Genetics to Breeding

Cross Assist (www.rosaceae.org/breeders_toolbox/cross_assist) is a decision-support tool for breeders to plan crosses. This software was developed as a component of the RosBREED project’s Breeding Information Management System to support U.S. breeders of crops in the Rosaceae family, and the underlying database is housed on the Genome Database for Rosaceae. Cross Assist identifies efficient pairwise parental combinations from a breeder’s available parent pool. This efficiency is determined by the number of seedlings needed to result in a target number predicted to each perform within specified trait thresholds. Efficient crosses achieve all trait thresholds with relatively few seedlings. The software uses available information on the breeding value of each candidate parent to predict how well resulting seedlings would perform. Calculations can be made on three increasing levels of breeding value information. The first method, “Phenotype”, uses only phenotypic information in the database. “+Pedigree” adds information provided by pedigree – estimated breeding value. “+Ped+DNA” further adds information provided by functional genotypes for Mendelian trait loci and QTLs. Where such additional information is not available, calculations behind the latter two methods revert to use of phenotypes only. Users supply two specifications as input: target number of seedlings and target trait thresholds. Cross Assist’s output provides a list of crosses, sorted initially by the estimated number of seedlings required. Further functionalities are being added, such as incorporation of linkage information, output addressing the trait loci involved and their available DNA tests, and integration with our sister software, Seedling Select.

A fascinating region of the apple genome is the proximal third of chromosome 16. Features detected in breeding germplasm using resources generated by the RosBREED project include a hotspot for heterozygosity, a hotspot for recombination, an introgressed segment from an enigmatic crab apple source, and a hotspot for QTLs covering all major categories of fruit quality: flavor via acidity and sweetness, texture via firmness and crispness, appearance via fruit size and bitter pit incidence, and even nutritional composition via phenolics content! The region was genetically dissected into several haplotype blocks (“haploblocks”). Focusing on U.S. apple breeding germplasm, breeding utility was assigned to each haploblock. Breeding utility was primarily characterized by effects on trait levels and the relative economic value of trait levels differentiated and the allele frequencies and sources in breeding germplasm. Often, alleles associated with desirable levels of one trait are linked in repulsion phase with those of another trait (e.g., acidity and phenolics; crispness and bitter pit incidence). However, most such linkages can be broken. Ideally, breeding parents contain the most valuable haploblocks in coupling phase and in homozygosity for use in efficiently enriching subsequent breeding families with alleles for superior performance in multiple traits. The best haploblock combinations identified within rare individuals of RosBREED’s reference U.S. germplasm represent valuable parents for imparting superior genetic potential. As collaborative research efforts rapidly accumulate DNA information across the genome for apple and related rosaceous crops, useful genetic stocks can be developed by targeted haploblock assembly into desired configurations.

The acidity content of an apple contributes to its delicious taste and overall enjoyment. Two major loci are known to contribute to the bulk of the heritability of acidity levels in apple. The Ma locus on chromosome 16, with its underlying malic acid transporter gene, accounts for ~30% of the phenotypic variance in Washington apple breeding program (WABP) germplasm. The recently discovered “A” locus on chromosome 8, hypothesized to be responsible for malic acid production, explains another ~20% of observed variation. A DNA test is available and in use for the Ma locus (“Ma-indel”) but not for the A locus. At Washington State University, a new DNA test (“LG8A-SSR”) was developed to help differentiate between apple fruit acidity levels. Primers were developed for microsatellites near the “A” QTL peak. From ten sets of primers, several were chosen that matched functional haplotype patterns obtained from RosBREED’s high-resolution SNP data. By pairing outcomes of LG8A-SSR and Ma-indel, differentiation of five levels of acidity could be made, allowing for a more accurate prediction. In combination, these two tests explain > 50% of phenotypic variation within WABP germplasm and were used in the 2014 marker-assisted seedling selection. The predictive power of these two tests should be verified on breeding germplasm of other regions. The double DNA testing strategy creates a powerful predictive tool helping breeders select for the desired acidity levels.

DNA tests that predict valuable trait levels are essential for widespread adoption of marker-assisted breeding (MAB) of rosaceous tree fruits. The RosBREED project has facilitated development of DNA tests for important traits in peach, apple, and cherry, including peach sweetness and acidity. Based on a quantitative trait locus (QTL), discovered on peach chromosome 7 by RosBREED collaborators, explaining ~20% of phenotypic variation for titratable acidity in normal acid peaches and ~10% of variation in soluble solids content, a DNA test (“G7Flav-SSR”) was developed at Washington State University. Standard cultivars and University of Arkansas (UA) and Clemson University (CU) breeding germplasm were used to confirm predictiveness of the DNA test, where G7Flav-SSR clearly differentiated low:low, low:high, and high:high allelic combinations. Validation of this new DNA test was conducted on unselected families of CU and AR germplasm. G7Flav-SSR results were used to guide 2014 crossing decisions in the UA peach breeding program. This advance in DNA-informed breeding represents an example of successful collaboration among institutions and across disciplines. Other DNA tests emerging from RosBREED include those for the prediction of peach maturity time, bacterial spot resistance, firmness, and blush, apple sweetness, acidity, and firmness, and sweet cherry maturity time and firmness. By harnessing the power of collaboration, specifically the integration of pedigree, phenotypic, and genotypic data generated by RosBREED team members for QTL discovery, the development of these DNA tests was possible. Tools such as G7Flav-SSR are now available to make DNA-based predictions a routine part of tree fruit breeding.

Plant metabolites including volatile organic compounds (VOCs) play a major role in apple flavor notes. Breeding for improved flavor is a major objective in many breeding programs although there is no systematic selection for specific VOCs. Knowledge of the genetic systems controlling these compounds will facilitate breeding for enhanced concentration and combinations of these compounds in apple. This study profiled individual VOCs in ~200 individuals including commercial cultivars and their progenies from the Washington State University’s (WSU) apple breeding program, maintained at the WSU Sunrise orchard, Wenatchee. Fruit was collected, juiced and frozen during the harvest season of 2012, then assayed for VOCs via GC-MS. The accessions were genotyped using ~1000 polymorphic SNPs of a total of 9K SNPs on the Infinium® II array developed in the RosBREED (www.rosbreed.org) project. The integration of phenotypic (VOCs) and genotypic data (SNPs) to identify quantitative trait loci (QTL) linked to these traits was done using FlexQTL™ software. In all accessions, pentyl acetate showed the least concentration (0.1-102.1 ng/ml) while hexanal had the highest concentration (55.8-3390.1 ng/ml). QTL were identified for precursors such as 1-butanol on LG 2 and LG 16, and for 2-hexenal on LG 4 (2 QTL) and LG 7.Methyl-butanol, 1-pentanol and hexanal each had one QTL on LG 13, LG 10 and LG 13, respectively. Among the esters assayed, QTL were identified for butyl-acetate on LG 5 and LG 8, on LG 4 for hexyl-acetate, on LG 2, LG 4, LG 10 and LG 16 for methylbutylacetate and on LG 5 for pentylacetate. The implications of these results are discussed in the context of breeding for enhanced flavors in apple.

The USDA Specialty Crop Research Initiative-funded RosBREED project has the objective of enabling marker-assisted breeding (MAB) in the economically important agricultural family of the Rosaceae. To standardize and increase the accuracy of MAB in Rosaceae, it is necessary to characterize many horticultural and fruit quality traits in representative germplasm. A well-developed comprehensive phenotyping protocol for productivity traits, fruit quality traits and horticulturally objectionable traits, developed for use in the Washington State University (WSU) Sweet Cherry Breeding Program, is described and selected correlations among traits observed and quantified. The protocol facilitates standardization of data among researchers working with sweet cherry across various environments and institutions. Data collected from sweet cherry ‘Crop Reference Set’ and ‘Breeding Pedigree Set’ between 2010 and 2012 was evaluated for correlation among sweet cherry phenotypic traits, and the results are presented. Selected results indicate significant moderate correlations for harvest date with fruit weight and fruit firmness (r=0.26, p<0.0001 and r=0.39, p<0.0001, respectively), with later- maturing varieties tending towards larger and firmer cherries, in general. Also, fruit weight had a positive significant association with pedicel-fruit retention force with r=0.43 (p<0.0001). However, soluble solid content showed a negative relationship with fruit weight and firmness (r=-0.34 and r=-0.20, respectively). Progress in breeding for multiple traits simultaneously will be faster with the significant positive correlations obtained in this study.

Powdery mildew (PM), caused by Podosphaera clandestina, and bacterial canker (BC), caused by Pseudomonas syringae pv. syringae, are the major diseases of sweet cherry in the USA. Incorporation of natural resistance into elite cultivars would be an effective way to reduce reliance on fungicide and pesticide use and facilitate the transition to sustainable production systems. This study was designed to identify quantitative trait loci (QTL) underlying PM and BC infection to facilitate development of new resistant cultivars. Six hundred pedigree-linked germplasm were used in this study. PM was scored in the field on a 0-5 scale (0 = no visible symptoms and 5 = very severe infection on leaves) from 2011 to 2013. BC phenotyping was performed by inoculating five healthy and newly emerging leaves with 10 µl of 100,000,000 CFU/ml bacteria suspension mixed with 0.5% surfactant by mid-rib wound method in a detached leaf assay. Disease was scored in the lab on a 0-4 scale (0 = no necrosis and 4 = total necrosis). Approximately 1100 single nucleotide polymorphism (SNP) and four simple sequence repeat (SSR) markers were used for determining genome-wide marker-locus-trait associations. One PM QTL mapped on top of linkage group (LG) 5 in the three years while others mapped on LGs 1, 3 and 6 in a single year. For BC, one major QTL was identified on top of LG 5. The co-location of QTL for both diseases on LG 5 will be explored further to develop a breeding strategy for the two diseases combined.

Sweetness and acidity are important fruit quality traits that drive consumer acceptability of sweet cherry. Developing new sweet cherry cultivars with superior taste attributes is a major breeding objective in fruit breeding programs. This study was designed to identify quantitative trait loci (QTL) associated with soluble solids content (SSC) and titratable acidity (TA) in sweet cherry to facilitate development of new cultivars with exceptional taste characteristics. A total of 601 pedigree-linked individuals were used in this study. Five largest fruits from each individual were selected at maturity for SSC measurement. Juice from a bulked sample of 25 fruit was used for TA measurement. One thousand and ninety one (1091) single nucleotide polymorphism (SNP) and four simple sequence repeat (SSR) markers were used to provide genome-wide markers for determining marker-locus-trait associations. QTL analyses were performed in FLexQTL™ for SSC and TA using phenotypic data collected in 2010, 2011 and 2012. Three QTL for SSC were mapped on the ‘Texas’ x ‘Earlygold’ Prunus reference map on linkage group (LG) 2 (in 2011 and 2012), on LG 4 (in 2012) and on LG 7 (a minor one in 2011). Three QTL were identified for TA; one mapped on LG 2 in 2010, the other on LG 4 in 2011 and 2012 and the third on LG 6 in 2012. The haplotypes for these QTL are discussed in relation to breeding for SSC and TA in sweet cherry.

Over 60 years ago, the existence of different races of Venturia inaequalis was demonstrated using inoculation experiments with monospore isolates of the pathogen on the red-fleshed scab-resistant apple cultivar ‘Geneva’. The findings suggested the presence of at least three scab resistance genes in this host. More recently, our genetic studies with five monospore isolates of V. inaequalis have indicated the presence of at least four genes. Three of these, two dominant genes and one recessive gene, have been mapped to a 5 cM region on linkage group 4, which corresponds to a 2 Mbp region containing nine candidate NBS-LRR resistance genes on the physical map of ‘Golden Delicious’. Primers specific to the candidate genes were designed for the fine-mapping of the resistance genes and used to construct a detailed genetic map defining the loci. The consequences of the complexity of the ‘Geneva’ scab resistance for breeding and the selection of a differential host (3) for V. inaequalis pathotype monitoring (www.vinquest.ch) will be discussed.

Pears (Pyrus spp.) are one of the most important tree fruit crops. They are an excellent source of fiber and vitamin C. The genus Pyrus is native to the Northern Hemisphere and consists of over 20 species. Little work has been done to analyze the genetic relationships among pears. It is important for breeders to understand pedigree relationships among pear germplasm in order to make informed crosses. To assess the genetic diversity and relationships among ~200 advanced pear genotypes, target region amplification polymorphism (TRAP) markers were used. Six primer combinations (three sets of polymerase chain reaction) produced a total of 86 polymorphic loci. Scoring information was subsequently analyzed with the software programs STRUCTURE, CLUMPP, and DISTRUCT in order to assign individuals into populations and determine admixture within individuals. STRUCTURE output indicates that these particular individuals fall into three populations and that there is admixture among the individuals. In addition to analyzing the genetic diversity and relationships among these individuals, a virtual representation of the data was created using the custom software seeDNA™. The software generates a unique genetic identity number based upon the loci amplified, this format is converted to a two dimensional barcode. seeDNA™ is compatible with all types of marker output including SNPs generated via next generation sequencing platforms.