Carica papaya L. (Caricaceae) originated in Mesoamerica. This species is an important commercial fruit cultivated in tropical and subtropical regions of the world1. Seminal propagation is the main system used for C. papaya cultivation, resulting in individuals of three biological sexes typically following a Mendelian inheritance2. Carica papaya possesses a relatively small nuclear genome size showing ~ 1C = 0.32 pg, equivalent to ~ 318 Mbp3. This species is diploid with karyotype exhibiting 2n = 2x = 18 chromosomes, which have been classified as metacentric and submetacentric3,4.
Sex chromosome evolution and sexual differentiation mechanisms have been investigated from different plant species, and C. papaya stands out as an important model species5. Sex differentiation in C. papaya was initially based on one gene with at least three alleles (M1, M2 and m). Considering this proposal, male, hermaphrodite and female plants differentiated from the genotypes M1 m, M2 m and m m, respectively6,7. The genotypes M1 M1, M2 M2 and M2 M1 are lethal, promoting the zygotic embryo death6,7. Storey revised his hypothesis, suggesting that the sex is not differentiated by the expression of one gene with three alleles, but by linked genes located in a region of the sex chromosomes where recombination is suppressed8. Corroborating to this Storey’s hypothesis, further evidence showed that the recombination is severely suppressed in the region close to the sexual differentiation locus9, and at least seven genes were identified in the sexual specific portion10. Furthermore, structural chromosomal rearrangements (inversions, deletions and translocations), which may be the outcomes of the recombination suppression, were detected in the sex differentiation region10.
Sex differentiation region in C. papaya genome was genetically mapped to linkage group 1, which is related to the chromosome 111,12. C. papaya biological sex is expressed by a XY chromosome sex differentiation system, from which females are homogametic (XX), and males (XY) and hermaphrodites (XYh) are heterogametic. Y and Yh chromosomes have a specific sexual differentiation region that shows few genetic differences13. Male-specific region (MSY) and hermaphrodite-specific region (HSY) are about 9.8 Mbp, while X-specific region (XX) has 6.0 Mbp14. MSY and HSY chromosome portions have a larger genome size in relation to the corresponding region on the X chromosome, mainly due to the DNA sequence duplications and insertion of mobile elements (retrotransposons) in this region13. HSY possesses more repetitive sequences than X. In addition, 121 pseudoautosomal genes occur between HSY and X chromosomes, 56 specific HSY genes, and 74 specific X genes14.
In association to cytogenomics, genomic sequencing data makes possible the physical mapping in mitotic and meiotic chromosomes of different DNA sequences, including single-copy and/or low-copy genes. Fluorescent in situ hybridization (FISH) is a cytogenomics technique that maps DNA sequences on chromosomes. FISH contributes to the construction of physical maps, and, consequently, the integration of these maps with genetic maps15,16. FISH in C. papaya ‘Solo’ and ‘Maradol’ mitotic chromosomes from 18S and 5S rDNA sequences evidenced the 18S rDNA in the pericentromeric portion of one chromosome pair, while the 5S rDNA was mapped in the pericentromeric portion of three chromosome pairs17. In contrast, pachytene chromosomes of C. papaya ‘SunUp’ showed 5S rDNA on chromosomes 3, 5, 8, 9 and Y, and the 45S rDNA was mapped in the pericentromeric region of the chromosome 4 short arm15. BAC-FISH mapped the BAC clones associated with the 12 linkage groups (LG) on the C. papaya pachytene chromosomes: BAC 96C17 (LG 1), BAC 39C20 (LG 9) and BAC 39P03 (LG 11), BAC 23B18 (LG 6 ), BAC 15O14 (LG 2), BAC 57E17 (LG 5), BAC 07H21 (LG 3), BAC 12M21 (LG 7) and BAC 01P02 (LG 12), BAC 43N18 (LG 4), BAC 78D03 (LG 8) and BAC 99D21 (LG 10), respectively on chromosomes 1 (XY), 2, 3, 4, 5, 6, 7, 8 and 9, integrating the LG to the individual chromosomes15.
In plants, physical mapping has contributed to the characterization of the sex chromosomes. The mapping of a repetitive DNA sequence, named HSR1, in Humulus lupulus L. showed that this sequence is located in the subtelomeric region of the X and Y chromosome long arm. However, the HSR1 was also mapped in the pericentromeric portion of the chromosome X18. In Cannabis sativa L., the repetitive sequence named CS-1 was mapped in the subtelomeric region of the Y chromosome short arm, while both arms of the X chromosome showed this sequence in the subtelomeric region19. The mapping of repetitive sequences in Hippophae rhamnoides L. revealed the HRTR 12 repetitive sequence only in the Y chromosome20. Repetitive DNA sequences, named RAYS, were only mapped in the Y chromosome of Rumex acetosa L.21. Therefore, the sex chromosome characterization in plants has been conducted mainly from the repeatome sequences. In addition to physical mapping, FISH has been accomplished to assists in the early identification of sporophyte sex. For example, a polymorphic molecular marker named NAPF-2 showed fluorescent signals in leaf nuclei of hermaphrodite plants, however, no signal was detected in leaf nuclei of female plants of C. papaya ‘Golden’ and ‘Rubi’22.
In addition to these sequences (mainly from the repeatoma), the physical mapping of single-copy and/or low-copy genes is needed to expand the knowledge about the C. papaya genome, contributing to knowing and understanding its organization and evolution. Moreover, these genes also have potential as cytomolecular markers. In this context, some sequences that occur in the sexual differentiation region were explored here, including: somatic embryogenesis receptor kinase gene (serk 2), short vegetative phase gene (svp-like) and monodehydroascorbate reductase gene (mdar 4)12,23.
The serk 2 gene, which was sequenced in the X and Yh chromosomes14, encodes a protein that belongs to the plasma membrane receptor kinase family. SERK protein has leucine-rich repeats, acting on signal transduction23 and male sporogenesis24. The svp-like gene encodes a transcription factor that regulates the transition from the vegetative to the reproductive phase, activating the classes B and C genes of the floral morphogenesis25,26. Gene expression and sequencing approaches showed that the svp-like gene present on the Y chromosome encodes a wild-type protein with all domains. Due to a Copia-like retrotransposon insertion, a mutant allele of this gene occurs on the Yh chromosome, encoding a protein that has only the K-box domain12,13,27,28. In addition, svp-like gene was also identified on the chromosome 414. The mdar 4 gene, which was also sequenced in X and Yh chromosomes14, encodes an enzyme that has antioxidant activity, eliminating reactive oxygen species and, consequently, increasing tolerance against oxidative stress in some plant species29,30. Genetic analyzes have shown that the mdar 4 gene has a wild-type allele on the X chromosome, while the allele of the Y and Yh chromosomes contains a LTR-retroelement sequence30. Y and Yh mutant alleles encode a shorter truncated MDAR 4 enzyme in flower tissues28.
Considering the previous genomic and cytogenomic data, we hypothesize that C. papaya sex chromosomes differ in several DNA sequences31, including svp-like and mdar 4 genes. Based on this, we aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya mitotic chromosomes. So, we start the mapping of single-copy and/or low-copy genes, differing from the previous C. papaya cytogenomics that were accomplished from rDNA genes and BAC sequences.
