Identification of early fruit development reference genes in plum

Authors: Galimba, Kelsey; TOSETTI, ROBERTA - Cranfield University; LOERICH, KAREN - Hagerstown Community College; MICHAEL, LEANN - Hagerstown Community College; PABHAKAR, SAVITA - Hagerstown Community College; DOVE, CYNTHIA - Hagerstown Community College; Dardick, Christopher - Chris; Callahan, Ann

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2020
Publication Date: 4/17/2020
Citation: Galimba, K.D., Tosetti, R., Loerich, K., Michael, L., Pabhakar, S., Dove, C., Dardick, C.D., Callahan, A.M. 2020. Identification of early fruit development reference genes in plum. PLoS One. https://doi.org/10.1371/journal.pone.0230920.
DOI: https://doi.org/10.1371/journal.pone.0230920

Interpretive Summary: One way to look for genes that affect different traits in breeding is to see if the gene’s RNA expression is different in the plants with that trait. To measure that change in RNA expression, you look at differences in the ratio of the gene of interest and a reference gene that doesn’t change expression. There are over 26,000 genes in plum and they express RNA at different times, in different tissues, and under different environmental conditions. We are interested in fruit development and how to eliminate the stone in stone fruit like plums. To pick out a set of reference genes, we sequenced all of the RNAs at different stages of early plum fruit development (0-12 days after bloom) and in the stone tissues, and chose nine genes that were expressed at the same levels at all times and tissues to test as reference genes. We tested these using qPCR on a fruit RNA development series including stone tissues. Three genes were expressed at the same level in all of the tissues and times. We can now use these to test genes that we think affect specific traits, like lack of stones, to determine if their RNA varies with that trait.

Technical Abstract: An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2-fold amongst the 20 libraries of early fruit development and endocarp tissues. These were tested on a plum series of developmental fruit samples to determine if any could be used as a reference gene in the analyses of fruit-based tissues in plum. The three most stable genes as determined using RefFinder, imidazole glycerol-phosphate dehydratase, histone acetyltransferase and a sorting nexin 1, were further tested to standardize genes expressed differentially in endocarp tissue between normal and minimal endocarp cultivars. To determine the universality of those nine genes as fruit development reference genes, three other data sets of RNAseq from peach and apple were analyzed to determine the reference gene expression. Multiple genes exhibited tissue specific patterns of expression, while one gene, the sorting nexin 1, emerged as possessing a universal pattern between species, developmental stages, and tissue types. The results suggest that the use of existing RNAseq data to identify standard genes can provide references for a specific tissues or experimental conditions under exploration.