Paul W. Skroch

Towards an integrated linkage map of common bean. 4. Development of a core linkage map and alignment of RFLP maps

Abstract Three RFLP maps, as well as several RAPD maps have been developed in common bean (Phaseolus vulgaris L.). In order to align these maps, a core linkage map was established in the recombinant inbred population BAT93×Jalo EEP558 (BJ). This map has a total length of 1226 cM and comprises 563 markers, including some 120 RFLP and 430 RAPD markers, in addition to a few isozyme and phenotypic marker loci. Among the RFLPs mapped were markers from the University of California, Davis (established in the F2 of the BJ cross), University of Paris-Orsay, and University of Florida maps. These shared markers allowed us to establish a correspondence between the linkage groups of these three RFLP linkage maps. In total, the general map location (i.e., the linkage group membership and approximate location within linkage groups) has been determined for some 1070 markers. Approaches to align this core map with other current or future maps are discussed.

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes.

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.

Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance

RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Neis average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.

Species boundaries and interrelationships of two closely related sympatric diploid wild potato species, Solanum astleyi and S. boliviense, based on RAPDs

Abstract The more than 200 wild and cultivated species relatives of potato (Solanum sect. Petota) present a valuable germplasm base for cultivar improvement. However, species boundaries and interrelationships within sect. Petota are controversial, inhibiting the efficient organization of the many germplasm collections of these species. One controversy involves questions of species boundaries and interrelationships of S. astleyi and S. boliviense. Solanum boliviense is narrowly endemic to two Departments in southern Bolivia, and S. astleyi is known only from one site entirely within the range of this species, where they co-occur. Both species are diploid and morphologically very similar. Artificial hybrids between them are fully fertile, and the species putatively hybridize naturally. These data have been interpreted to designate them as separate species or as S. astleyi an ecotype of S. boliviense. Putative progenitors of S. astleyi are S. boliviense, S. megistacrolobum subsp. megistacrolobum, and S. megistacrolobum subsp. toralapanum. We evaluated interrelationships among these species with random amplified polymorphic DNA’s (RAPDs) generated for 2 accessions of S. astleyi and 14 accessions of S. boliviense. These represent the entire geographic range of the former species and nearly the entire range of the latter. We also analyzed 1 accession each of S. acaule subsp. acaule, S. acaule subsp. aemulans, S. albicans, S. berthaultii, S. megistacrolobum subsp. megistacrolobum, S. megistacrolobum subsp. toralapanum, S. raphanifolium, S. sogarandinum, and S. sparsipilum. Phenetic analyses of the RAPD data show S. astleyi and S. boliviense to form two distinct groups and to be more similar to each other than to any of the other species investigated, suggesting that S. astleyi and S. boliviense are sister taxa. The divergence of S. astleyi and S. boliviense relative to other species examined suggests that they are worthy of taxonomic recognition at the subspecies, rather than species level, and we propose the new combination S. boliviense subsp. astleyi.

Phaseolus vulgaris — The common bean integration of RFLP and RAPD-based linkage maps

Gregor Mendel (1866) conducted the first genetic analysis of common beans. Mendel studied the inheritance of growth habit, and pod color and shape in a progeny between P. vulgaris and P. nanus (= P. vulgaris, bush type) in order to confirm his findings with peas. Unfortunately, further studies on the inheritance of flower and seed coat color were hampered by his use of interspecific hybrids between P. nanus and P. multiflorus (= P. coccineus), which are now known to yield aberrant ratios. Later, Shaw and Norton (1918) used intraspecific crosses and determined that pigmentation and pigmentation patterns of the seed coat are controlled by multiple independent factors. A few years later Sax (1923) began to identify the multiple components that determine the inheritance of these traits. A single factor was identified as responsible for pigmentation, while two linked factors were identified to control mottling; this appears to be the first report of linkage in beans. Furthermore, Sax (1923) was the first to report linkage between a Mendelian character (seed coat pigmentation) and a QTL (for seed size). Although the common bean was used as experimental material at the inception of genetics, its genetic characterization has lagged behind that of many other crop species.

Polymorphism of the leghemoglobin gene in Phaseolus demonstrated by polymerase chain reaction amplification

SummaryGenetic variance within Phaseolus vulgaris or among Phaseolus species for leghemoglobin composition may be useful in breeding for enhanced nitrogen fixation. Using primers constructed from conserved regions of the leghemoglobin gene, polymerase chain reaction was used to specifically amplify the Lba gene in total DNA samples from 17 lines and 10 species of Phaseolus. These primers are 100% homologous with the 5′- and 3′-ends of the leghemoglobin-encoding genes Lba of Phaseolus vulgaris, Lbc2 and Lbc3 of Glycine max, and 90% homologous to the G. max Lba gene. With one exception, only a single band was amplified using this approach with DNA isolated from 11 species of Phaseolus. The species of Phaseolus used in these experiments can be grouped into six classes based on the size of the amplified product which corresponded to their presumed genetic relatedness. Arranged in decreasing order by size these classes are: (1) Phaseolus lunatus and Phaseolus polystacius; (2) Phaseolus anisotrichus; (3) Phaseolus acutifolius, Phaseolus filiformis, Phaseolus angustissimus acc. # 16, and Phaseolus oligospermus; (4) Phaseolus angustissimus acc # 166, which had two major bands; (5) Phaseolus polyanthus, Phaseolus microspermus, and Phaseolus coccineus; and (6) Phaseolus vulgaris. No significant heterogeneity of amplified product within Phaseolus vulgaris was observed among 12 lines examined, but heterogeneity was observed between lines within Phaseolus acutifolius and Phaseolus angustissimus. Polymerase chain reaction amplification of conserved genes may be a useful method to facilitate the introgression of desirable genes from wild and exotic germplasm.