Phillip D. Griffiths

Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats

Sequence databases were screened to identify simple sequence repeats (SSRs) in Brassica oleracea sequences. A total of 512 B. oleracea DNA sequences were screened and 43 potential SSRs were identified. Thirty-six primer pairs were designed to amplify target sequences. Of the 36 primer pairs, six failed to amplify fragments of expected sizes, and 17 primer pairs failed to generate polymorphisms. Thirteen SSRs were used to assess genetic similarity between 54 B. oleracea cultivars, belonging to 3 variteal groups (cabbage, cauliflower, and broccoli). Pairwise genetic similarities were calculated for cultivars, and a dendrogram of relationships was produced. All cabbage cultivars were distinguished from each other and clustered in two separate groups. Five cauliflower cultivars could not be distinguished with SSR markers used in the study. Three broccoli cultivars clustered with cauliflower cultivars, and two cauliflower cultivars grouped with broccoli cultivars. The varietal group with the narrowest genetic variation in the study was cauliflower (B. oleracea var. botrytis) followed by broccoli (B. oleracea var. italica) and cabbage (B. oleracea var. capitata) groups. Polymorphism information content (PIC) values and number of alleles produced per marker ranged between 0.25 to 0.86 and 1 to 8, respectively, for database derived SSR markers.

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F2 populations were developed by self-pollinating 3 black rot resistant F1 plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F2 plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F2 plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production.

Development of black rot resistant interspecific hybrids between Brassica oleracea L. cultivars and Brassica accession A 19182, using embryo rescue

Black rot is a bacterial disease of Brassica oleracea caused by Xanthomonas campestris pv. campestris. Resistance to the major black rot races 1 or 4 has been identified in related Brassica species including B. carinata and B. napus. In this study, two B. juncea accessions (A 19182 and A 19183) that are resistant to races 1 and 4 of Xcc were used as maternal and paternal parents to generate interspecific hybrids with B. oleracea cultivars. Interspecific hybrids were recovered using the embryo rescue technique and confirmed through inheritance of paternal molecular markers. Twenty-six interspecific hybrid plants were obtained between A 19182 and B. oleracea cultivars, but no interspecific hybrids were obtained using A 19183. Although interspecific hybrid plants were male sterile, they were used successfully as maternal parents to generate backcross plants using embryo rescue. All hybrid and BC1 plants were resistant to black rot races 1 and 4.

Genotyping-by-Sequencing Enabled Mapping and Marker Development for the By-2 Potyvirus Resistance Allele in Common Bean

Since its emergence in 2001, an aphid‐transmitted virus disease complex has caused substantial economic losses to snap bean (Phaseolus vulgaris L.) production and processing in the Great Lakes Region of the United States. The general ineffectiveness of chemical control measures for nonpersistently transmitted viruses established an urgent need for the development and deployment of cultivars with resistance to the component viruses. Our objectives were to further characterize the inheritance of resistance to Bean yellow mosaic virus (BYMV), which is conditioned by the By‐2 allele, to adapt genotyping‐by‐sequencing (GBS) to common bean to discover and genotype genome‐wide single nucleotide polymorphisms (SNPs) in a set of recombinant inbred lines (RILs) derived from an introgression program, and to enable and validate marker‐assisted selection for By‐2. We optimized ApeKI for GBS in common bean and retained 7530 high‐quality SNPs that segregated in our introgression RILs. A case–control genome‐wide association study (GWAS) was used to discover 44 GBS SNPs that were strongly associated with the resistance phenotype and which delimited a 974 kb physical interval on the distal portion of chromosome 2. Seven of these SNPs were converted to single‐marker Kompetitive Allele‐Specific Polymerase chain reaction (KASP) assays and were demonstrated to be tightly linked to BYMV resistance in an F2 population of 185 individuals. This research enables marker‐assisted selection of By‐2, provides enhanced resolution for fine mapping, and demonstrates the potential of GBS as a highly efficient, high‐throughput genotyping platform for common bean breeding and genetics.

Diallel analysis of yield components of snap beans exposed to two temperature stress environments

Ten snap beans (‘Barrier’, ‘Brio’, ‘Carson’, ‘Cornell 502’, ‘CT 70’, ‘HB 1880’, ‘Hystyle’, ‘Labrador’, ‘Opus’ and ‘Venture’) were selected for differential temperature tolerance and used as parents in a complete diallel mating design. The 45 F1 hybrid lines (with reciprocals) and parents were screened at 32 ∘C day/28 ∘C night, and in a separate experiment, 16 ∘C day/10 ∘C night, during reproductive development in replicated controlled environments. Variation for yield under temperature treatments was observed among parents and hybrids, with certain hybrids exceeding parental performance. Significant (P≤ 0.0001) general combining ability (GCA), and significant (P≤ 0.05) specific combining ability (SCA) were observed for yield components including pod number, seed number, and seeds per pod. There was evidence that pod number and seeds per pod under temperature stress are under separate genetic control. Reciprocal effects and heterosis were not significant. GCA could not be predicted from parental performance. The breeding line ‘Cornell 502’ had the highest GCA under high temperature, and the cultivar ‘Brio’ had the highest GCA under low temperature. The cross ‘Brio’ × ‘Venture’ was high yielding in both temperature treatments. Heat tolerance and chilling tolerance were associated in certain parents and hybrids. However, performance under high and low temperature treatments was not generally correlated in the parents and hybrids, indicating that these traits should be selected separately.

A series of eIF4E alleles at the Bc-3 locus are associated with recessive resistance to Clover yellow vein virus in common bean

Clover yellow vein virus (ClYVV) is capable of causing severe damage to common bean (Phaseolus vulgaris L.) production worldwide. The snap bean market class is particularly vulnerable because infection may lead to distortion and necrosis of the fresh green pods and rejection of the harvest. Three putatively independent recessive genes (cyv, desc, bc-3) have been reported to condition resistance to ClYVV; however, their allelic relationships have not been resolved. We identified, evaluated, and characterized the phenotypic and molecular genetic variation present in 21 informative common bean genotypes for resistance to ClYVV. Allelism testing phenotypes from multiple populations provided clear evidence that the three genes were a series of recessive alleles at the Bc-3 locus that condition unique potyvirus strain- and species-specific resistance spectra. Candidate gene analysis revealed complete association between the recessive resistance alleles and unique patterns of predicted amino acid substitutions in P. vulgaris eukaryotic translation initiation factor 4E (PveIF4E). This led to the discovery and characterization of two novel PveIF4E alleles associated with resistance to ClYVV, PveIF4E3, and PveIF4E4. We developed KASPar allele-specific SNP genotyping assays and demonstrated their ability to accurately detect and differentiate all of the PveIF4E haplotypes present in the germplasm, allelism testing, and in three separate segregating populations. The results contribute to an enhanced understanding and accessibility of the important potyvirus resistance conditioned by recessive alleles at Bc-3. The KASPar assays should be useful to further enable germplasm exploration, allelic discrimination, and marker-assisted introgression of bc-3 alleles in common bean.

Effects of Growth Temperature and Postharvest Cooling on Anthocyanin Profiles in Juvenile and Mature Brassica oleracea

The effects of growth temperatures on anthocyanin content and profile were tested on juvenile cabbage and kale plants. The effects of cold storage time were evaluated on both juvenile and mature plants. The anthocyanin content in juvenile plants ranged from 3.82 mg of cyanidin-3,5-diglucoside equivalent (Cy equiv)/g of dry matter (dm) at 25 °C to 10.00 mg of Cy equiv/g of dm at 16 °C, with up to 76% diacylated anthocyanins. Cold storage of juvenile plants decreased the total amount of anthocyanins but increased the diacylated anthocyanin content by 3-5%. In mature plants, cold storage reduced the total anthocyanin content from 22 to 12.23 mg/g after 5 weeks of storage in red cabbage, while the total anthocyanin content increased after 2 weeks of storage from 2.34 to 3.66 mg of Cy equiv/g of dm in kale without having any effect on acylation in either morphotype. The results obtained in this study will be useful for optimizing anthocyanin production.