Ppe.RPT/SSC-1: from QTL mapping to a predictive KASP test for ripening time and soluble solids concentration in peach.

Genomic regions associated with ripening time (RPT) and soluble solids concentration (SSC) were mapped using a pedigreed population including multiple F1 and F2 families from the Clemson University peach breeding program (CUPBP). RPT and SSC QTLs were consistently identified in two seasons (2011 and 2012) and the average datasets (average of two seasons). A target region spanning 10,981,971-11,298,736 bp on chromosome 4 of peach reference genome used for haplotype analysis revealed four haplotypes with significant differences in trait values among different diplotype combinations. Favorable alleles at the target region for both RPT and SSC were determined and a DNA test for predicting RPT and SSC was developed. Two Kompetitive Allele Specific PCR (KASP) assays were validated on 84 peach cultivars and 163 seedlings from the CUPBP, with only one assay (Ppe.RPT/SSC-1) needed to predict between early and late-season ripening cultivars and low and high SSC. These results advance our understanding of the genetic basis of RPT and SSC and facilitate selection of new peach cultivars with the desired RPT and SSC.

Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate.

The accumulation of metabolic wastes in cell cultures can diminish product quality, reduce productivity, and trigger apoptosis. The limitation or removal of unintended waste products from Chinese hamster ovary (CHO) cell cultures has been attempted through multiple process and genetic engineering avenues with varied levels of success. One study demonstrated a simple method to reduce lactate and ammonia production in CHO cells with adaptation to extracellular lactate; however, the mechanism behind adaptation was not certain. To address this profound gap, this study characterizes the phenotype of a recombinant CHO K-1 cell line that was gradually adapted to moderate and high levels of extracellular lactate and examines the genomic content and role of extrachromosomal circular DNA (eccDNA) and gene expression on the adaptation process. More than 500 genes were observed on eccDNAs. Notably, more than 1000 genes were observed to be differentially expressed at different levels of lactate adaptation, while only 137 genes were found to be differentially expressed between unadapted cells and cells adapted to grow in high levels of lactate; this suggests stochastic switching as a potential stress adaptation mechanism in CHO cells. Further, these data suggest alanine biosynthesis as a potential stress-mitigation mechanism for excess lactate in CHO cells.

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

Chilling requirement (CR) is an important agronomic trait controlling the floral bud break for proper flowering in peach. Even though it has been widely researched and several peach CR quantitative trait loci (QTLs) have been identified, no diagnostic DNA tests validated in the U.S. peach breeding germplasm are available for this trait. Breeders and growers need a simple DNA test to predict the CR of peach cultivars for their particular environment. Therefore, we developed a quick and reliable Kompetitive Allele Specific PCR (KASP) DNA test using haplotype information from 9K IPSC genotype data of the U.S. peach germplasm integrating four CR-associated SNP markers from the previously reported CR QTL region on linkage group 1. Four KASP assays (Ppe.CR.1-1 to -4) were developed and validated on 77 peach cultivars, and nine accessions from two F2 populations, with 96 and 74% accuracy in determining expected CR genotype (compared to SNP array) and predicting phenotype, respectively. Furthermore, the Ppe.CR.1 showed 80% accuracy in predicting the precise CR phenotype in the Clemson University peach breeding material. Only one Ppe.CR.1 KASP assay (Ppe.CR.1-1) is needed to distinguish between haplotypes with CR lower and higher than 800 chilling hours, and two Ppe.CR.1 assays (Pp.CR.1-1 and -4), are capable of distinguishing low, moderate, and high CR alleles. Coupled with the crude DNA extraction, the Ppe.CR.1 DNA test provides a low-cost option for breeders and growers to predict CR in peach material with more than 70% accuracy.

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

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.

Corrigendum: Multi-Locus Genome-Wide Association Studies Reveal Fruit Quality Hotspots in Peach Genome.

[This corrects the article DOI: 10.3389/fpls.2021.644799.].

Genome-Wide Association Study of Brown Rot (Monilinia spp.) Tolerance in Peach.

Brown rot, caused by Monilinia spp., is one of the most important diseases on stone fruit worldwide. Severe yield loss can be caused by pre- and post-harvest fruit decay. Although some degree of tolerance has been reported in peach and almond, the genetic resistance in peach cultivars is still lacking. To date, only few genomic regions associated with brown rot response in fruit skin and flesh have been detected in peach. Previous studies suggested brown rot tolerance in peach being a polygenic quantitative trait. More information is needed to uncover the genetics behind brown rot tolerance in peach. To identify the genomic regions in peach associated with this trait, 26 cultivars and progeny from 9 crosses with 'Bolinha' sources of tolerance, were phenotyped across two seasons (2015 and 2016) for brown rot disease severity index in wounded and non-wounded fruits and genotyped using a newly developed 9+9K peach SNP array. Genome wide association study using single- and multi-locus methods by GAPIT version 3, mrMLM 4.0, GAPIT and G Model, revealed 14 reliable SNPs significantly associated with brown rot infection responses in peach skin (10) and flesh (4) across whole genome except for chromosome 3. Candidate gene analysis within the haplotype regions of the detected markers identified 25 predicted genes associated with pathogen infection response/resistance. Results presented here facilitate further understanding of genetics behind brown rot tolerance in peach and provide an important foundation for DNA-assisted breeding.

Multi-Locus Genome-Wide Association Studies Reveal Fruit Quality Hotspots in Peach Genome.

Peach is one of the most important fruit crops in the world, with the global annual production about 24.6 million tons. The United States is the fourth-largest producer after China, Spain, and Italy. Peach consumption has decreased over the last decade, most likely due to inconsistent quality of the fruit on the market. Thus, marker-assisted selection for fruit quality traits is highly desired in fresh market peach breeding programs and one of the major goals of the RosBREED project. The ability to use DNA information to select for desirable traits would enable peach breeders to efficiently plan crosses and select seedlings with desired quality traits early in the selection process before fruiting. Therefore, we assembled a multi-locus genome wide association study (GWAS) of 620 individuals from three public fresh market peach breeding programs (Arkansas, Texas, and South Carolina). The material was genotyped using 9K SNP array and the traits were phenotyped for three phenological (bloom date, ripening date, and days after bloom) and 11 fruit quality-related traits (blush, fruit diameter, fruit weight, adherence, fruit firmness, redness around pit, fruit texture, pit weight, soluble solid concentration, titratable acidity, and pH) over three seasons (2010, 2011, and 2012). Multi-locus association analyses, carried out using mrMLM 4.0 and FarmCPU R packages, revealed a total of 967 and 180 quantitative trait nucleotides (QTNs), respectively. Among the 88 consistently reliable QTNs detected using multiple multi-locus GWAS methods and/or at least two seasons, 44 were detected for the first time. Fruit quality hotspots were identified on chromosomes 1, 3, 4, 5, 6, and 8. Out of 566 candidate genes detected in the genomic regions harboring the QTN clusters, 435 were functionally annotated. Gene enrichment analyses revealed 68 different gene ontology (GO) terms associated with fruit quality traits. Data reported here advance our understanding of genetic mechanisms underlying important fruit quality traits and further support the development of DNA tools for breeding.