Chloroplast Genome Mapping and Plastid Ulrastructure Analysis of Chlorophyll Deficient Mutants of Alfalfa

Abstract

Physical maps of chloroplast genomes allow comparison of genome structure and organization and can provide physical markers useful for evaluating plastid inheritance. Physical maps of the chloroplast DNA (ctDNA) from phenotypically normal alfalfa (Medicago sativa L.) genotypes and two independently isolated cytoplasmically and biparentally inherited chlorophyll‐deficient mutants were developed and compared. Both mutants were observed as somatic sectors but each had a different phenotype, one producing albino and the other yellow‐green tissue. Maps of overlapping restriction fragments were constructed using both purified chloroplast DNA and total DNA digested with the restriction endonucleases Bam HI, Xho I, and Hind III and utilizing a complete set of chloroplast probes. A total of four points of variation, two changes in restriction endonuclease recognition sites and two small addition/deletion events were observed among the genotypes studied. Combinations of these polymorphisms defined four distinct ctDNA genotypes. Only one addition/deletion polymorphism was unique to the mutant chloroplast DNA (ctDNAs) and the albino and yellow‐green chloroplast genomes were indistinguishable from each other by this analysis. Electron microscopy revealed differences in development between the two chlorophyll‐deficient mutants. Plastids from albino tissue showed envelope degradation, no internal membrane development, sparse stroma with vacuoles, and osmiophyllic globuli. Plastids from yellow‐green tissue showed limited thylakoid membrane development, no grana, relatively dense stroma with ribosome‐like particles, osmiophyllic globules, and various degrees of vacuolization. This study identified molecular markers with which to assess plastid inheritance in alfalfa and demonstrated that the dramatic arrest in plastid development found in both mutants was not paralleled by large changes in their chloroplast genomes.