Characterisation, Genetic Mapping and Development of Marker Selection Strategies for Resistance to the Papaya Ringspot Virus Type P (PRSV-P) in 'Highland Papaya'

Abstract

The Papaya ringspot virus Type P (PRSV-P) is a major constraint on cultivation of papaya (Carica papaya L.) world wide. Trees infected with the virus become stunted, have low fruiting yield and the quality of fruit from infected trees is poor. Losses of up to 70% of expected yield have been reported in some infected areas. The virus has had a significant impact on papaya production in Australia, effectively derailing the papaya growing industry in SE Queensland since it was first reported in 1991. Naturally occurring resistance to PRSV-P has not been identified in any papaya cultivar to date. Development of resistant papaya is considered the best strategy for long-term virus control. Several species from the related genus Vasconcellea exhibit complete resistance to PRSVP, and present a valuable resource for developing new PRSV-P resistant papaya varieties. To date, the only other resistance strategy has been transgenic, and although this has been successful it does not meet all requirements of the different papaya growing regions. Consequently, characterisation and application of resistance genes identified in Vasconcellea is an important step toward developing new strategies for broad spectrum resistance to PRSV-P. In this study an interspecific hybrid population of Vasconcellea cundinamarcensis (PRSV-P resistant) and V. parviflora (susceptible) was used for genetic characterisation of PRSV-P resistance. This is the first cross reported for these two species and to our knowledge the only population to segregate for PRSV-P resistance donated from V. cundinamarcensis. This provided a rare opportunity to examine heredity of valuable traits identified in the parents, which included resistance to three important diseases of papaya. 246 F2 plants were manually exposed to PRSV-P. Analysis of segregation of the resistant phenotype and several morphological markers revealed normal Mendelian inheritance of all traits, implying relative stability of the hybrid genome. Importantly, segregation of PRSV-P resistance adhered to a 1:3 ratio, indicating single dominant gene regulation of this trait. Genetic mapping of the PRSV-P resistance gene was achieved by recombination analysis of the resistant phenotype and randomly amplified DNA fingerprint (RAF) markers in the F2 generation. Discrete framework maps ranging in size from 630 - 745 cM were generated for both parents, accounting for up to 55 % of the total genome size. The PRSV-P resistance gene, prsv-1, mapped to linkage group 7 in V. cundinamarcensis within 4 cM of adjacent RAF markers. Segregation analysis of 242 RAF markers in the F2 revealed 32% of markers exhibited non-Mendelian segregation, indicative of a moderate level of genetic instability in the hybrid genome. This result was consistent with decreased fertility and seed viability observed in the F1 and F2. Although several markers close to prsv-1 were identified by linkage mapping, it was desirous to develop additional markers more tightly linked to the resistance gene that could be used for reliable selection of resistant genotypes. Bulked segregant analysis using ca. 4200 RAF markers revealed 12 candidates that associated with PRSV-P resistance. Of these, 5 were found autonomously in the resistant bulk individuals. Genetic mapping of these markers in the F2 population confirmed their linkage with prsv-1, four mapping within 9.7 cM of the resistance gene, and one (Opa11_5r) collocating with it. All 4 markers mapped closer to prsv-1 than those identified in the first mapping experiment. Sequence characterisation of the two most tightly linked candidates, Opk4_1r and Opa11_5r, revealed their homology to annotated proteins, a small nuclear ribonuclear protein and a serine/threonine protein kinase respectively. The latter class of protein has been associated with virus disease resistances, and as such is an appealing candidate for resistance gene isolation. Comparative analysis of the Opk4_1r sequence characterised amplified region (SCAR) in the parents of this cross permitted development of a codominant cleavage amplified polymorphic sequence (CAPS) marker that correctly diagnosed resistant genotypes 99% of the time, and mapped within 2 cM of prsv-1. Availability of this marker will enhance efficiency of artificial selection in breeding programs to introgress PRSV-P resistance into hybrids of V. cundinamarcensis by reducing the duration of breeding cycles and increasing the genetic gain per cycle. The main obstacle facing application of this CAPS marker in papaya is the omnipotent genetic barriers that have largely impeded hybridisation of papaya and V. cundinamarcensis to date. However, by utilising in vitro embryo rescue techniques, and applying sexually compatible species such as V. parviflora as intermediaries, we hope to bridge hybridisation with the less compatible species.