Chromosome Sorting and PCR-Based Physical Mapping in Pea (Pisum Sativum L.)

Abstract

Three pea lines with reconstructed karyotypes were used for analysis and subsequent purification of individual chromosome types using flow cytometry and sorting. The lines JI 145, JI 146, and JI 148 possess defined chromosomal translocations allowing discrimination of three to four chromosome types from each line based on the different sizes of translocation chromosomes. Whereas only two chromosomes could be sorted from standard (wild-type) karyotype, a combined use of these lines allowed sorting of six out of the seven types of pea chromosomes. Chromosomes were identified and purity of flow-sorted fractions was assessed using fluorescence in-situ hybridization with a PisTR-B probe that was previously shown to give labelling patterns characteristic for each chromosome types. The fractions of flow-sorted chromosomes were of very high purity (> 95%) and proved to be suitable for detection of gene and marker sequences using PCR with specific primers. Three fractions containing chromosomes 2⁷, 7² and a pool of all remaining chromosomes (1, 3, 4, 5, 6) flow-sorted from the line JI 148 were then used for PCR-based physical localization of genetic markers selected from linkage groups IV and VII. These experiments enabled assignment of the linkage groups IV and VII to chromosomes 4 and 7, respectively.