Chromosomal location and gene paucity of the male specific region on papaya Y chromosome

Sex chromosomes in flowering plants evolved recently and many of them remain homomorphic, including those in papaya. We investigated the chromosomal location of papaya’s small male specific region of the hermaphrodite Y (Y^h) chromosome (MSY) and its genomic features. We conducted chromosome fluorescence in situ hybridization mapping of Y^h-specific bacterial artificial chromosomes (BACs) and placed the MSY near the centromere of the papaya Y chromosome. Then we sequenced five MSY BACs to examine the genomic features of this specialized region, which resulted in the largest collection of contiguous genomic DNA sequences of a Y chromosome in flowering plants. Extreme gene paucity was observed in the papaya MSY with no functional gene identified in 715 kb MSY sequences. A high density of retroelements and local sequence duplications were detected in the MSY that is suppressed for recombination. Location of the papaya MSY near the centromere might have provided recombination suppression and fostered paucity of genes in the male specific region of the Y chromosome. Our findings provide critical information for deciphering the sex chromosomes in papaya and reference information for comparative studies of other sex chromosomes in animals and plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accumulation of interspersed and sex-specific repeats in the non-recombining region of papaya sex chromosomes

Background

The papaya Y chromosome has undergone a degenerative expansion from its ancestral autosome, as a consequence of recombination suppression in the sex determining region of the sex chromosomes. The non-recombining feature led to the accumulation of repetitive sequences in the male- or hermaphrodite-specific regions of the Y or the Y^h chromosome (MSY or HSY). Therefore, repeat composition and distribution in the sex determining region of papaya sex chromosomes would be informative to understand how these repetitive sequences might be involved in the early stages of sex chromosome evolution.

Results

Detailed composition of interspersed, sex-specific, and tandem repeats was analyzed from 8.1 megabases (Mb) HSY and 5.3 Mb corresponding X chromosomal regions. Approximately 77% of the HSY and 64% of the corresponding X region were occupied by repetitive sequences. Ty3-gypsy retrotransposons were the most abundant interspersed repeats in both regions. Comparative analysis of repetitive sequences between the sex determining region of papaya X chromosome and orthologous autosomal sequences of Vasconcellea monoica, a close relative of papaya lacking sex chromosomes, revealed distinctive differences in the accumulation of Ty3-Gypsy, suggesting that the evolution of the papaya sex determining region may accompany Ty3-Gypsy element accumulation. In total, 21 sex-specific repeats were identified from the sex determining region; 20 from the HSY and one from the X. Interestingly, most HSY-specific repeats were detected in two regions where the HSY expansion occurred, suggesting that the HSY expansion may result in the accumulation of sex-specific repeats or that HSY-specific repeats might play an important role in the HSY expansion. The analysis of simple sequence repeats (SSRs) revealed that longer SSRs were less abundant in the papaya sex determining region than the other chromosomal regions.

Conclusion

Major repetitive elements were Ty3-gypsy retrotransposons in both the HSY and the corresponding X. Accumulation of Ty3-Gypsy retrotransposons in the sex determining region of papaya X chromosome was significantly higher than that in the corresponding region of V. monoica, suggesting that Ty3-Gypsy could be crucial for the expansion and evolution of the sex determining region in papaya. Most sex-specific repeats were located in the two HSY expansion regions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-335) contains supplementary material, which is available to authorized users.     

Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities.

Abbreviations

ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - – Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - – Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

Carpeloidy in flower evolution and diversification: a comparative study in Carica papaya and Arabidopsis thaliana

Background and Aims

Bisexual flowers of Carica papaya range from highly regular flowers to morphs with various fusions of stamens to the ovary. Arabidopsis thaliana sup1 mutants have carpels replaced by chimeric carpel–stamen structures. Comparative analysis of stamen to carpel conversions in the two different plant systems was used to understand the stage and origin of carpeloidy when derived from stamen tissues, and consequently to understand how carpeloidy contributes to innovations in flower evolution.

Methods

Floral development of bisexual flowers of Carica was studied by scanning electron microscopy and was compared with teratological sup mutants of A. thaliana.

Key Results

In Carica development of bisexual flowers was similar to wild (unisexual) forms up to locule initiation. Feminization ranges from fusion of stamen tissue to the gynoecium to complete carpeloidy of antepetalous stamens. In A. thaliana, partial stamen feminization occurs exclusively at the flower apex, with normal stamens forming at the periphery. Such transformations take place relatively late in development, indicating strong developmental plasticity of most stamen tissues. These results are compared with evo-devo theories on flower bisexuality, as derived from unisexual ancestors. The Arabidopsis data highlight possible early evolutionary events in the acquisition of bisexuality by a patchy transformation of stamen parts into female parts linked to a flower axis-position effect. The Carica results highlight tissue-fusion mechanisms in angiosperms leading to carpeloidy once bisexual flowers have evolved.

Conclusions

We show two different developmental routes leading to stamen to carpel conversions by late re-specification. The process may be a fundamental aspect of flower development that is hidden in most instances by developmental homeostasis.     

Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans

Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of ~500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (~160 kDa) and M2 (~250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 5′ seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.     

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.     

In vitro expression of partial resistance toPhytophthora palmivora by shoot cultures of papaya

Shoot apices ofCarica papaya were multiplied in vitro on solidified nutrient media supplemented with -naphthyl-acetic acid and 6-benzylaminopurine. The micropropagated shoots were inoculated in vitro, through a stem wound, with a sporangial suspension (1.2×10⁴ sporangia ml^-1) ofPhytophthora palmivora. The symptoms exhibited by the shoots in vitro were similar to those described previously for infection of the whole plant in the field. The time taken for the host tissue to become brown and to wilt and the time to sporulation of the pathogen were all recorded for each shoot of four varieties of papaya challenged with each of ten isolates ofP. palmivora. Significant differences were observed between host-pathogen combinations for these variables and host-specificity was detected amongst the isolates ofP. palmivora. The time taken for the shoot to wilt was positively correlated with the time to sporulation of the isolated but both these variables were negatively correlated with the time to browning of the shoot. In vitro selection for disease resistance will be useful during breeding programmes involving papaya genotypes which are maintained through clonal propagation.     

Detection of three wound-induced proteins in papaya latex

The effects of routine mechanical wounding for latex collection from unripe fruits of the tropical Carica papaya tree were investigated. For that purpose, the protein composition of three different latexes was analyzed. The first one, commercially available, was provided in the form of a spray-dried powder, the second one was harvested from fully grown but unripe papaya fruits that are regularly tapped for latex production and the last one, was obtained from similar fruits wounded for the first time. Repeated mechanical wounding was found to profoundly affect the protein content of the latex inducing, among others, activation of papain. Regularly tapped latexes also accumulated several low molecular weight proteins not yet identified, as well as three proteins identified as a trypsin inhibitor, a class-II chitinase and a glutaminyl cyclase on the basis of their enzymatic or inhibitory activities and chromatographic elution profiles. This latter was found here, for the first time, to be a wound-induced protein. The roles of these proteins in the plant defense mechanism are discussed.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

Analysis of papaya BAC end sequences reveals first insights into the organization of a fruit tree genome

Papaya (Carica papaya L.) is a major tree fruit crop of tropical and subtropical regions with an estimated genome size of 372 Mbp. We present the analysis of 4.7% of the papaya genome based on BAC end sequences (BESs) representing 17 million high-quality bases. Microsatellites discovered in 5,452 BESs and flanking primer sequences are available to papaya breeding programs at . Sixteen percent of BESs contain plant repeat elements, the vast majority (83.3%) of which are class I retrotransposons. Several novel papaya-specific repeats were identified. Approximately 19.1% of the BESs have homology to Arabidopsis cDNA. Increasing numbers of completely sequenced plant genomes and BES projects enable novel approaches to comparative plant genomics. Paired BESs of Carica, Arabidopsis, Populus, Brassica and Lycopersicon were mapped onto the completed genomes of Arabidopsis and Populus. In general the level of microsynteny was highest between closely related organisms. However, papaya revealed a higher degree of apparent synteny with the more distantly related poplar than with the more closely related Arabidopsis. This, as well as significant colinearity observed between peach and poplar genome sequences, support recent observations of frequent genome rearrangements in the Arabidopsis lineage and suggest that the poplar genome sequence may be more useful for elucidating the papaya and other rosid genomes. These insights will play a critical role in selecting species and sequencing strategies that will optimally represent crop genomes in sequence databases.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Chun Wan J. Lai and Qingyi Yu have contributed equally to this work.     

Monoclonal antibodies to the two most basic papaya proteinases

The proteinases fromCarica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.     

Clonal propagation of papaya in vitro

A procedure for the rapid tissue culture propagation of papaya is being developed. Tissue culture methods using apices of nursery and orchard trees of Carica papaya cv. Sunrise Solo were evaluated. The explants were established in a modified Murashige and Tucker (1969) basal medium with half-strength inorganic salts, 0.5mgl^-1 6-benzylaminopurine (BA) and 0.2mgl^-1 naphthaleneacetic acid (NAA). Established explants were transferred to a proliferation medium consisting of Murashige and Tucker (1969) basal medium, 0.5mgl^-1 BA and 0.1mgl^-1 NAA, which caused extensive multiplication of shoots. Rooting was induced at a higher frequency by subculturing plantlets onto media with indole-3-butyric acid (IBA) than with NAA.     

鱼类性别与性别鉴定

性别分化和性别决定相互联系又有所区别,具双向潜力的未分化性腺经过程序性发生的一系列事件,发育成精巢或卵巢,并出现第二性征的过程称为性别分化,而性别决定则是确定性分化方向的方式。       

A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings

Female plants of several dioecious angiosperms are commercially valued for production of fruits or seeds, viz. papaya, nutmeg, pistachio, kiwi fruit and jojoba. To make the cultivation profitable it is necessary to grow more female than male plants. To discriminate between male and female plants, sex-specific molecular markers have been identified in a few dioecious species such as Silene and pistachio. However, accurate and convenient sex diagnostic methods for early sexing of seedlings are not available to date. For the first time, we report here a PCR-based Seedling Sex Diagnostic Assay (SSDA) specially designed for early sexing of papaya seedlings. We have developed a male-specific SCAR marker in papaya by cloning a male-specific RAPD (831 bp) fragment and designing longer primers. The potential of this SCAR marker is further exploited to develop a simplified and highly accurate sex diagnostic assay by (1) including an internal PCR control, (2) following a single-step DNA extraction procedure and (3) optimising the PCR conditions to simultaneously amplify male-specific and control bands from the crude leaf extract. This diagnostic approach would be of great commercial significance to papaya growers as well as to seed companies and plant nurseries for early identification of female seedlings of dioecious species. In principle, this experimental design could be easily applied to molecular analysis of any agriculturally important trait for which specific DNA probes could be identified and hence opens new avenues of research in the field of genetic diagnostics of plants.     

番木瓜性别决定及其鉴定研究新进展

番木瓜有3种基本性别类型,性别遗传较为复杂.就其植株的多型性表现、性别决定及其鉴定研究、连锁遗传图的构建、分子标记辅助选择技术和花器的发育等方面的研究进展进行了综述,并对番木瓜性别鉴定的应用前景做了展望.     

The influences of PRG-1 on the expression of small RNAs and mRNAs

Background

In metazoans, Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that mutation of prg-1 causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.

Results

In this study, we wanted to systematically demonstrate the function of PRG-1 in the regulation of small RNAs and their targets. By analyzing small RNAs and mRNAs with and without a mutation in prg-1 during C. elegans development, we demonstrated that (1) mutation of prg-1 leads to a decrease in the expression of 21U-RNAs, and causes 35 ~ 40% of miRNAs to be down-regulated; (2) in C. elegans, approximately 3% (6% in L4) of protein-coding genes are differentially expressed after mutating prg-1, and 60 ~ 70% of these substantially altered protein-coding genes are up-regulated; (3) the target genes of the down-regulated miRNAs and the candidate target genes of the down-regulated 21U-RNAs are enriched in the up-regulated protein-coding genes; and (4) PRG-1 regulates protein-coding genes by down-regulating small RNAs (miRNAs and 21U-RNAs) that target genes that participate in the development of C. elegans.

Conclusions

In prg-1-mutated C. elegans, the expression of miRNAs and 21U-RNAs was reduced, and the protein-coding targets, which were associated with the development of C. elegans, were up-regulated. This may be the mechanism underlying PRG-1 function.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-321) contains supplementary material, which is available to authorized users.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

两栖动物性染色体的多样性及其进化机制的研究进展

两栖动物性别决定类型和性染色体具有多样性的特点。在已发现异形性染色体两栖动物中,大部分物种Y或W染色体大于其对应的X或Z染色体,少数物种具有高度分化的Y或W染色体。同时两栖动物类群内基因组大小差异大,性染色体间分子水平上也存在差异。高频转换、偶然重组和染色体重排可能是两栖动物性染色体进化过程中的关键机制。本综述通过对两栖动物性染色体进化的深入探讨,揭示其遗传性别决定的机理,有助于对两栖动物性别人工调控的进一步探索。