Development and application of microsatellite markers for genomic analysis of papaya

Papaya has a relatively small genome, displays high levels of phenotypic diversity, and is amenable to transformation, making it attractive as a fruit tree model system. The high level of phenotypic diversity seen among papaya cultivars in the field does not correlate with the low levels of genotypic polymorphism thus far elucidated. The highly mutable nature of microsatellites or simple sequence repeats (SSRs) make them potentially powerful markers for distinguishing deoxyribonucleic acid (DNA) polymorphisms between closely related genotypes. Genomic research for papaya has resulted in a significant quantity of sequence data. We mined 28.1 Mb of bacterial artificial chromosomes end sequences, 5.8 Mb of complementary DNA, and 1.6 Mb of random genomic sequences for SSRs. We generated 938 SSR markers and tested for polymorphism among seven varieties that had been used to produce five mapping populations. The level of polymorphism was highest for Kaek Dum × 2H94 with 210 markers, followed by UH928 × SunUp with 194, AU9 × SunUp with 189, UH918 × SunUp with 177, and Kapoho × SunUp displaying the lowest level with 97. Variation in levels of polymorphism, motif predominance, and motif length between the genomic and genic fractions indicated differential selection pressures acting on the microsatellites in these two fractions. The microsatellites developed in this study will greatly assist in the genetic and physical mapping of the papaya genome as well as enhance breeders’ ability to improve the crop. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monoclonal antibodies against staphylococcal enterotoxins

MAb anti-Staphylococcal enterotoxins (MAb-SE) were produced in mice with a mixture of reference SE types A, B, C₁ and D at a concentration of 1 g each per mouse; the last booster injection was by intrasplenic route. Nine clones were chosen, two produced anti-SEB and anti-SED, one anti-SEA and anti-SEB, two anti-SED, two anti-SEB and one produced anti-SEC_1. The MAb-SE were partially purified as judged by PAGE–SDS. The partially purified antibodies could demonstrate the presence of SE in milk samples containing 0.5 g of toxin m l^–1.     

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.     

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.     

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     

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.     

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.     

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.     

A genetic linkage map of papaya based on randomly amplified polymorphic DNA markers

A genetic linkage map of papaya (Carica papaya L.) was constructed using randomly amplified polymorphic DNA (RAPD) markers and a F₂ population derived from a University of Hawaii UH breeding line 356 x Sunrise cross. A total of 596 10-mer primers were screened, and 96 polymorphisms were detected. At LOD 4.0, 62 of these markers mapped to 11 linkage groups comprising 999.3 cM. About 80% of the markers conformed to expected Mendelian segregation ratios. We have mapped the locus that determines sex to a 14-cM region flanked by RAPD markers. The results demonstrate the usefulness of RAPD markers for developing a basic genetic linkage map in papaya.Journal series No. 4146 of the Hawaii Institute of Tropical Agriculture and Human Resources     

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.     

High frequency somatic embryogenesis and plant regeneration from papaya hypocotyl callus

High frequency somatic embryogenesis in papaya (Carica papaya L.) tissue cultures was achieved by culturing hypocotyl sections from ten-day-old seedlings on half-strength Murashige and Skoog salts (MS) medium containing modified MS vitamins, 2.3 to 112.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), 400 mg l^-1 glutamine, and 6% sucrose. Four hermaphroditic Hawaiian cultivars produced embryogenic calluses after ten to 14 weeks of culture at 27°C in the dark. Efficiency in embryogenic response of genotypes differed, Kapoho > Sunset > Sunrise > Waimanalo. The frequency of embryogenesis in induction medium containing 4.5 M 2,4-d was lowest with 3% sucrose and highest with 7% sucrose. Somatic embryos developed directly from embryogenic calluses on induction medium, or, more often, they differentiated from calluses subcultured on a medium devoid of growth regulators. Between 50 and 500 embryos were produced from each 2-mm hypocotyl section after at least two months on induction medium and two months on maturation medium. Embryos subsequently developed into normal-looking plants on MS medium. Shoot cuttings from germinated embryos and micropropagated plants were rooted with 5.0 M indole-3-butyric acid (IBA), grown in the greenhouse, and transferred to the field.Journal Series no. 3732 of the Hawaii Institute of Tropical Agriculture and Human Resourees     

Resolution of secondary alcohols via Carica papaya lipase-catalyzed enantioselective acylation

The Carica papaya lipase-catalyzed acylation of benzylcarbinols with vinyl hexanoate proceeded smoothly and enantiospecifically (E > 200), affording the R-esters and leaving the S-alcohols intact. Thus, this plant lipase proved to be a promising biocatalyst for the resolution of alcohols as well as for that of carboxylic acids reported earlier.     

Monoclonal antibodies againstWuchereria bancrofti microfilarial excretory-secretory antigens

A battery of monoclonal antibodies were produced againstWuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the 1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed antiWuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids showed 10 clones which were specifically positive only toWuchereria bancrofti microfilarial excretory-secretory antigens.     

Assessment of genetic and epigenetic changes following cryopreservation in papaya

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.     

Pythium aphanidermatum root rot of pawpaw (Carica papaya L.) in Nigeria

Root rot of pawpaw (Carica papaya L.) reported in Nigeria is caused byPythium aphanidermatum which was consistently isolated from diseased plant parts and highly pothogenic. Out of 16 different media tested, it grew best on corn-meal-agar (CMA) and CMA supplemented with cellulose and sucrose. The highest number of oospores/ml was on CMA with average diameter of 19.9±0.1 µm. The symptom is characterized by dark brown rot of roots, absence of secondary roots and disintegration of internal tissue of the main root. These cause the progressive decline of the aerial parts of the tree untill it dies.     

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.     

Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars

Summary We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.Journal series No. 3692 of the Hawaii Institute of Tropical Agriculture and Human Resources     

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.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.