A region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atpA) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3' ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3' coding and 3' flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5' end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.

The turf-type bermudagrasses include diploid Cynodon transvaalensis Burtt Davy, tetraploid C. dactylon (L.) Pers., and sterile triploid hybrids produced by crosses of these species. The objective of thiis study was to develop a set of mierosatellite markers that could distinguish among commercially important turf-type cultivars. A genomic library enriched for tfie [CA/GT]^ repeat motif was constructed from DNA of the Tifway' fiybrid and sequenced to identify mierosatellite regions. Twenty-five microsatellite-flanking primer sets were developed and used to genotype two plant introductions and 12 turf-type cultivars. These primer sets produced an average of 10 amplicons across tfie 14 DNA templates. Sequences of selected amplicons revealed polymorphism resulting from expansion and/or contraction of the microsatellite and from indel mutations in the microsatellite flanking regions. As few as two primer sets were sufficient to differentiate all unrelated introduction lines and cultivars. Tfie primer sets failed to distinguish among closely related cultivars developed by selection of natural variants, but one primer set uniquely distinguished the cultivar TifEagle from its irradiated parent cultivar. Tfiese genomic microsatellites were not derived from gene coding sequences and will supplement tfie existing expressed sequence tag (EST)-based bermudagrass microsatellites. Tfiey will be most useful for evaluating tfie genetic diversity of Cynodon accessions and distinguishing among cultivars that exploit thils diversity.

Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.

Restorer-of-fertility (Rf ) alleles for S-type cytoplasmic male sterility (CMS-S) are prevalent in Mexican races of maize and teosinte. Forty-five Rf alleles from 26 races of maize and 6 Rf alleles from different accessions of teosinte were found to be homozygous viable, consistent with the hypothesis that they are naturally occurring Rf alleles. Mapping and allelism studies were performed to assess the number of genes represented by these 51 alleles. Forty-two of the Rf alleles mapped to the long arm of chromosome 2 (2L), and 5 of these were further mapped to the whp1-rf3 region. The Rf3 restoring allele, found in some U.S. maize inbred lines, cosegregates with internal processing of CMS-S mitochondrial transcripts. Three of the 5 mapped Rf alleles were associated with a similar RNA processing event. Allelism or tight linkage was confirmed between Rf3 and 2 teosinte alleles (Rf K-69-6 and Rf 9477) and between Rf3 and the Cónico Norteño allele Rf C-N (GTO 22). The rf3 region of 2L potentially encodes a complex of linked rf genes. The prevalence of restoring alleles in this chromosomal region, among normal-cytoplasm accessions of Mexican maize and teosinte, supports the conclusion that these alleles have functions in normal mitochondrial gene expression that by chance allow them to restore male fertility in S cytoplasm.