Ppe.XapF: High throughput KASP assays to identify fruit response to Xanthomonas arboricola pv. pruni (Xap) in peach

Abstract

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious peach disease with symptoms that traverse severe defoliation and black surface pitting, cracking or blemishes on peach fruit with global economic impacts. A management option for control and meeting consumer demand for chemical-free, environmentally friendly fruit production is the development of resistant or tolerant cultivars. We developed simple, accurate, and efficient DNA assays (Ppe.XapF) based on SNP genotyping with KASP technology to quickly test for bacterial spot resistance alleles in peach fruit that allows breeders to cull seedlings at the greenhouse stage. The objective of this research was to validate newly developed DNA tests that target the two major QTLs for fruit resistance in peach with diagnostic utility in predicting fruit response to bacterial spot infection. Our study confirms that with only two Ppe.XapF DNA tests, Ppe.XapF1-1 and Ppe.XapF6-2, individuals carrying susceptible alleles can be identified. Use of these efficient and accurate Ppe.XapF KASP tests resulted in 44% reduction in seedling planting rate in the Clemson University peach breeding program.

Ppe.CR.1 DNA test for predicting chilling requirement in peach

“…Cycle 25 of real-time PCR was selected for the following analyses for each KASP assay to maximize separation between genotype clusters and minimize background ampli cation. The relative uorescence units (RFU) values of this cycle were transferred to the template developed by Fleming et al 33 , and the proposed protocol was followed to determine the nal genotype of each sample.…”

“…A template spreadsheet designed by Fleming et al 33 assigns genotypes automatically based on default or user-speci ed parameters (Supplementary Table S3-6). In this spreadsheet, delta value thresholds can be manually adjusted so that automatic genotype assignments correspond to what is seen in the scatterplot of relative FAM and HEX uorescence values.…”

“…Few DNA tests are available for peach, and most are associated with fruit quality 44 or disease resistance 33 . Only one attempt to develop a DNA test to predict CR in peach was reported 34 .…”

“…Five SNPs that distinguished twelve unique haplotypes on Chr1 between 43.58-43.78 Mbp 35 were chosen for KASP assay development. Primers were designed for each of the ve SNPs following Fleming et al 33 protocol: GC content between 30-55%; annealing temperature (Tm) of 62-66 ºC; 21-30 bp length; product size of 42-100 bp; secondary structure more positive than − 9 kcal/mole; no more than four di-nucleotides; no more than 4 or 5 identical nucleotides in a row; and no more than 3 Gs and/or Cs in the last 5 bp of the primer. Primers were validated in silico with the Primer-BLAST tool against the NCBI database.…”

“…Recently, the KASP technique has gained popularity because it can rapidly and reliably genotype SNPs cost-effectively better than other genotyping platforms [31][32][33] .…”