in all parental lines, including H238-107 (Figure 2).

To identify the factor(s) responsible for the segregation distortion, a genome-wide linkage analysis (GWLA) was performed using the whole-genome sequence of each seed and pollen parent. However, the seed parent of C100, M119-226, died after seeds were acquired, preventing it from being analyzed. Therefore, the genome sequence of Caprifig6085, which was an ancestral genotype of M119-226 and may have a genomic region associated with segregation distortion, was used for seed parent analysis as an alternative to M119-226 (Table 1 and Figure 3) (Segregation distortion was not observed in the mating with Masui Dauphine and Violette de Solies as parents). The 4,614,159 SNPs generated by Shirasawa et al. (2021, unpublished data) included 9,061 high-impact SNPs in the seed and pollen parents. The GWLA using the SNP panel indicated that 194 high-impact SNPs were specific to Caprifig6085 as the seed parent and 215 high-impact SNPs were specific to H238-107 as the pollen parent. The functional annotation of these SNPs on the basis of a BLAST analysis produced information for 201 SNPs (98 and 103 SNPs for the seed and pollen parents, respectively) (Supplementary Tables 1, 2). The genes with high-impact SNPs included a LEA gene (i.e., encoding a late embryogenesis abundant protein). A sequence analysis revealed that the fig LEA gene (designated as FcLEA1) is a single-copy gene located on chromosome Fc01a, which also includes the FcRAN1 locus (Fc01a: 181,985–187,609). Moreover, the polymorphic site in FcLEA1 corresponds to a non-synonymous substitution that changes a leucine to a proline (Figure 4).

The mapping of all GWLA SNPs to whole pseudomolecules indicated that high-impact SNPs were widely distributed throughout the genome in the seed and pollen parents, but there were no high-impact SNPs on chromosomes Fc03 and Fc06 of the seed parents (Figure 5).

It is unclear whether the observed segregation distortion is caused by gametophytic lethality, embryonic lethality, or specific genetic events. Accordingly, in this study, a GWLA was performed assuming that the causative factor is located on the chromosomes of either the seed parent (M119-226) or the pollen parent (H238-107) of the population with distorted segregation. Unfortunately, all M119-226 plants died before their genome sequence data could be obtained, making it impossible to further analyze line M119-226. However, there were four ancestral lines of M119-226, namely Caprifig6085, VC-119, Masui Dauphine, and Violette de Dauphine. Both Masui Dauphine and Violette de Dauphine are not associated with segregation distortion (Table 1). Therefore, during the analysis of the seed parents, it was assumed that the Caprifig6085 and VC-119 genomes include a causative factor of segregation distortion. In this study, Caprifig6085 was used as a substitute for M119-226.

Since it is assumed that there are a wide variety of factors that can cause segregation distortion, the number of candidate genes involved is considered to be large corresponding to be the number of factors. For example, in Arabidopsis thaliana, 510 EMBRYO-DEFECTIVE (EMB) genes (Meinke, 2020) and 129 genes associated with female gametophyte deficiency (Pagnussat et al., 2005) have been identified, all of which may contribute to segregation distortion. During the analysis of the seed parents, 98 genes were detected as specific to M119-226. Additionally, we identified several genes similar to the above-mentioned A. thaliana genes related to female gametophyte deficiency.

The fertilization of the A. thaliana mutant line UNE15, which has a mutation in a gene encoding a LEA protein, is reportedly abnormal because of defective pollen tube guidance (Pagnussat et al., 2005). Accordingly, the female gametophytes of the seed parent M119-226 may be infertile because of a mutation similar to that in UNE15. In addition to the LEA gene, other candidate genes may be responsible for segregation distortion, including genes encoding proteins with unknown functions.

If segregation distortion is due to male gametophytic lethality, the causal allele (gene) is likely located on the X chromosomes of the pollen parents because most of the genotyped alleles in the C100 population were the G/A allele derived from the Y chromosome. However, an examination of the pollen used for mating indicated there were no differences in the shapes and appearance of the pollen grains between the pollen parent lines with and without distorted segregation, implying the distortion was at least independent of the pollen physiological state. It remains possible there is a defect in the process after the pollination event (e.g., pollen tube elongation). The TATA element modulatory factor (TMF) (FCA_r2.3chr09: 8,140,218) may affect the segregation distortion. The A. thaliana TFIID factor AtTAF6 (i.e., a TATA-binding protein) controls pollen tube growth (Lago et al., 2005). Additionally, TMF binds to the HIV-1 TATA element and inhibits the transcriptional activation by the TATA-binding protein (Garcia et al., 1992).

Although the current study did not determine whether FcRAN1 is a true sex-determining gene in F. carica, a male-biased FcRAN1 genotype ratio suggests that Y chromosome drive (Voelker, 1972; Sweeny and Barr, 1978; Wood and Newton, 1991; Presgraves et al., 1997) is important (Figure 6). For a Y chromosome drive system to be functional, the distorter gene and FcRAN1 (i.e., potential sex-determining gene) should be at different loci on the same Y chromosome (Fc01a) of the pollen parent. Of the nine genes detected on chromosome Fc01a (Supplementary Table 2), the RNase H-encoding gene should be investigated for its potential role in segregation distortion. Simoni et al. (2020) introduced a sex-distorter (I-PpoI) and a CRISPR-based gene drive into mosquito (Anopheles gambiae), resulting in stable Y chromosome drive and decreases in the proportion of male individuals in successive generations. Thus, a site-specific DNA double-strand break system involving endonucleases, including I-PpoI, is necessary for a functional Y chromosome drive (Ohle et al., 2016).

The FcRAN1 KASP marker analysis indicated that some plants in the C100 population had the G/A genotype. There are two possible explanations for this observation. First, the effect of the distorter may have been incomplete. Second, specific events (e.g., chromosomal recombination) may have occurred in which the distorter gene was not activated. Future studies should further characterize the genetic background of the distortion via crossing experiments involving multiple seed parents, including H238-107, and the female C100 progeny. Moreover, because the candidate genes discussed herein were selected on the basis of bioinformatics data, their functions will need to be validated through genetic transformation or genome editing.

The high-impact SNPs in the pollen parents were within 5 Mbp from the end of Fc01a (Figure 5), suggestive of a relationship between the sex-linked region and the segregation distortion. Because the sex-determining gene (i.e., FcRAN1 or a nearby gene) basically affects the population sex ratio, the distorter gene and FcRAN1 are likely located close together and behave similarly.

The biological or evolutionary significance of the segregation distortion observed in figs is unclear. However, the occurrence of segregation distortion can change the genetic composition of the species population and may maintain or increase the genetic diversity of the population. In fact, segregation distortion reportedly leads to stable large-scale genomic differences between males and females in hybrid ants (Kulmuni et al., 2010).

Because the FcRAN1 genotype is strongly correlated with the sex phenotype, the genotypic distortion could influence the evolution of sexual reproduction and sex chromosomes in various ways (Kozielska et al., 2010). Fig X and Y chromosomes are homomorphic, with no significant differences in their molecular structure (Storey, 1975), indicating that fig sex chromosomes are like autosomal chromosomes and are not well differentiated. Therefore, clarifying the segregation distortion of the FcRAN1 genotype may help elucidate the differentiation of fig sex and autosomal chromosomes.

Seven seed parents (M119-226, V180-13, Masui Dauphine, H180-2, HIG5, Toyomitsuhime, and Burjassotte Greece) and six pollen parents (H238-107, M238-1, VD238-83, VS238-53, NG60, and CH13) were used for hybridizations, which were completed between 1989 and 2018 according to Storey (1975). The obtained seeds were sown in a seedbed and the resulting young seedlings were transplanted to 9-cm pots containing dedicated soil. Leaf samples were collected from the seedlings for KASP and CAPS marker analyses as described below.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

HI and HY designed the study. HI, KS, HY, SY, TH, and HN completed the analyses. HI, KS, HY, SY, MS, TH, KT, and HN interpreted the data. HI, KS, and HY wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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