Nuclear DNA contents and ploidy levels of North American Vaccinium species and interspecific hybrids

Authors: REDPATH, LAUREN - North Carolina State University; ARYAL, RISHI - North Carolina State University; LYNCH, NATHAN - North Carolina State University; SPENCER, JESSICA - North Carolina State University; Hulse-Kemp, Amanda; BALLINGTON, JAMES - North Carolina State University; Green, Jaimie; Bassil, Nahla; Hummer, Kim; RANNEY, THOMAS - North Carolina State University; ASHRAFI, HAMID - North Carolina State University

Submitted to: Scientia Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/28/2022
Publication Date: 2/9/2022
Citation: Redpath, L., Aryal, R., Lynch, N., Spencer, J.A., Hulse-Kemp, A.M., Ballington, J.R., Green, J., Bassil, N.V., Hummer, K.E., Ranney, T., Ashrafi, H. 2022. Nuclear DNA contents and ploidy levels of North American Vaccinium species and interspecific hybrids. Scientia Horticulturae. 297. Article 110955. https://doi.org/10.1016/j.scienta.2022.110955.
DOI: https://doi.org/10.1016/j.scienta.2022.110955

Interpretive Summary: Blueberry breeding involves the crossing of many different species that have very different genome structures. Understanding these different genome structures is essential for the modern blueberry breeder to know what is possible within the scope of the breeding materials. This project focuses of utilizing high-throughput methods to analyze the genome structure of a diverse set of breeding materials in the collections at North Carolina State University and at the National Clonal Germplasm Repository in Corvallis, Oregon. A method called flow cytometry, which measures the size of DNA by visualizing the size of nucleus in each sample, was used along with a second method called kmer analysis, which is a sophisticated method for looking at unique word strings in DNA sequence data, in order to calculate the genome sizes. We found that there is generally a varying range about the mean for different levels of ploidy from two copies of the genome in diploids, to six copies of the genome in hexaploids in different species. This data will help breeders in each of the programs as well as stakeholders that request materials from the germplasm collection for their own research and breeding purposes better use the materials available to produce the next generation of blueberry cultivars.

Technical Abstract: Breeding strategies for improving blueberry (Vaccinium corymbosum and V. virgatum) cultivars often include introgressing regionally adapted species into the cultivated gene pools through interspecific hybridization. However, these approaches are complicated in blueberry due to variation in ploidy, triploid blocks and infertility, production of unreduced gametes, and aneuploidy. Having knowledge of ploidy and reproductive pathways in germplasm collections and breeding programs is foundational for developing breeding strategies, genome-wide association studies (GWAS), and genomic selection studies. The objective of this study was to use flow cytometry, k-mer distribution analysis, and known pedigree information to evaluate genome sizes (2C nuclear and 1Cx monoploid), and ploidy of diverse accessions from Vaccinium sections and species that comprise an inter- and intra-specific diversity panel (DP). Plants were sampled from the North Carolina State University blueberry breeding program and the National Clonal Germplasm Repository collections, and included a DP of 358 accessions that are simultaneously being used for related genomic studies. The nuclear DNA content was analyzed via flow cytometry. The mean (range) DNA content of diploid, tetraploid, and hexaploid reference species was 1.20 pg (0.99 pg in V. crassifolium ‘Well’s Delight’ to 1.41 pg in V. caesariense NC79-24), 2.37 pg (2.11 pg in V. corymbosum ‘Concord’ to 3.01 pg in V. corymbosum DE599), and 3.64 pg (3.24 in V. constablaei NC83-21-2 to 3.80 in V. virgatum ‘Premier’ and NC4790), respectively. Of the 358 unique accessions analyzed for ploidy, 225 were tetraploid, 28 were diploid, 2 were triploid, 77 were pentaploid or aneuploid with 2C values between tetraploid and hexaploid values, and 26 were hexaploid. Pedigree analysis of hybridization in the tetraploid accessions primarily consisted of interspecific crosses within tetraploid species or between diploid and tetraploid species producing tetraploid offspring. Diploid species that readily hybridized with tetraploids, producing tetraploids, included V. caesariense, V. darrowii, V. elliottii, V. ovatum, V. pallidum, and V. tenellum, indicating that they produce unreduced gametes. Tetraploid hybrid pedigrees, which involved hexaploid crosses within three prior generations, had a 2C value range between 2.22 pg and 2.59 pg. Anticipated pentaploid 2C DNA content is ~3 pg; however, the interspecific pentaploid and aneuploid progeny 2C DNA content ranged from 2.61 pg to 3.15 pg. We speculate some of these progeny to be near tetraploids with extra chromosomes from hexaploid progenitors. Further karyotyping of these individuals is necessary to ascertain aneuploidy anomalies. This research provides an expanded knowledge base of genome sizes, ploidy, and reproductive pathways for diverse species and hybrids to enhance future breeding, improvement, and the genomic study of blueberry.