Origin and domestication of papaya Yh chromosome

Gender in papaya is genetically controlled by a sex-linked region that behaves like an XY sex chromosome, and maleness versus hermaphroditism is controlled by slightly different sex-specific Y chromosome regions, Yh (HSY) in hermaphrodites and Y (MSY) in males. Both the HSY and MSY are ∼8.1 Mb (∼15% of the largest papaya chromosome, Chromosome 1), and recombination with the X is suppressed, so that hermaphrodite- and male-specific regions can be defined (Liu et al. 2004; Wang et al. 2012). The corresponding region of the X is only 3.5 Mb, and both the Y and Yh have increased repeat sequence content, changed physical structure, and different gene content (Wang et al. 2012). Any combination of the Y and Yh chromosomes (YY, YYh, or YhYh) is inviable, and the embryos abort 25–50 d after pollination, suggesting that the Y chromosome types are similar and that both are missing an essential gene that is functional in the X. Wild papaya populations are dioecious, with one-half male and one-half female plants, whereas cultivated papaya is predominantly gynodioecious, with two-thirds hermaphrodite and one-third female plants, though dioecious varieties do exist. There is no direct archaeological evidence for the center of origin of papaya, but the presence of natural populations in Mexico and Central America and the cultivation in Mexico and Belize predating the Spaniards suggest a Mesoamerican origin (Colunga-GarciaMarin and Zizumbo-Villarreal 2004). William Storey (1976) wrote, “Since dioecism seems to be the evolutionary norm in Caricaceae, it is possible that ambisexual forms owe their continued existence to human selection.” This hypothesis was previously rejected after analysis of a pair of X- and Y-specific bacterial artificial chromosomes (BACs) from an improved (but not cultivated) dioecious variety, AU9, and their homologous BAC from the gynodioecious cultivar SunUp. The resulting molecular dating estimate suggested that the Y chromosomes of males and hermaphrodites diverged ∼73,000 yr ago (Yu et al. 2008), long before the origin of agriculture in Mesoamerica ∼6200 yr ago (Pope et al. 2001). It is worth further testing to establish the age of the HSY, because if the HSY diverged very recently from the MSY, papaya could offer the opportunity to identify the gene or genes responsible for the gender difference. Such genes would be candidates for the female suppressor involved in the early stages of sex chromosome evolution in this species. The sex chromosomes in other organisms, such as mammals, are ancient (Veyrunes et al. 2008; Bellott et al. 2014; Cortez et al. 2014), and the genes involved in their initial evolution cannot be identified, because many subsequent changes, including gene gains and losses, have occurred (Hughes et al. 2010, 2012; Zhou et al. 2014). The younger sex chromosomes of some species of plants, fish, and insects may provide insights into the mechanisms involved in the early stages of the evolution of separate sexes and of sex chromosomes (Delph et al. 2010; Zhou and Bachtrog 2012). To understand the origin and accurately estimate the divergence time of the HSY and MSY, sequencing of HSY and MSY sequences is needed. Here, we describe complete sequencing of the AU9 MSY. Moreover, sequencing multiple individuals of both males and hermaphrodites is necessary, because the origin or origins of hermaphrodites are unknown, and the AU9 MSY might not be closely related to the ancestor of the HSY, as we indeed show to be the case. Moreover, sequences from multiple individuals are needed, because the HSY and MSY of any single varieties may include mutations in genes that are not responsible for the phenotypic difference in their gender; only fixed differences between the Yh and Y are candidates for causing the functional difference (though variants in individual varieties can help exclude candidate genes because the female-suppressor is dominant, causing maleness or hermaphroditism in the heterozygous XY or XYh state, respectively). The objectives of this study were to (1) sequence the MSY as a necessary step toward identifying the genes distinguishing the Y and Yh chromosome; (2) determine which Y chromosome, the Y or Yh, is ancestral in papaya and identify the origin of the derived MSY or HSY by resequencing male and hermaphrodite genomes sampled from wild and domesticated populations; (3) use the sequences from wild populations to estimate diversity and thus test the domestication hypothesis independently of the molecular dating based on MSY–HSY divergence; and (4) use the sequences from wild populations to discover genes with fixed differences between the sets of Y-specific sequences of males and hermaphrodites, where the differences may affect gene functions, to generate candidate genes for the Y-linked carpel suppressor. The Yh chromosome of hermaphrodites differs from the Y of males by lacking a female suppressor. The finding that the papaya Yh and Y diverged very recently is therefore important, because it suggests that the number of fixed differences between the two chromosomes may be small. Sequences from papaya males from natural populations therefore offer the opportunity to identify the gene or genes responsible for the gender difference.