ShinyFruit: interactive fruit phenotyping software and its application in blackberry.

Introduction: Horticultural plant breeding programs often demand large volumes of phenotypic data to capture visual variation in quality of harvested products. Increasing the throughput potential of phenomic pipelines enables breeders to consider data-hungry molecular breeding strategies such as genome-wide association studies and genomic selection. Methods: We present an R-based web application called ShinyFruit for image-based phenotyping of size, shape, and color-related qualities in fruits and vegetables. Here, we have demonstrated one potential application for ShinyFruit by comparing its estimates of fruit length, width, and red drupelet reversion (RDR) with ImageJ and analogous manual phenotyping techniques in a population of blackberry cultivars and breeding selections from the University of Arkansas System Division of Agriculture Fruit Breeding Program. Results: ShinyFruit results shared a strong positive correlation with manual measurements for blackberry length (r = 0.96) and ImageJ estimates of RDR (r = 0.96) and significant, albeit weaker, correlations with manual RDR estimation methods (r = 0.62 - 0.70). Neither phenotyping method detected genotypic differences in blackberry fruit width, suggesting that this trait is unlikely to be heritable in the population observed. Discussion: It is likely that implementing a treatment to promote RDR expression in future studies might strengthen the documented correlation between phenotyping methods by maximizing genotypic variance. Even so, our analysis has suggested that ShinyFruit provides a viable, open-source solution to efficient phenotyping of size and color in blackberry fruit. The ability for users to adjust analysis settings should also extend its utility to a wide range of fruits and vegetables.

A multiplexed plant-animal SNP array for selective breeding and species conservation applications.

Reliable and high-throughput genotyping platforms are of immense importance for identifying and dissecting genomic regions controlling important phenotypes, supporting selection processes in breeding programs, and managing wild populations and germplasm collections. Amongst available genotyping tools, single nucleotide polymorphism arrays have been shown to be comparatively easy to use and generate highly accurate genotypic data. Single-species arrays are the most commonly used type so far; however, some multi-species arrays have been developed for closely related species that share single nucleotide polymorphism markers, exploiting inter-species cross-amplification. In this study, the suitability of a multiplexed plant-animal single nucleotide polymorphism array, including both closely and distantly related species, was explored. The performance of the single nucleotide polymorphism array across species for diverse applications, ranging from intra-species diversity assessments to parentage analysis, was assessed. Moreover, the value of genotyping pooled DNA of distantly related species on the single nucleotide polymorphism array as a technique to further reduce costs was evaluated. Single nucleotide polymorphism performance was generally high, and species-specific single nucleotide polymorphisms proved suitable for diverse applications. The multi-species single nucleotide polymorphism array approach reported here could be transferred to other species to achieve cost savings resulting from the increased throughput when several projects use the same array, and the pooling technique adds another highly promising advancement to additionally decrease genotyping costs by half.

Genome-wide association identifies key loci controlling blackberry postharvest quality.

Introduction: Blackberry (Rubus subgenus Rubus) is a soft-fruited specialty crop that often suffers economic losses due to degradation in the shipping process. During transportation, fresh-market blackberries commonly leak, decay, deform, or become discolored through a disorder known as red drupelet reversion (RDR). Over the past 50 years, breeding programs have achieved better fruit firmness and postharvest quality through traditional selection methods, but the underlying genetic variation is poorly understood. Methods: We conducted a genome-wide association of fruit firmness and RDR measured in 300 tetraploid fresh-market blackberry genotypes from 2019-2021 with 65,995 SNPs concentrated in genic regions of the R. argutus reference genome. Results: Fruit firmness and RDR had entry-mean broad sense heritabilities of 68% and 34%, respectively. Three variants on homologs of polygalacturonase (PG), pectin methylesterase (PME), and glucan endo-1,3-ß-glucosidase explained 27% of variance in fruit firmness and were located on chromosomes Ra06, Ra01, and Ra02, respectively. Another PG homolog variant on chromosome Ra02 explained 8% of variance in RDR, but it was in strong linkage disequilibrium with 212 other RDR-associated SNPs across a 23 Mb region. A large cluster of six PME and PME inhibitor homologs was located near the fruit firmness quantitative trait locus (QTL) identified on Ra01. RDR and fruit firmness shared a significant negative correlation (r = -0.28) and overlapping QTL regions on Ra02 in this study. Discussion: Our work demonstrates the complex nature of postharvest quality traits in blackberry, which are likely controlled by many small-effect QTLs. This study is the first large-scale effort to map the genetic control of quantitative traits in blackberry and provides a strong framework for future GWAS. Phenotypic and genotypic datasets may be used to train genomic selection models that target the improvement of postharvest quality.

High-density linkage maps and loci for berry color and flower sex in muscadine grape (Vitis rotundifolia).

KEY MESSAGE: Linkage maps of muscadine grape generated using genotyping-by-sequencing (GBS) provide insight into genome collinearity between Muscadinia and Euvitis subgenera and genetic control of flower sex and berry color. The muscadine grape, Vitis rotundifolia, is a specialty crop native to the southeastern USA. Muscadine vines can be male, female, or perfect-flowered, and berry color ranges from bronze to black. Genetic linkage maps were constructed using genotyping-by-sequencing in two F1 populations segregating for flower sex and berry color. The linkage maps consisted of 1244 and 2069 markers assigned to 20 linkage groups (LG) for the 'Black Beauty' × 'Nesbitt' and 'Supreme' × 'Nesbitt' populations, respectively. Data from both populations were used to generate a consensus map with 2346 markers across 20 LGs. A high degree of collinearity was observed between the genetic maps and the Vitis vinifera physical map. The higher chromosome number in muscadine (2n = 40) compared to V. vinifera (2n = 38) was accounted for by the behavior of V. vinifera chromosome 7 as two independently segregating LGs in muscadine. The muscadine sex locus mapped to an interval that aligned to 4.64-5.09 Mb on V. vinifera chromosome 2, a region which includes the previously described V. vinifera subsp. sylvestris sex locus. While the MYB transcription factor genes controlling fruit color in V. vinifera are located on chromosome 2, the muscadine berry color locus mapped to an interval aligning to 11.09-11.88 Mb on V. vinifera chromosome 4, suggesting that a mutation in a different gene in the anthocyanin biosynthesis pathway determines berry color in muscadine. These linkage maps lay the groundwork for marker-assisted breeding in muscadine and provide insight into the evolution of Vitis species.

Non-destructive Phenotyping to Identify Brachiaria Hybrids Tolerant to Waterlogging Stress under Field Conditions.

Brachiaria grasses are sown in tropical regions around the world, especially in the Neotropics, to improve livestock production. Waterlogging is a major constraint to the productivity and persistence of Brachiaria grasses during the rainy season. While some Brachiaria cultivars are moderately tolerant to seasonal waterlogging, none of the commercial cultivars combines superior yield potential and nutritional quality with a high level of waterlogging tolerance. The Brachiaria breeding program at the International Center for Tropical Agriculture, has been using recurrent selection for the past two decades to combine forage yield with resistance to biotic and abiotic stress factors. The main objective of this study was to test the suitability of normalized difference vegetation index (NDVI) and image-based phenotyping as non-destructive approaches to identify Brachiaria hybrids tolerant to waterlogging stress under field conditions. Nineteen promising hybrid selections from the breeding program and three commercial checks were evaluated for their tolerance to waterlogging under field conditions. The waterlogging treatment was imposed by applying and maintaining water to 3 cm above soil surface. Plant performance was determined non-destructively using proximal sensing and image-based phenotyping and also destructively via harvesting for comparison. Image analysis of projected green and dead areas, NDVI and shoot biomass were positively correlated (r ≥ 0.8). Our results indicate that image analysis and NDVI can serve as non-destructive screening approaches for the identification of Brachiaria hybrids tolerant to waterlogging stress.

Mapping of powdery mildew resistance gene Pm53 introgressed from Aegilops speltoides into soft red winter wheat.

KEY MESSAGE: A powdery mildew resistance gene was introgressed from Aegilops speltoides into winter wheat and mapped to chromosome 5BL. Closely linked markers will permit marker-assisted selection for the resistance gene. Powdery mildew of wheat (Triticum aestivum L.) is a major fungal disease in many areas of the world, caused by Blumeria graminis f. sp. tritici (Bgt). Host plant resistance is the preferred form of disease prevention because it is both economical and environmentally sound. Identification of new resistance sources and closely linked markers enable breeders to utilize these new sources in marker-assisted selection as well as in gene pyramiding. Aegilops speltoides (2n = 2x = 14, genome SS), has been a valuable disease resistance donor. The powdery mildew resistant wheat germplasm line NC09BGTS16 (NC-S16) was developed by backcrossing an Ae. speltoides accession, TAU829, to the susceptible soft red winter wheat cultivar 'Saluda'. NC-S16 was crossed to the susceptible cultivar 'Coker 68-15' to develop F2:3 families for gene mapping. Greenhouse and field evaluations of these F2:3 families indicated that a single gene, designated Pm53, conferred resistance to powdery mildew. Bulked segregant analysis showed that multiple simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers specific to chromosome 5BL segregated with the resistance gene. The gene was flanked by markers Xgwm499, Xwmc759, IWA6024 (0.7 cM proximal) and IWA2454 (1.8 cM distal). Pm36, derived from a different wild wheat relative (T. turgidum var. dicoccoides), had previously been mapped to chromosome 5BL in a durum wheat line. Detached leaf tests revealed that NC-S16 and a genotype carrying Pm36 differed in their responses to each of three Bgt isolates. Pm53 therefore appears to be a new source of powdery mildew resistance.