Development of plants resistant to Papaya leaf distortion mosaic virus by intergeneric hybridization between Carica papaya and Vasconcellea cundinamarcensis

Abstract

In this study, we confirmed that Vasconcellea cundinamarcensis resists Papaya leaf distortion mosaic virus (PLDMV), and used it to produce intergeneric hybrids with Carica papaya. From the cross between C. papaya and V. cundinamarcensis, we obtained 147 seeds with embryos. Though C. papaya is a monoembryonic plant, multiple embryos were observed in all 147 seeds. We produced 218 plants from 28 seeds by means of embryo-rescue culture. All plants had pubescence on their petioles and stems characteristic of V. cundinamarcensis. Flow cytometry and PCR of 28 plants confirmed they were intergeneric hybrids. To evaluate virus resistance, mechanical inoculation of PLDMV was carried out. The test showed that 41 of 134 intergeneric hybrid plants showed no symptoms and were resistant. The remaining 93 hybrids showed necrotic lesions on the younger leaves than the inoculated leaves. In most of the 93 hybrids, the necrotic lesions enclosed the virus and prevented further spread. These results suggest that the intergeneric hybrids will be valuable material for PLDMV-resistant papaya breeding.

Introduction

Papaya (Carica papaya, 2n = 18), belonging to the Caricaceae family, is one of the most important fruit trees in tropical and subtropical areas (Pereira et al. 2014, Rockinger et al. 2016). In many papaya-producing countries, viruses cause the most destructive diseases of this species. For instance, the Papaya ringspot virus (PRSV) has become a serious problem in production areas such as Hawaii, Brazil, Thailand and Taiwan (Davidson 2008, Tripathi et al. 2008). In Hawaii, PRSV was discovered in 1992, and had severely affected nearly 100% of the papaya plants within 3 years (Ferreira et al. 2002). While, in Okinawa Island, Japan, the Papaya leaf distortion mosaic virus (PLDMV) was first reported in 1954. Thereafter, PLDMV was spread from Okinawa Island to Ryukyu Islands and Taiwan (Bau et al. 2008, Kawano and Yonaha 1992, Maoka et al. 1996). PLDMV-infected papaya plants show distorted and discolored leaves and fruits, together with thin stems, and exhibit reduced fruit production. Although these symptoms are similar to those caused by PRSV infection and both viruses belong to the Potyvirus genus, comparisons of their amino acid sequences and serological tests have confirmed that they are not the same virus (Maoka and Hayata 2005). Because PLDMV is easily transmitted by aphid vectors and any PLDMV-resistant C. papaya resources are unknown, commercial cultivation of papaya on Okinawa is carried out in greenhouses to prevent viral infections. Therefore, PLDMV is a major constraint to papaya production, rather than PRSV, in Okinawa.

To control PRSV in papaya, two strategies have been employed. The first is the use of transgenic plants. Two transgenic papaya cultivars, ‘Rainbow’ and ‘SunUp’, which show resistance to PRSV by overexpressing its coat protein gene, have been commercialized in Hawaii (Tennant et al. 2001). In Taiwan, since both PRSV and PLDMV are serious problems, transgenic papaya plants that are resistant to both two viruses were developed by overexpressing the truncated coat protein genes of PRSV and PLDMV (Bau et al. 2008, Kung et al. 2009). Another strategy for controlling PRSV is the use of Vasconcellea species (2n = 18, Pereira et al. 2014, Rockinger et al. 2016), which are PRSV-resistant genetic resources belonging to the Caricaceae family. Several Vasconcellea spp. (V. cauliflora, V. cundinamarcensis, and V. quercifolia) exhibit complete resistance to PRSV (Conover 1964) and their resistance gene has been introduced into papaya by means of intergeneric crosses. Although C. papaya and Vasconcellea species are cross-incompatible (Magdalita et al. 1996), embryo-rescue techniques can overcome the barriers, leading to successful introduction of PRSV resistance into papaya (Chen et al. 1991, Manshardt and Wenslaff 1989). In the PRSV resistance in F1 and F2 progeny derived from the cross between V. parviflora (susceptible) and V. cundinamarcensis (resistant), PRSV resistance was regulated by a single dominant gene (prsv-1). Polymorphic randomly amplified DNA fingerprint markers (Dillon et al. 2005) and a cleaved amplified polymorphic sequence marker linked to prsv-1 (Dillon et al. 2006) were developed. Although several approaches for utilizing PRSV resistance in V. cundinamarcensis have been reported, its PLDMV resistance was not investigated.

In this study, to support the development of PLDMV-resistant papaya, we tested its resistance to the virus, produced intergeneric hybrids between C. papaya and V. cundinamarcensis by means of embryo rescue, and evaluated PLDMV resistance in the hybrids.

Materials and Methods

Evaluation of PLDMV resistance

Before production of intergeneric hybrid plants between C. papaya and V. cundinamarcensis, we evaluated the PLDMV resistance of V. cundinamarcensis and C. papaya cultivar ‘Sunrise Solo’ by mechanical inoculation in a greenhouse. The V. cundinamarcensis plants used were a progeny obtained by the cross between female parent MP8 and male parent MP17, which maintained in Okinawa Prefectural Agricultural Research Center. The inoculum was prepared by homogenizing 0.2 g of PLDMV infected papaya leaf in 20 ml of 0.02 M potassium phosphate, pH 7.0. A fully expanded leaf of young V. cundinamarcensis (66 plants) and C. papaya (27 plants) 20 to 30 cm in height was dusted with 600-mesh carborundum and rubbed with the inoculum. Controls (rubbing with carborundum but inoculation with 0.02 M potassium phosphate), young V. cundinamarcensis (50 plants) and C. papaya (23 plants) 20 to 30 cm in height were also maintained in the greenhouse. All plants were inoculated three times at 2-week intervals. Symptom development in the inoculated and control plants was observed for 6 months after the first inoculation.

In the PLDMV resistance of the intergeneric hybrids by mechanical inoculation. 134 and 84 young intergeneric hybrid plants 20 to 30 cm in height were used for virus-inoculation and control, respectively. Conditions for the inoculum preparation, inoculation, maintenance of the plants, and observation of symptom development were the same as those described above.

Production of intergeneric hybrids by embryo rescue

A female parent of the C. papaya cultivar ‘Sunrise Solo’ and a male parent of V. cundinamarcensis, MP17, were used for the intergeneric hybridization. Intergeneric cross was carried out following the method developed by Dinesh et al. (2007). Anthers were collected from the 20 male flowers of V. cundinamarcensis, MP17, and soaked in a 50-ml polypropylene tube containing 10 ml of 5% sucrose solution. Then, by shaking the tube, pollens were extracted and homogenized. For intergeneric crosses, the pollens prepared were pollinated to seven female flowers of a C. papaya cultivar ‘Sunrise Solo’ by a brush. Intergeneric hybrids were produced following the method developed by Magdalita et al. (1996) with the following minor modifications. Embryos were obtained from seeds of 90 to 100 days after pollination (C. papaya × V. cundinamarcensis) and transferred to embryo-rescue culture under a stereomicroscope. We used gellan gum for solidification of the Murashige-Skoog (MS) medium (Murashige and Skoog 1962). Embryos were cultured at 26°C with a day length of 16 h. MS medium supplemented with a combination of 3 mg/L of gibberellic acid (GA₃), 0.05 mg/L of 6-benzylaminopurine (BAP) and 0.05 mg/L of α-naphthalene acetic acid (NAA) was used for the primary culture of embryos for 2 weeks. Subsequently, embryos were transferred to regulator-free MS medium and cultured for 3 months. To promote shoot elongation, somatic embryos were then transferred to MS medium supplemented with a combination of 0.2 mg/L BAP and 0.02 mg/L NAA. To induce rooting, individual shoots with two to three leaves (>0.5 cm in length) were transferred to MS medium that contained 0.5 mg/L of indole-3-butyric acid and cultured in the dark for 1 week. Then shoots were transferred to vermiculite medium containing half-strength liquid MS medium and cultured for 3 weeks under the same conditions used to culture the embryos. Finally, individual plants with roots were acclimatized in polyethylene bags with vermiculite and peat moss medium (Yu et al. 2000). Of these acclimatized plants, 28 representative plants derived from 28 seeds were grown in a greenhouse and applied to analyses for confirmation of intergeneric hybridization. After flowering of the 28 plants, we determined the flower sex and peduncle length.

Confirmation of intergeneric hybridization by means of flow cytometry

We used 25-mm² leaf samples from the rescued plants, C. papaya, and V. cundinamarcensis to confirm hybridization by using the CyStain UV Precise P Kit and Partec CyFlow PA flow cytometer (Partec GmbH, Munster, Germany). In these analyses, we tested the parent plants and hybrids separately, but also tested combinations of the two parent plants and of the parent plants with the hybrids. Leaf samples were chopped with a razor blade in extraction buffer. After digestion, the samples were filtered through Partec CellTrice (30 μm nylon mesh) and stained using 4, 6-diamidino-2-phenylindol. Following staining at room temperature, the relative nuclear DNA contents of the samples were measured. The fluorescence intensity for C. papaya was adjusted to 100 to provide a standard for comparison. The fluorescence intensities were then determined for all materials and expressed as ratio relative to the fluorescence intensity of C. papaya. Hybridization of the 28 rescued plants grown in the greenhouse was confirmed by relative fluorescence intensities intermediate between those of C. papaya and V. cundinamarcensis.

Nucleic acid extraction and cDNA synthesis

Genomic DNAs and total RNAs from the leaves of the putative hybrids and their parents were extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and RNeasy Plant Mini Kit (Qiagen), respectively. The extractions were carried out according to the manufacturer’s instructions. Nucleic acid concentrations and integrity were determined using a NanoDrop 2000c (Thermo Scientific, Waltham, MA, USA) and agarose gel electrophoresis. To perform accurate RT-PCR, 1 μg of total RNA was treated with 70 units of DNase I (Takara Bio, Shiga, Japan) at 37°C for 30 min in a 25-μL reaction volume. The DNase I was then inactivated by adding 1.3-μL of 0.5 M EDTA (pH 8.0) and incubating the solution at 80°C for 10 min. We directly applied 3-μL of the DNase I-treated total RNA in cDNA synthesis using the TaKaRa RNA PCR Kit (AMV) Ver. 3.0 (Takara Bio). The oligo dT-adaptor primer was used for reverse transcription according to the manufacturer’s instruction.

Confirmation of intergeneric hybridization by PCR

Chloroplast and nuclear DNA regions were amplified to confirm hybridization. In non-coding chloroplast DNA sequences, the existence of polymorphisms at the inter- and intra-specific levels are known (Shiraishi et al. 2001, Taberlet et al. 1991, Urasaki et al. 2005). Therefore, in this study, amplification of the trnD-trnY intergenic spacer region in chloroplast DNA (cpDNA) was conducted using the CS4U and CS4L primer set designed by Shiraishi et al. (2001). For amplification of the nuclear DNA region, we targeted the monodehydroascorbate reductase (MDAR) gene, which is located in the non-recombining region of the sex chromosomes in C. papaya (Urasaki et al. 2012). To amplify a fragment of MDAR gene, we designed the primer set HF3 (5′-TGAGAGGTCTGATTCCCACT-3′) and MSY32R1 (5′-GTGATGCAATCTTGCACTGG-3′). One μL of genomic DNA solution was used for amplification. PCR was performed using a 25-μL reaction volume with the DNA polymerase KOD FX (TOYOBO LIFE SCIENCE, Osaka, Japan). Amplifications were performed under the following conditions: preheating at 94°C for 2 min, followed by 35 cycles of amplification at 98°C for 10 sec, annealing at 56°C for 30 sec, and extension at 68°C for 1 min, and with a final extension at 68°C for 5 min. PCR products were separated using 1.5% agarose gel electrophoresis, and visualized by ethidium bromide staining. In the trnD-trnY intergenic spacer region, PCR products were directly sequenced by an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems; Supplemental Fig. 2). In MDAR gene, two PCR products from an intergeneric hybrid plant were separated in the agarose gel and each fragment was sequenced (Supplemental Fig. 2). Accession numbers for the trnD-trnY intergenic spacer regions in C. papaya and V. cundinamarcensis are EU431223 and LC152431, respectively. The accession numbers for an allele of MDAR gene in C. papaya and two alleles of MDAR gene in V. cundinamarcensis are AC238599, LC152880, and LC152881, respectively.

Detection of PLDMV by RT-PCR

For detection of PLDMV, we used a younger leaf than the inoculated leaf of the plant 3 months after the first inoculation. In each younger leaves of C. papaya with severe mosaic symptoms, V. cundinamarcensis with no symptoms and the intergeneric hybrid with no symptoms, five cDNAs from five leaf segments including veins and five cDNAs from five leaf segments selected at random were used for RT-PCR. In the younger leaf of intergeneric hybrid with necrotic lesions, five cDNAs from five leaf segments including veins and five cDNAs from five leaf segments including necrotic lesions were used for RT-PCR. We amplified the coat protein (CP) gene of PLDMV by RT-PCR. To amplify a fragment of CP gene, we designed the primer set, CPF3 (5′-GAATAGCGCATCTGGTGCTA-3′, nt 9191 to 9210) and CPR2 (5′-ATATCGAACGCCGGTGAATG-3′, nt 9919 to 9938) from the complete nucleotide sequence of PLDMV (Maoka and Hayata 2005; accession number AB088221). We used actin as the control, with the primer set Cp47F and Cp47R designed by Porter et al. (2008). One μL of cDNA solution was used for RT-PCR, and the other RT-PCR, electrophoresis, and sequencing conditions were the same as those described in Confirmation of intergeneric hybridization by PCR. Accession numbers for actin gene in C. papaya and V. cundinamarcensis are EL784289 and LC152432, respectively.

Results

PLDMV resistance of V. cundinamarcensis and RT-PCR for PLDMV detection

PLDMV resistance of V. cundinamarcensis plants was evaluated by mechanical inoculation of the leaves. As shown in Table 1, controls showed no symptoms throughout the 6-month observation period. In C. papaya, all 27 of the PLDMV-inoculated plants showed typical PLDMV disease symptoms within 3 weeks after the first inoculation, demonstrating that C. papaya was susceptible (Fig. 1A, 1E). On the contrary, none of the 66 PLDMV-inoculated V. cundinamarcensis plants showed symptoms throughout the 6-months period after inoculation (Fig. 1B), representing that V. cundinamarcensis plants were completely resistant. To monitor virus multiplication in the PLDMV-infected plants, RT-PCR analysis was carried out. In the virus-infected C. papaya, coat protein (CP) gene of PLDMV was amplified from cDNAs obtained from a leaf with severe mosaic symptoms (Fig. 2A). In V. cundinamarcensis plants with no symptoms, no viral amplification products were observed (Fig. 2B). Therefore, we proceeded to intergeneric hybrids production for introducing PLDMV resistance in V. cundinamarcensis to papaya.

Table 1 Evaluation of PLDMV resistance in the parent plants and intergeneric hybrids by means of mechanical inoculation

Plant material	No. of plants tested	No symptomsa	Typical symptomsb	Necrotic lesionsc
C. papaya, control	23	23	0	0
C. papaya, PLDMV	27	0	27	0
V. cundinamarcensis, control	50	50	0	0
V. cundinamarcensis, PLDMV	66	66	0	0
Intergeneric hybrid, control	84	84	0	0
Intergeneric hybrid, PLDMV	134	41	0	93

^a  No. of plants showing no symptoms 3 months after the inoculation.

^b  No. of plants showing typical symptoms (mosaic, distortion and discoloration) of PLDMV 3 months after the inoculation.

^c  No. of plants showing necrotic lesions on the younger leaves than the inoculated leaves 3 months after the inoculation.

Fig. 1

Symptoms on the inoculated leaves. (A) C. papaya with severe mosaic symptoms. (B) V. cundinamarcensis with no symptoms. (C) Intergeneric hybrid with no symptoms. (D) Intergeneric hybrid with necrotic lesions. (A) to (D) are the younger leaves than the inoculated leaves 3 months after inoculation with PLDMV. Boxes (5 mm × 5 mm) indicate the locations used for detection of PLDMV by RT-PCR. In (A) to (C), positions 1 to 5 and positions 6 to 10 represent veins and random positions, respectively. In (D), positions 1 to 5 and positions 6 to 10 represent veins and necrotic lesions, respectively. (E) C. papaya with leaf distortion and mosaic symptoms 6 months after the inoculation. (F) Intergeneric hybrid with no symptoms 6 months after the inoculation.

Fig. 2

Detection of the partial coat protein gene of PLDMV (748-bp) by RT-PCR. Lanes 1 to 10 correspond to positions 1 to 10, respectively, in Fig. 1. In (D), positions 1 to 5 represent veins and positions 6 to 10 represent necrotic lesions. As a control, the partial cDNA fragment of the actin gene (632-bp) was amplified.

Production of intergeneric hybrids

For intergeneric crosses, pollens of a male V. cundinamarcensis plant were pollinated to seven female flowers of a C. papaya plant. After pollination, fruit growth was observed in a papaya tree and seven enlarged fruits, after 90 to 100 days of pollination, were obtained. In total, we harvested 402 seeds from the seven fruits, while no seeds from the fruits of unpollinated flowers. In 147 of the seeds, embryos were found and aseptically transferred onto embryo-rescue medium for primary culture. All of the embryos were less than 2 mm, which was smaller than the embryos of C. papaya at the same stage. In all of the 147 seeds from intergeneric crosses, multiple embryos were observed per seed (Fig. 3A), even though C. papaya is a monoembryonic plant. After primary culture, somatic embryogenesis was observed in multiple embryos from 28 seeds. By transferring them to growth regulator-free MS medium, multiple shoots were produced (Fig. 3B). To promote shoot elongation, each shoot with a green tip was transferred onto elongation medium (Fig. 3C). After 3 weeks, individual shoots with a few leaves developed were transferred to root induction medium and cultured for 1 week in the dark. Subsequently, individual shoots were transferred onto vermiculite medium for root growth (Fig. 3D). After 3 weeks, normal roots with root hairs had emerged (Fig. 3E). In total, 218 plantlets were obtained from the 28 seeds and acclimatized. All 218 plants had pubescence on their petioles and stems that is characteristic of V. cundinamarcensis (Fig. 3F).

Fig. 3

Production of intergeneric hybrid plants of C. papaya × V. cundinamarcensis. (A) Multiple embryos from a seed 90 to 100 days after pollination. (B) Somatic embryogenesis from a zygotic embryo. (C) Shoot elongation. (D) Root development in vermiculite medium. (E) An intergeneric hybrid plant. (F) Pubescent petioles of an intergeneric hybrid plant.

Of these 218 plants, 28 representative plants derived from 28 seeds were grown in a greenhouse. After flowering, we determined their sex phenotype and inflorescence characteristics. Though the flowers were female in all 28 plants, 11 and 17 plants had long (male type) and short (female type) peduncles, respectively (Supplemental Fig. 1, Supplemental Table 1).

Flow cytometry analysis and PCR to confirm hybridization

To confirm hybridization of the plants developed by intergeneric crosses and embryo-rescue culture, flow cytometry was carried out. Fig. 4 shows histograms of the relative fluorescence intensity (RFI) of nuclei isolated from leaf tissues of C. papaya, V. cundinamarcensis and the rescued plants. We adjusted the peak RFI for C. papaya to be at 100 (Fig. 4A) to standardize the values, and analyzed RFI of V. cundinamarcensis and the 28 rescued plants planted in the greenhouse. The peaks for V. cundinamarcensis were concentrated at 125 (n = 4, Fig. 4B). The peaks for 26 of the 28 rescued plants were located at 110, with RFI intermediate between those of the parental species (Fig. 4C), and the rest of the plants showed ambiguous peaks at around RFI 210 (data not shown). In simultaneous analyses with two leaf samples (C. papaya and V. cundinamarcensis) and three leaf samples (C. papaya, V. cundinamarcensis, and intergeneric hybrid), two and three peaks (respectively) were observed at around RFI 100 and 125 (Fig. 4D) and RFI 100, 125, and 110 (Fig. 4E).

Fig. 4

Confirmation of intergeneric hybridization by flow cytometry to determine the relative nuclear DNA content. Peaks for C. papaya were standardized to a value of 100. (A) C. papaya. (B) V. cundinamarcensis. (C) Intergeneric hybrid plant. (D) Mixture of C. papaya and V. cundinamarcensis. (E) Mixture of C. papaya, V. cundinamarcensis, and the intergeneric hybrid. Peaks I, II, and III originated from C. papaya, V. cundinamarcensis and the intergeneric hybrid, respectively.

As an alternative way to confirm hybridization, the trnD-trnY intergenic spacer region in cpDNA were amplified. In the female parent (C. papaya) and the 28 rescued plants, 577-bp fragments were amplified, while a 533-bp fragment was amplified in the male parent (V. cundinamarcensis) (Fig. 5A, Supplemental Fig. 2). As a nuclear gene, MDAR (monodehydroascorbate reductase) gene (Supplemental Fig. 2) on the papaya sex chromosomes was amplified and a 480-bp fragment was identified in the female parent (C. papaya) and, two fragments (529 and 531-bp), which presumably represent alleles from the X and Y chromosomes, were amplified in the male parent (V. cundinamarcensis). In the 28 plants from intergeneric cross, two combinations of amplified fragments were identified: 480- and 529-bp, or 480- and 531-bp (Fig. 5B, Supplemental Table 1), suggesting that the nuclear genome of each plant was a hybrid. In the 11 hybrid plants with long peduncles, the 531-bp fragment was amplified. On the other hand, in the 17 plants with short peduncles, the 529-bp fragment was obtained (Supplemental Fig. 1, Supplemental Table 1).

Fig. 5

Confirmation of intergeneric hybridization by PCR. M and N are 100-bp DNA Ladder Marker (New England BioLabs, Ipswich, MA, USA) and a negative control, respectively. C represents the female parent (C. papaya, cultivar ‘Sunrise Solo’). V represents the male parent (V. cundinamarcensis). (A) The PCR results for the trnD-trnY intergenic spacer region (Supplemental Fig. 2) showed amplification of a 577-bp fragment from the female parent and the intergeneric hybrids, and a 533-bp fragment from the male parent. (B) The PCR results for MDAR gene (Supplemental Fig. 2) showed amplification of a 480-bp fragment from C. papaya, 529- and 531-bp fragments from V. cundinamarcensis. From the intergeneric hybrids, 480- and 529-bp fragments or, 480- and 531-bp fragments were amplified.

PLDMV resistance of the intergenic hybrids and RT-PCR for PLDMV detection

PLDMV resistance of the intergenic hybrid plants was evaluated by mechanical inoculation of the leaves. As shown in Table 1, the control hybrid plants had no symptoms throughout the 6-months observation period. Of the 134 PLDMV-inoculated intergeneric hybrids, 41 plants showed no symptoms during this period and PLDMV was not detected by RT-PCR (Figs. 1C, 2C). The other 93 hybrid plants showed necrotic lesions in the younger leaves than the infected leaves 3 months after the first inoculation (Fig. 1D), and no apparent disease symptoms were observed. In the infected intergeneric hybrid plant that showed necrotic lesions, amplification of CP genes was detected in the five cDNA samples from the lesions, whereas in the five cDNA samples from leaf areas outside of the lesions, viral RNA was not detected (Fig. 2D). At 3 months after the development of necrotic lesions (i.e., after 6 months from the first inoculation), further development of necrotic lesions were not observed on younger leaves in 84 of the 93 hybrid plants. However, the other 9 of the hybrid plants developed further severe lesions not only on younger leaves than the leaves inoculated but also onto non-expanded leaves and died.

Discussion

Although the production of intergeneric hybrids between C. papaya and Vasconcellea species is known to be difficult due to their cross-incompatibility, several researchers have reported that embryo rescue could overcome their cross-incompatibility. For example, Manshardt and Wenslaff (1989) produced intergeneric hybrids by culturing embryos 90 to 180 days after pollination from several combinations of crosses: C. papaya × V. pubescens and a reciprocal cross, C. papaya × V. quercifolia, and C. papaya × V. stipulata. Chen et al. (1991) also obtained hybrids by culturing embryos 60 days after pollination in C. papaya × V. cauliflora. Furthermore, in C. papaya × V. cauliflora, Magdalita et al. (1998) found that embryos 90 to 120 days after pollination were most efficient for rescue culture. In this study, our intergeneric cross using the sucrose-treated pollens to break the intergeneric crossing barrier showed 100% of fruit set, which is usually observed in intraspecific cross of C. papaya. We also obtained embryos from seven fruits 90 to 100 days after pollination, as described previously, and embryo-rescue culture promoted to develop plantlets as shown by Magdalita et al. (1996). Because all intergeneric hybrid plants from the embryo-rescue culture had pubescence on their petioles and stems, this phenotype appears to be a useful marker for discriminating hybrid plants.

The flow cytometry analysis showed that the relative DNA content of V. cundinamarcensis was 1.25 times that of C. papaya. According to a previous report by Gschwend et al. (2013), the genome sizes of C. papaya and V. cundinamarcensis were 442.8 Mb and 566.7 Mb, respectively (i.e., a ratio of 1.28), which agreed well with the present results. Most of the plants from intergeneric crosses showed intermediate nuclear DNA content (1.1 times the papaya genome size) between the parental species, and therefore represented diploid intergeneric hybrids. Meanwhile, two hybrid plants had relative DNA contents of 2.1 times the papaya genome size, suggesting that they were tetraploid plants.

In the PCR analysis to confirm the hybridization, we detected amplification of C. papaya originated trnD-trnY (577-bp), and C. papaya and V. cundinamarcensis originated two MDAR gene fragments from all 28 rescued plants. In addition to quantification of nuclear DNA contents, PCR analysis of organelle DNA (trnD-trnY) and alleles of nuclear MDAR gene demonstrated that all the analyzed plants from intergeneric crosses including two tetraploid plants were intergeneric hybrids between C. papaya and V. cundinamarcensis. All of the intergeneric hybrids had cytoplasm of C. papaya (the maternal plant), as previously reported by Van Droogenbreck et al. (2005). Between C. papaya and Vasconcellea spp., chromosome number was conserved as n = 9 (Pereira et al. 2014, Rockinger et al. 2016), and Wu et al. (2010) suggested that their sex chromosomes originated from the same autosome. According to Urasaki et al. (2012), MDAR gene was located on the papaya sex chromosome, and its allele was presented in both X and Y chromosomes. Its PCR analysis in the hybrids showed that either the 531-bp fragment or the 529-bp fragment from V. cundinamarcensis were segregated, whereas the 480-bp fragment from C. papaya was observed in all of the analyzed plants. Since long and short peduncles are distinctive characteristics of male and female plants, respectively, in V. cundinamarcensis and C. papaya, they are suggested to be sex-dependent phenotype markers. Of the 28 analyzed hybrids, this phenotype co-segregated with alleles of MDAR gene from V. cundinamarcensis. Therefore, the 531-bp and 529-bp fragments of MDAR gene in V. cundinamarcensis were probably located on the Y and X chromosomes, respectively. However, all 28 of the mature intergeneric hybrid plants bloomed female flowers, as in a previous report (O’Brien and Drew 2009). Further studies are therefore necessary for confirming sex chromosome type and elucidating sex expression mechanism in these hybrids.

By the mechanical inoculation of PLDMV in the intergeneric hybrid plants, three phenotypes were observed. The first phenotype showed no leaf symptoms, as in the PLDMV-infected V. cundinamarcensis plants. The second phenotype exhibited the formation of necrotic lesions on the younger leaves than the inoculated leaves. In the hybrid plants with no symptoms, PLDMV was not detected by RT-PCR, which suggests that they were completely resistant to PLDMV. The phenotype with necrotic lesions did not show the typical symptoms of PLDMV in susceptible C. papaya plants, such as distortion and discoloration of the leaves. In the observed necrotic lesions in the younger leaves, PLDMV was present, but it was absent in the areas outside the necrotic lesions. In these plants, the leaves with necrotic lesions continued to fall until 6 months from the first inoculation, and no additional development of necrotic lesions was observed in younger leaves than the leaves with lesions. Therefore, PLDMV infection was established and moved to the younger leaves in these plants. But the necrotic lesion prevented the virus from further spread in these plants, as in the hypersensitive response observed in resistance mediated by R-gene in other plant-pathogen combination (Yang et al. 1997). The third phenotype was the sequential formation of necrotic lesions from the younger leaves than the inoculated leaves to non-expanded leaves, which was observed in the 9 hybrid plants. The all 9 hybrids showing this phenotype died. In the susceptible mutants, ryc1-2 and rcy1-4, of Arabidopsis thaliana ecotype C24 resistant to an yellow strain of Cucumber mosaic virus, the delayed necrotic lesion development was observed and allowed virus spread (Sekine et al. 2006). In these 9 hybrids, delayed hypersensitive response may occur and does not prevent virus spread. O’Brien and Drew (2009) produced 300 hybrids between C. papaya and V. cundinamarcensis, and confirmed that all hybrid plants resisted PRSV. However, in our PLDMV inoculation test, we newly found three different phenotypes. Still, genes responsible for these differences in the resistance phenotype are unknown, and further genetic study will be necessary for elucidating their genetic mechanisms.

In this study, we produced intergeneric hybrids between C. papaya and V. cundinamarcensis that exhibited resistance to PLDMV, using the embryo rescue technique. Since the hybrid plants showed PLDMV-resistant phenotypes, they will be valuable materials for breeding PLDMV-resistant papaya in Ryukyu Islands, Japan, where the virus threatens papaya crops. We are currently performing backcrosses between the intergeneric hybrids and C. papaya.

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number 26450014.

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