Ploidy of USDA (United States Department of Agriculture) world pear germplasm collection determined by flow cytometry

Authors: Postman, Joseph; Bassil, Nahla; Bell, Richard

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2015
Publication Date: 9/20/2015
Citation: Postman, J.D., Bassil, N.V., Bell, R.L. 2015. Ploidy of USDA (United States Department of Agriculture) world pear germplasm collection determined by flow cytometry. Acta Horticulturae. 1094:75-81.

Interpretive Summary: The USDA National Clonal Germplasm Repository maintains a collection of wild and cultivated pears from around the world in a living collection located outside of Corvallis, Oregon. All wild pear species have two sets of chromosomes (diploid), but some cultivated varieties have more than two sets. Some varieties are known to have three sets of chromosomes (triploid) and a few have four (tetraploid) or more. Flow cytometry is a technique used to determine the total amount of DNA in a cell by disolving the cells, staining the DNA and quickly passing the cell nuclei through a detector that measures the amount of stain and therefore the relative amount of DNA per cell. A cell nucleus that is tetraploid (4 chromosomes) with have twice as much DNA as a normal diploid cell. A triploid (3 chromosomes) cell will have an amount of DNA intermediate between a diploid and a tetraploid. We measured the amount of DNA in the cells of 1,284 different pear trees representing species and cultivars. All wild pears were diploid, as expected. Some pear varieties that were supposed to be tetraploid, according to information received with the trees, were actually diploid. Eighty eight triploid pears were detected, and nearly all of these were European rather than Asian cultivars. A DNA fingerprinting technique was used to confirm the number of chromosomes in some of the triploid pears, and the results agreed with the flow-cytometry results. The results of the flow-cytometry confirmed the identities of some pears varieties that are known to be triploid, and allowed us to discard some trees that supposed to be tetraploid and were not. We discovered that dozens of trees were triploid that had not previously been know to be. The pollen from triploid trees is usually sterile, and knowing the number of chromosomes in each tree in the collection helps plant breeders to avoid using trees that will not be successful at producing seeds.

Technical Abstract: Living germplasm collections representing world diversity of pear (Pyrus L.) are maintained by the U.S. Department of Agriculture at the National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, USA. Flow cytometry was performed on young leaf tissue from 1,284 genebank accessions to assess ploidy, and 93% were found to be diploid (2n = 2x = 34). All pear wild relative species examined were diploid. The European and interspecific hybrid cultivar collections included 85 triploid trees. Only three Asian cultivars were found to be triploid. One European cultivar was tetraploid and one was aneuploid (2.3x). Six pear clones received as purported tetraploids were determined to be diploid, and two were chimeral, having both tetraploid and diploid tissue. Many of the pear cultivars identified as triploid were not previously known as such. Simple sequence repeat (SSR) or microsatellite-based profiles were generated for 19 triploid accessions and reference pear cultivars using a standard fingerprinting set of 12 SSR markers. Two to nine of the 12 SSRs generated three alleles in the triploid accessions, thus supporting the triploid determination. The use of flow cytometry to determine ploidy combined with SSR markers for genetic fingerprinting helped to confirm synonymy in several pear accessions. Ploidy results permitted the elimination of misidentified accessions that should have represented triploid or tetraploid genotypes. Knowledge about the ploidy of genebank accessions will help breeders avoid using sterile pollen when making crosses. The NCGR genebank provides propagation material for breeding and genetic research by on-line request through the USDA Germplasm Resources Information Network (GRIN).