Fox hunting in wild apples: searching for novel genes in Malus sieversii

Authors: WISNIEWSKI, MICHAEL - Retired ARS Employee; Artlip, Timothy - Tim; LIU, JIA - Chongqing University; Ma, Jing; Burchard, Erik; NORELLI, JOHN - Retired ARS Employee; Dardick, Christopher - Chris

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2020
Publication Date: 12/14/2020
Citation: Wisniewski, M., Artlip, T.S., Liu, J., Ma, J., Burchard, E.A., Norelli, J., Dardick, C.D. 2020. Fox hunting in wild apples: searching for novel genes in Malus sieversii. International Journal of Molecular Sciences. http://dx.doi.org/10.3390/ijms21249516.
DOI: https://doi.org/10.3390/ijms21249516

Interpretive Summary: Commercial apple cultivars are vulnerable to many diseases, insect pests, and erratic weather conditions. These problems potentially impact supply and reliability along with grower profitability. The main ancestor to modern apples (Malus sieversii) arose in Central Asia and has been shown resistant to harsh weather conditions and to a variety of diseases. Unfortunately, the genome and genetics are poorly known. Simply breeding commercial cultivars with these wild apples would take decades to find progeny with desirable fruit and horticultural qualities. As a first step, winter bark tissues were collected from Malus sieversii trees, genetic material was isolated, and introduced the genetic material into the model plant species Arabidopsis, which has a short life span and is easily manipulated. We then sequenced over 4000 Arabidopsis plants harboring Malus sieversii genes. We screened Arabidopsis for enhanced freezing tolerance and for genes unknown in modern apples or other plant species. We found nearly 150 genes with no similarity to other genes. We didn’t find any Arabidopsis with enhanced freezing tolerance, but plan on screening for disease tolerance and other environmental stresses. This is important because we now have an extensive Arabidopsis seed bank with Malus sieversii genes that could serve as a valuable genetic resource for future rapid cycle breeding efforts with apple that can speed up the breeding process.

Technical Abstract: Malus sieversii is the main progenitor of modern apple (Malus pumila) cultivars and is considered a valuable resource for genetic diversity, especially since modern apple cultivars are derived from a small pool of founder genotypes. Despite the importance of M. sieversii as a source for disease resistance, stress tolerance, and novel fruit characteristics little is known about gene function in the genus of Malus. Notably, a genome sequence for this species is not available. To address this situation, we have constructed a genetic library of transgenic lines of Arabidopsis using the FOX (Full-length cDNA Over-eXpressing) gene hunting system where each transgenic line is overexpressing a full-length gene obtained from cDNA of the PI619283 selection of M. sieversii. The cDNA library was constructed from mRNA obtained from bark tissues collected in late fall – early winter, a time at which many abiotic and biotic stress-adaptative genes are expressed. PI613981 was previously used to identify a source of resistance in a mapping population (PI613981 X ‘Royal Gala’) to the postharvest disease, blue mold, caused by Penicillium expansum. A large selection of M. sieversii genotypes was collected as seed or budwood by the USDA-ARS and maintained at the USDA-ARS Apple Germplasm Repository, Geneva, NY. FOX hunting is an alternative gain-of-function gene hunting system used to study gene function and identify novel genes in a high-throughput system such as Arabidopsis. In the current study, full-length cDNA library from winter-acclimated M. sieversii accession PI613981 bark was constructed by conducting a mass transformation of apple genes into Arabidopsis. Over 4000 apple-FOX Arabidopsis lines have been currently established with the eventual objective of generating approximately 10,000 independent lines. Individual lines are being grown in several generations to enhance homozygosity of the inserted gene and to generate a store of seeds for screening for functional genotypes and for distribution to other researchers interested in apple gene function. Transformation is being confirmed by screening for antibiotic resistance. Once confirmed, inserted genes are being sequenced and annotated by BLAST analysis using the sequences resident at the Genome Database for Rosaceae, apple genome version 3 (GDDH) and NCBI. Phenotypic information (morphology) is also being added to an in-house database. Inserts from various GO categories were identified including cell cycle and dormancy genes (cyclins, methyl transferases, vernalization5, remorin). Abiotic stress response genes such as Late Embryogenesis Abundant (LEA), Heat Shock Protein (HSP)-20 chaperones and redox-homeostasis genes were found, along with biotic stress response genes such as mitogen-activated kinases and disease resistance/ receptors. Numerous lines have also been screened for freezing resistance. The transgenic lines being constructed represent a valuable genetic resource for exploring gene function and diversity in Malus sieversii.