The Cissus quadrangularis genome reveals its adaptive features in an arid habitat.

Cissus quadrangularis is a tetraploid species belonging to the Vitaceae family and is known for the Crassulacean acid metabolism (CAM) pathway in the succulent stem, while the leaves perform C3 photosynthesis. Here, we report a high-quality genome of C. quadrangularis comprising a total size of 679.2 Mb which was phased into two subgenomes. Genome annotation identified 51 857 protein-coding genes, while approximately 47.75% of the genome was composed of repetitive sequences. Gene expression ratios of two subgenomes demonstrated that the sub-A genome as the dominant subgenome played a vital role during the drought tolerance. Genome divergence analysis suggests that the tetraploidization event occurred around 8.9 million years ago. Transcriptome data revealed that pathways related to cutin, suberine, and wax metabolism were enriched in the stem during drought treatment, suggesting that these genes contributed to the drought adaption. Additionally, a subset of CAM-related genes displayed diurnal expression patterns in the succulent stems but not in leaves, indicating that stem-biased expression of existing genes contributed to the CAM evolution. Our findings provide insights into the mechanisms of drought adaptation and photosynthesis transition in plants.

A multiplexed plant-animal SNP array for selective breeding and species conservation applications.

Reliable and high-throughput genotyping platforms are of immense importance for identifying and dissecting genomic regions controlling important phenotypes, supporting selection processes in breeding programs, and managing wild populations and germplasm collections. Amongst available genotyping tools, single nucleotide polymorphism arrays have been shown to be comparatively easy to use and generate highly accurate genotypic data. Single-species arrays are the most commonly used type so far; however, some multi-species arrays have been developed for closely related species that share single nucleotide polymorphism markers, exploiting inter-species cross-amplification. In this study, the suitability of a multiplexed plant-animal single nucleotide polymorphism array, including both closely and distantly related species, was explored. The performance of the single nucleotide polymorphism array across species for diverse applications, ranging from intra-species diversity assessments to parentage analysis, was assessed. Moreover, the value of genotyping pooled DNA of distantly related species on the single nucleotide polymorphism array as a technique to further reduce costs was evaluated. Single nucleotide polymorphism performance was generally high, and species-specific single nucleotide polymorphisms proved suitable for diverse applications. The multi-species single nucleotide polymorphism array approach reported here could be transferred to other species to achieve cost savings resulting from the increased throughput when several projects use the same array, and the pooling technique adds another highly promising advancement to additionally decrease genotyping costs by half.

Genome-wide association identifies key loci controlling blackberry postharvest quality.

Introduction: Blackberry (Rubus subgenus Rubus) is a soft-fruited specialty crop that often suffers economic losses due to degradation in the shipping process. During transportation, fresh-market blackberries commonly leak, decay, deform, or become discolored through a disorder known as red drupelet reversion (RDR). Over the past 50 years, breeding programs have achieved better fruit firmness and postharvest quality through traditional selection methods, but the underlying genetic variation is poorly understood. Methods: We conducted a genome-wide association of fruit firmness and RDR measured in 300 tetraploid fresh-market blackberry genotypes from 2019-2021 with 65,995 SNPs concentrated in genic regions of the R. argutus reference genome. Results: Fruit firmness and RDR had entry-mean broad sense heritabilities of 68% and 34%, respectively. Three variants on homologs of polygalacturonase (PG), pectin methylesterase (PME), and glucan endo-1,3-ß-glucosidase explained 27% of variance in fruit firmness and were located on chromosomes Ra06, Ra01, and Ra02, respectively. Another PG homolog variant on chromosome Ra02 explained 8% of variance in RDR, but it was in strong linkage disequilibrium with 212 other RDR-associated SNPs across a 23 Mb region. A large cluster of six PME and PME inhibitor homologs was located near the fruit firmness quantitative trait locus (QTL) identified on Ra01. RDR and fruit firmness shared a significant negative correlation (r = -0.28) and overlapping QTL regions on Ra02 in this study. Discussion: Our work demonstrates the complex nature of postharvest quality traits in blackberry, which are likely controlled by many small-effect QTLs. This study is the first large-scale effort to map the genetic control of quantitative traits in blackberry and provides a strong framework for future GWAS. Phenotypic and genotypic datasets may be used to train genomic selection models that target the improvement of postharvest quality.

A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. "Hillquist").

Blackberries (Rubus spp.) are the fourth most economically important berry crop worldwide. Genome assemblies and annotations have been developed for Rubus species in subgenus Idaeobatus, including black raspberry (R. occidentalis), red raspberry (R. idaeus), and R. chingii, but very few genomic resources exist for blackberries and their relatives in subgenus Rubus. Here we present a chromosome-length assembly and annotation of the diploid blackberry germplasm accession "Hillquist" (R. argutus). "Hillquist" is the only known source of primocane-fruiting (annual-fruiting) in tetraploid fresh-market blackberry breeding programs and is represented in the pedigree of many important cultivars worldwide. The "Hillquist" assembly, generated using Pacific Biosciences long reads scaffolded with high-throughput chromosome conformation capture sequencing, consisted of 298 Mb, of which 270 Mb (90%) was placed on 7 chromosome-length scaffolds with an average length of 38.6 Mb. Approximately 52.8% of the genome was composed of repetitive elements. The genome sequence was highly collinear with a novel maternal haplotype-resolved linkage map of the tetraploid blackberry selection A-2551TN and genome assemblies of R. chingii and red raspberry. A total of 38,503 protein-coding genes were predicted, of which 72% were functionally annotated. Eighteen flowering gene homologs within a previously mapped locus aligning to an 11.2 Mb region on chromosome Ra02 were identified as potential candidate genes for primocane-fruiting. The utility of the "Hillquist" genome has been demonstrated here by the development of the first genotyping-by-sequencing-based linkage map of tetraploid blackberry and the identification of possible candidate genes for primocane-fruiting. This chromosome-length assembly will facilitate future studies in Rubus biology, genetics, and genomics and strengthen applied breeding programs.

A genome for Cissus illustrates features underlying its evolutionary success in dry savannas.

Cissus is the largest genus in Vitaceae and is mainly distributed in the tropics and subtropics. Crassulacean acid metabolism (CAM), a photosynthetic adaptation to the occurrence of succulent leaves or stems, indicates that convergent evolution occurred in response to drought stress during species radiation. Here we provide the chromosomal level assembly of Cissus rotundifolia (an endemic species in Eastern Africa) and a genome-wide comparison with grape to understand genome divergence within an ancient eudicot family. Extensive transcriptome data were produced to illustrate the genetics underpinning C. rotundifolia's ecological adaption to seasonal aridity. The modern karyotype and smaller genome of C. rotundifolia (n = 12, 350.69 Mb/1C), which lack further whole-genome duplication, were mainly derived from gross chromosomal rearrangements such as fusions and segmental duplications, and were sculpted by a very recent burst of retrotransposon activity. Bias in local gene amplification contributed to its remarkable functional divergence from grape, and the specific proliferated genes associated with abiotic and biotic responses (e.g. HSP-20, NBS-LRR) enabled C. rotundifolia to survive in a hostile environment. Reorganization of existing enzymes of CAM characterized as diurnal expression patterns of relevant genes further confer the ability to thrive in dry savannas.

Physiological and biochemical responses of strawberry crown and leaf tissues to freezing stress.

BACKGROUND: In northern Iran and other cold regions, winter freezing injury and resultant yield instability are major limitations to strawberry production. However, there is scarcity of information on the physiological and biochemical responses of strawberry cultivars to freezing stress. This study aimed to investigate the physiological and biochemical responses of strawberry cultivars (Tennessee Beauty, Blakemore, Kurdistan, Queen Elisa, Chandler, Krasnyy Bereg, and Yalova) to different freezing temperature treatments (- 5, - 10, - 15, - 20, and - 25 °C) under controlled conditions. RESULTS: All measured physiological and biochemical features were significantly affected by the interaction effect between low temperatures and cultivars. Tennessee Beauty showed the highest RWC at - 25 °C. The highest Fv/Fm was observed in Queen Elisa. Krasnyy Bereg had the least freezing injury (FI) in crown and leaf, while Yalova and Chandler showed the highest crown and leaf FI, respectively. At - 20 to - 25 °C, the highest carbohydrates contents of crown and leaf were noted in Blakemore and Krasnyy Bereg cultivars, respectively. The Yalova showed the highest protein content in both crown and leaf tissues at - 25 °C. The Tennessee Beauty and Blackmore cultivars showed the highest proline in crowns and leaves at - 15 °C, respectively. The highest ThioBarbituric Acid Reactive Substances (TBARS) contents in the crown and leaf were observed in Kurdistan and Queen Elisa, respectively. Queen Elisa and Krasnyy Bereg cultivars showed SOD and POD peaks in the crown at - 15 °C, respectively. CONCLUSION: Freezing stress was characterized by decreased Fv/Fm and RWC, and increased FI, TBARS, total carbohydrates, total proteins, proline content, and antioxidant enzyme activity. The extent of changes in above mentioned traits was cultivar dependent. FI and TBARS were the best traits among destructive parameters for evaluating freezing tolerance. Moreover, maximum quantum yield of PSII (Fv/Fm index), as non-destructive parameters, showed a significant efficiency in rapid assessment for screening of freezing tolerant strawberry cultivars. The cultivars Krasnyy Bereg, Queen Elisa, and Kurdistan were the most tolerant cultivars to freezing stress. These cultivars can be used as parents in breeding programs to develop new freezing tolerant cultivars.

Reproductive developmental transcriptome analysis of Tripidium ravennae (Poaceae).

BACKGROUND: Tripidium ravennae is a cold-hardy, diploid species in the sugarcane complex (Poaceae subtribe Saccharinae) with considerable potential as a genetic resource for developing improved bioenergy and ornamental grasses. An improved understanding of the genetic regulation of reproductive processes (e.g., floral induction, inflorescence development, and seed development) will enable future applications of precision breeding and gene editing of floral and seed development. In particular, the ability to silence reproductive processes would allow for developing seedless forms of valuable but potentially invasive plants. The objective of this research was to characterize the gene expression environment of reproductive development in T. ravennae. RESULTS: During the early phases of inflorescence development, multiple key canonical floral integrators and pathways were identified. Annotations of type II subfamily of MADS-box transcription factors, in particular, were over-represented in the GO enrichment analyses and tests for differential expression (FDR p-value < 0.05). The differential expression of floral integrators observed in the early phases of inflorescence development diminished prior to inflorescence determinacy regulation. Differential expression analysis did not identify many unique genes at mid-inflorescence development stages, though typical biological processes involved in plant growth and development expressed abundantly. The increase in inflorescence determinacy regulatory elements and putative homeotic floral development unigenes at mid-inflorescence development coincided with the expression of multiple meiosis annotations and multicellular organism developmental processes. Analysis of seed development identified multiple unigenes involved in oxidative-reductive processes. CONCLUSION: Reproduction in grasses is a dynamic system involving the sequential coordination of complex gene regulatory networks and developmental processes. This research identified differentially expressed transcripts associated with floral induction, inflorescence development, and seed development in T. ravennae. These results provide insights into the molecular regulation of reproductive development and provide a foundation for future investigations and analyses, including genome annotation, functional genomics characterization, gene family evolutionary studies, comparative genomics, and precision breeding.

RNA-Seq and Gene Regulatory Network Analyses Uncover Candidate Genes in the Early Defense to Two Hemibiotrophic Colletorichum spp. in Strawberry.

Two hemibiotrophic pathogens, Colletotrichum acutatum (Ca) and C. gloeosporioides (Cg), cause anthracnose fruit rot and anthracnose crown rot in strawberry (Fragaria × ananassa Duchesne), respectively. Both Ca and Cg can initially infect through a brief biotrophic phase, which is associated with the production of intracellular primary hyphae that can infect host cells without causing cell death and establishing hemibiotrophic infection (HBI) or quiescent (latent infections) in leaf tissues. The Ca and Cg HBI in nurseries and subsequent distribution of asymptomatic infected transplants to fruit production fields is the major source of anthracnose epidemics in North Carolina. In the absence of complete resistance, strawberry varieties with good fruit quality showing rate-reducing resistance have frequently been used as a source of resistance to Ca and Cg. However, the molecular mechanisms underlying the rate-reducing resistance or susceptibility to Ca and Cg are still unknown. We performed comparative transcriptome analyses to examine how rate-reducing resistant genotype NCS 10-147 and susceptible genotype 'Chandler' respond to Ca and Cg and identify molecular events between 0 and 48 h after the pathogen-inoculated and mock-inoculated leaf tissues. Although plant response to both Ca and Cg at the same timepoint was not similar, more genes in the resistant interaction were upregulated at 24 hpi with Ca compared with those at 48 hpi. In contrast, a few genes were upregulated in the resistant interaction at 48 hpi with Cg. Resistance response to both Ca and Cg was associated with upregulation of MLP-like protein 44, LRR receptor-like serine/threonine-protein kinase, and auxin signaling pathway, whereas susceptibility was linked to modulation of the phenylpropanoid pathway. Gene regulatory network inference analysis revealed candidate transcription factors (TFs) such as GATA5 and MYB-10, and their downstream targets were upregulated in resistant interactions. Our results provide valuable insights into transcriptional changes during resistant and susceptible interactions, which can further facilitate assessing candidate genes necessary for resistance to two hemibiotrophic Colletotrichum spp. in strawberry.

A Rosaceae Family-Level Approach To Identify Loci Influencing Soluble Solids Content in Blackberry for DNA-Informed Breeding.

A Rosaceae family-level candidate gene approach was used to identify genes associated with sugar content in blackberry (Rubus subgenus Rubus). Three regions conserved among apple (Malus × domestica), peach (Prunus persica), and alpine strawberry (Fragaria vesca) were identified that contained previously detected sweetness-related quantitative trait loci (QTL) in at least two of the crops. Sugar related genes from these conserved regions and 789 sugar-associated apple genes were used to identify 279 Rubus candidate transcripts. A Hyb-Seq approach was used in conjunction with PacBio sequencing to generate haplotype level sequence information of sugar-related genes for 40 cultivars with high and low soluble solids content from the University of Arkansas and USDA blackberry breeding programs. Polymorphisms were identified relative to the 'Hillquist' blackberry (R. argutus) and ORUS 4115-3 black raspberry (R. occidentalis) genomes and tested for their association with soluble solids content (SSC). A total of 173 alleles were identified that were significantly (α = 0.05) associated with SSC. KASP genotyping was conducted for 92 of these alleles on a validation set of blackberries from each breeding program and 48 markers were identified that were significantly associated with SSC. One QTL, qSSC-Ruh-ch1.1, identified in both breeding programs accounted for an increase of 1.5 °Brix and the polymorphisms were detected in the intron space of a sucrose synthase gene. This discovery represents the first environmentally stable sweetness QTL identified in blackberry. The approach demonstrated in this study can be used to develop breeding tools for other crops that have not yet benefited directly from the genomics revolution.

Differential methylation and expression of HUA1 ortholog in three sex types of papaya.

Papaya is trioecious and an excellent system for studying sex determination and differentiation in plants. An ortholog of HUA1, CpHUA1, a gene controlling stamen and carpel development in Arabidopsis, was cloned and characterized in papaya. CpHUA1 consists of 12 exons with full genomic length of 19,313 bp in male AU9 and 19,312 bp in hermaphrodite SunUp, whereas the Arabidopsis HUA1 consists of 12 exons with full genomic length of 4300 bp. All the 324 SNPs between male and hermaphrodite varieties are in the 11th intron, which spans 8.5 kb. Quantitative RT-PCR revealed that CpHUA1 expression is highly elevated in carpels, suggesting that CpHUA1 may be involved in sex differentiation gene network. Southern blot analysis revealed a distinct restriction pattern in male AU9 compared to hermaphrodite Kapoho and SunUp, despite high DNA sequence identity and sharing of all but two EcoR I restriction sites in genomic CpHUA1 sequences of AU9 and SunUp. The methylation of cytosine at one restriction site in male but not in other two sex types may result in distinct restriction pattern of EcoR I in southern blot result. Bisulfite sequencing showed differential methylation of CpHUA1 among sex types, particularly the enrichment of sex-specific methylation in 9th and 11th intron. The methylation difference in cold stress induced male to hermaphrodite mutant mostly observed in the CHH context of CpHUA1, but no methylation difference detected in CHH context in other sex types, which may indicate the role of methylation in CHH context of CpHUA1 in temperature-related stress response and sex reversal.

Biochemical composition and antioxidant activity affected by spraying potassium sulfate in black grape (Vitis vinifera L. cv. Rasha).

BACKGROUND: The physiological and metabolic processes involved with grapevine growth and production are influenced by key macro- and micronutrients. Potassium is an essential plant nutrient that affects growth and fruit quality. In this study, the impact of foliar spraying of potassium sulfate (K2 SO4 ) on qualitative characteristics of grape berries was evaluated in the cultivar 'Rasha', a commonly cultivated cultivar in Kurdistan province of Iran. Leaves of the fully grown vines were sprayed with each of the 1.5 and 3 g L-1 K2 SO4 solutions once (1 month after petal senescence) and twice (15 days after first spraying). The control plants were sprayed with distilled water. Various biochemical contents and enzyme activities on the ripe berries were analyzed. RESULTS: Significant increases in anthocyanin, total protein content, and antioxidant enzyme activities were observed in the berries treated twice with 3 g L-1 K2 SO4 . Concentrations of total carbohydrate, phenol, and antioxidant activity in berries sprayed with K2 SO4 were higher than in the controls. We observed a strong correlation between antioxidant activity and different phenolic compounds. CONCLUSION: These findings suggest that K2 SO4 treatment influences biosynthesis of phenolic compounds and antioxidant enzymes. Thus, treatment by K2 SO4 could improve nutritional and qualitative attributes of grape. © 2018 Society of Chemical Industry.

Origin and domestication of papaya Yh chromosome.

Sex in papaya is controlled by a pair of nascent sex chromosomes. Females are XX, and two slightly different Y chromosomes distinguish males (XY) and hermaphrodites (XY(h)). The hermaphrodite-specific region of the Y(h) chromosome (HSY) and its X chromosome counterpart were sequenced and analyzed previously. We now report the sequence of the entire male-specific region of the Y (MSY). We used a BAC-by-BAC approach to sequence the MSY and resequence the Y regions of 24 wild males and the Y(h) regions of 12 cultivated hermaphrodites. The MSY and HSY regions have highly similar gene content and structure, and only 0.4% sequence divergence. The MSY sequences from wild males include three distinct haplotypes, associated with the populations' geographic locations, but gene flow is detected for other genomic regions. The Y(h) sequence is highly similar to one Y haplotype (MSY3) found only in wild dioecious populations from the north Pacific region of Costa Rica. The low MSY3-Y(h) divergence supports the hypothesis that hermaphrodite papaya is a product of human domestication. We estimate that Y(h) arose only ∼ 4000 yr ago, well after crop plant domestication in Mesoamerica >6200 yr ago but coinciding with the rise of the Maya civilization. The Y(h) chromosome has lower nucleotide diversity than the Y, or the genome regions that are not fully sex-linked, consistent with a domestication bottleneck. The identification of the ancestral MSY3 haplotype will expedite investigation of the mutation leading to the domestication of the hermaphrodite Y(h) chromosome. In turn, this mutation should identify the gene that was affected by the carpel-suppressing mutation that was involved in the evolution of males.

Sex determination in flowering plants: papaya as a model system.

Unisexuality in flowering plants evolved from a hermaphrodite ancestor. Transition from hermaphrodite to unisexual flowers has occurred multiple times across the different lineages of the angiosperms. Sexuality in plants is regulated by genetic, epigenetic and physiological mechanisms. The most specialized mechanism of sex determination is sex chromosomes. The sex chromosomes ensure the stable segregation of sexual phenotypes by preventing the recombination of sex determining genes. Despite continuous efforts, sex determining genes of dioecious plants have not yet been cloned. Concerted efforts with various model systems are necessary to understand the complex mechanism of sex determination in plants. Papaya (Carica papaya L.) is a tropical fruit tree with three sex forms, male, hermaphrodite, and female. Sexuality in papaya is determined by an XY chromosome system that is in an early evolutionary stage. The male and hermaphrodite of papaya are controlled by two different types of Y chromosomes: Y and Y(h). Large amounts of information in the area of genetics, genomics, and epigenetics of papaya have been accumulated over the last few decades. Relatively short lifecycle, small genome size, and readily available genetic and genomic resources render papaya an excellent model system to study sex determination and sex chromosomes in flowering plants.

Sex specific expression and distribution of small RNAs in papaya.

BACKGROUND: Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored. RESULTS: We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot. CONCLUSION: By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya.

BACKGROUND: The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. RESULTS: We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff's purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. CONCLUSIONS: We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants.

Rapid divergence and expansion of the X chromosome in papaya.

X chromosomes have long been thought to conserve the structure and gene content of the ancestral autosome from which the sex chromosomes evolved. We compared the recently evolved papaya sex chromosomes with a homologous autosome of a close relative, the monoecious Vasconcellea monoica, to infer changes since recombination stopped between the papaya sex chromosomes. We sequenced 12 V. monoica bacterial artificial chromosomes, 11 corresponding to the papaya X-specific region, and 1 to a papaya autosomal region. The combined V. monoica X-orthologous sequences are much shorter (1.10 Mb) than the corresponding papaya region (2.56 Mb). Given that the V. monoica genome is 41% larger than that of papaya, this finding suggests considerable expansion of the papaya X; expansion is supported by a higher repetitive sequence content of the X compared with the papaya autosomal sequence. The alignable regions include 27 transcript-encoding sequences, only 6 of which are functional X/V. monoica gene pairs. Sequence divergence from the V. monoica orthologs is almost identical for papaya X and Y alleles; the Carica-Vasconcellea split therefore occurred before the papaya sex chromosomes stopped recombining, making V. monoica a suitable outgroup for inferring changes in papaya sex chromosomes. The papaya X and the hermaphrodite-specific region of the Y(h) chromosome and V. monoica have all gained and lost genes, including a surprising amount of changes in the X.

Sequencing papaya X and Yh chromosomes reveals molecular basis of incipient sex chromosome evolution.

Sex determination in papaya is controlled by a recently evolved XY chromosome pair, with two slightly different Y chromosomes controlling the development of males (Y) and hermaphrodites (Y(h)). To study the events of early sex chromosome evolution, we sequenced the hermaphrodite-specific region of the Y(h) chromosome (HSY) and its X counterpart, yielding an 8.1-megabase (Mb) HSY pseudomolecule, and a 3.5-Mb sequence for the corresponding X region. The HSY is larger than the X region, mostly due to retrotransposon insertions. The papaya HSY differs from the X region by two large-scale inversions, the first of which likely caused the recombination suppression between the X and Y(h) chromosomes, followed by numerous additional chromosomal rearrangements. Altogether, including the X and/or HSY regions, 124 transcription units were annotated, including 50 functional pairs present in both the X and HSY. Ten HSY genes had functional homologs elsewhere in the papaya autosomal regions, suggesting movement of genes onto the HSY, whereas the X region had none. Sequence divergence between 70 transcripts shared by the X and HSY revealed two evolutionary strata in the X chromosome, corresponding to the two inversions on the HSY, the older of which evolved about 7.0 million years ago. Gene content differences between the HSY and X are greatest in the older stratum, whereas the gene content and order of the collinear regions are identical. Our findings support theoretical models of early sex chromosome evolution.