Development of a pepper (Capsicum annuum L.) hybrid variety with resistance to potato virus Y (PVY) using molecular breeding.
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Pepper (Capsicum annuum L.) is an important vegetable crop grown and consumed worldwide. Potato virus Y (PVY) is a globally economically important pathogen which significantly reduces the yield and quality of cultivated pepper. The virus is considered as a major limiting factor to the economic production of pepper in the province of KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). Many applied practices to control the spread of PVY are ineffective to mitigate the losses incurred by many farming communities across the KZN province. Therefore, the objectives of this study was to determine the full genome sequence of a PVY isolate from KZN, to identify resistance alleles in commercially available pepper varieties in KZN and to develop a pepper hybrid variety with resistance to PVY using a molecular breeding strategy The first part of the study was conducted to determine the first full genome sequence of a PVY isolate (JVW-186) infecting pepper from KZN. The complete genome sequence of JVW-186 was assembled from overlapping RT-PCR clones using MEGA 5 software. Individual ORFs were identified using the nucleotide data base NCBI and aligned using CLUSTALW. RDP4 software was used to identify recombination junctions in the sequence alignment of JVW-186. CLC Main Workbench 6 software was used to determine the nucleotide sequence similarity of recombinant and non-recombinant fragments of JVW-186 in conjunction with ten PVY parental isolates. Based on sequence data, virus morphology and the coat protein size as determined by SDS-PAGE analysis, the identity of the isolate JVW-186 was confirmed as PVY. Phylogenetic trees were constructed from all recombinant and non-recombinant segments of the sequence by the maximum likelihood method using MEGA 5 software. The full length sequence of JVW-186 consisted of 9700bp. Two ORF’s were identified at position 186 and 2915 of the sequence alignment encoding the viral polyprotein and the frameshift translated protein P3N-PIPO, respectively. RDP4 software confirmed two recombination breakpoints at position 343 and 9308 of the sequence resulting in four segments of the genome. At each recombination event, a 1021-bp fragment at the 5’ end in the region of the P1/HC-Pro protein and a 392-bp fragment in the region of the coat protein shared a high sequence similarity of 91.8 % and 98.89 % to the potato borne PVYC parental isolate PRI-509 and the PVYO parental isolate SASA-110 respectively. The non-recombinant fragment 1 clustered within the C clade of PVY isolates; however the large 7942-bp fragment 3 did not cluster within any of the clades although it shared > 80% nucleotide sequence similarity to other PVY isolates used in this study. Our results suggest that isolate JVW-186 is a novel recombinant strain of PVY that could have evolved due to the dynamics of selection. The second part of the study aimed to evaluate different pepper lines for resistance to PVY. Two recessive alleles (pvr21 and pvr22) located on the pvr2-elF4E locus are known to confer resistance to the virus. To this end, six pepper lines were challenged with PVY infected Nicotiana tabacum cv. Xanthi leaf material using mechanical inoculation under greenhouse conditions. Each line was assessed for resistance to PVY by visual screening for disease severity and quantitative enzyme linked immunosorbent assay (ELISA) for virus load. Pepper lines were further characterized using tetra-primer ARMS-PCR (amplification refractory mutation system polymerase chain reaction) to identify and differentiate the presence of homozygous/heterozygous resistance alleles that confer PVY resistance. Evaluations revealed two resistant pepper lines (Double Up and Cecelia) and varying levels of susceptibility in the other four pepper lines challenged with PVY. The most susceptible pepper line was Benno, although high levels of susceptibility were observed in three other lines (IP, Mantenga and Excellence). The pvr2+ allele was positively identified in all the susceptible pepper lines using the T200A tetra-primer which confirms that the presence of this allele is dominant for PVY susceptibility. Double Up and Cecelia were genotyped homozygous pvr21/pvr21 and pvr22/pvr22 respectively, and remained asymptomatic throughout the trial which indicates that these alleles confer resistance to the isolate of PVY used in this study. The information generated in this study can be incorporated into breeding programs intended to control PVY on pepper in KZN. The final part of the study focused on the development of resistant varieties as the best alternative to manage PVY diseases on pepper. Homozygous F2 pepper lines were developed from local germplasm carrying PVY resistance genes (pvr21 and pvr22) using marker assisted selection (MAS). The F1 progeny was obtained by crossing a homozygous pvr21 (resistant) ‘Double Up’ cultivar with a heterozygous susceptible (pvr2+/pvr22) ‘Benno’ cultivar. F1 and F2 generations were assessed for the presence of PVY resistance/susceptibility alleles (pvr2+/pvr21/pvr22) at the pvr2-elF4e locus using the tetra primer amplification refractory mutation system – polymerase chain reaction (ARMS-PCR) procedure. Negative selection was carried out using the tetra-primer T200A marker to detect the pvr2+ (susceptible) allele. All F1 progeny displaying the pvr2+ allele were eliminated from further study. All 302 plants belonging to 29 F2 families expressing homozygous recessive traits were tested via mechanical inoculation for their response to PVY infection and resistance to PVY was confirmed in all selected families based on symptomatology in greenhouse house screens using double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). These results show that ARMS-PCR can be used to successfully screen pepper genotypes for alleles that confer PVY resistance thereby contributing to the improvement of pepper production using molecular breeding approaches.