Biochemical marker genes for molecular genetics and plant breeding in pisum sativum L

Abstract

Three isoenzyme systems (amylase, esterase and glutamate oxalo acetate transaminase) were examined in seeds of pea ( Pisum sativum L.) and showed clear variations in their band patterns on gel electro phoresis between different lines. The inheritance of these isoenzyme systems, and the location of their structural genes on the pea genome were investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase ( Amy ) was found to be on chromosome 2, linked to the loci k and wb ( wb.. .9-k. . .25.. .Amy ). The gene for esterase ( Est ) was found to be linked to the gene Br (chromosome 4) but the exact location is uncertain because of a lack of morphological markers. The gene for glutamate oxaloacetate transaminase ( Got ) was found to be on chromosome 1 linked to the loci a and d ( a...24...Got...41.. d). Gel electrophoresis techniques have also been used to investigate genetically controlled variation in the major subunits (50,000 Mr) of vicilin, a storage protein of Pisum sativum L. The Fl protein band patterns were shown to be additive with respect to those of the parental lines and to be identical in reciprocal crosses. Genetic analysis of the F2 plants indicated that the 50,000 Mr vicilin subunits band pattern is controlled by a pair of co-dominant genes at a single locus. The F2 data were used to locate this major vicilin gene locus ( Vc-1 ) to chromoscane 7, closely linked to the r locus (for round and wrinkled seed surface). A third member of pea legumin gene family, denoted legB, has been sequenced using the "dideoxy chain termination" method with the M1 3 sequencing system. The complete nucleotide sequence showed that this gene has a general form typical of an eukaryotic gene. The homolgies between this gene and the previously published gene "legA" 'were estimated and showed strong homology between the two genes with eight amino acid substitutions and deletion of 14 bp in the third intron (IVS-3).The inheritance of ribosomal RNA (rRNA) genes in ( Pisum sativum L.) was investigated in a cross between two different lines, where length variation in rDNA fragments of Eco RI digests was observed. The results obtained showed that the rRNA genes are controlled by simple Mendelian system with "co-dominance" between alleles. In order to locate the rRNA gene sites to positions on the chromosomes, the segregation of ECO. RI restriction fragments of rDNA from F2 plants with respect to genes for selected morphological markers on chromosomes 4 and 7 (the chromosomes known to have nucleolus organizer regions) were tested. The F2 data showed no linkages between the selected markers and rRNA genes, therefore, in situ hybridization using rDNA radioactive probe ((^3)H- labelled rDNA clone, pHAI) and physical mapping procedures were used. The results obtained have located the rRNA gene sites to nucleolus organizer regions (satellite constrictions) at 138 and 60 map units from the centromeres of chromosomes 4 and 7, respectively.