Genetic Variability Analysis Of Common Bean Phaseolus Vulgaris L Using Various Molecular Markers PDF Download

Common bean (Phaseolus vulgaris) plants exhibit two distinct stem termination growth habit types: indeterminate and determinate. Plants with indeterminate growth habit have a terminal meristem that remains vegetative throughout the production of lateral vegetative and reproductive growth. Contrarily, in determinate plants, the terminal shoot meristem switches from a vegetative to reproductive state, resulting in a terminal inflorescence. While only indeterminate growth is observed in wild-type common bean populations, both growth habits can be observed in domesticated varieties. Determinate growth habit has been artificially selected, in combination with photoperiod insensitivity, to obtain varieties with a shortened and synchronized flowering period and earlier maturation, allowing mechanized harvest. In the early 1990s, researchers in Arabidopsis thaliana identified a gene, Terminal Flowering 1 (TFL1), as a locus controlling inflorescence meristem development. Three homologs of TFL1 were identified in common bean: Phaseolus vulgaris Terminal Flowering 1 x, y, and z (PvTFL1x, PvTFL1y, and PvTFL1z). PvTFL1y was the only candidate gene to contain natural genetic variation potentially correlated with determinacy and also co-segregate with the phenotypic locus for determinate growth habit (fin) on chromosome 01. In chapter one, our goal was to verify the role of PvTFL1y in determining growth habit. Using quantitative PCR, we found that two distinct mutant PvTFL1y haplotypes associated with determinacy caused mRNA abundance to decrease 20-133 fold; consistent with the recessive nature of fin. Furthermore, we were able to rescue mutant determinate (tfl1-1) Arabidopsis plants by transformation with the wild-type PvTFL1y gene. This work validates that the candidate gene, PvTFL1y, is the functional homolog of TFL1 and is the molecular locus underlying the fin phenotypic locus. In chapter two, we use the pervasive synteny among legumes species in a comparative genomics approach to identify growth habit homologs of PvTFL1y in 16 different Phaseolus species; two of which include accessions with a determinate growth habit: P. coccineus and P. lunatus. Neighbor-joining analyses of amplified homologs verified previous genetic relationships in the Phaseolus species. Additionally, a 2bp insertion/deletion (indel) in the P. lunatus homolog, PlTFL1, was found to segregate with growth habit. We created a Cleaved Amplified Polymorphic sequence (CAPs) marker for this indel that can be utilized in future co-segregation studies. No mutation could yet be found that could be causal to determinacy in P. coccineus. Several unique mutant alleles for determinate growth habit have been identified at the PvTFL1y locus and it is of interest whether they confer differences in flowering and plant architecture. In chapter three, we analyzed dissimilarities between four mutant PvTFL1y haplotypes by transferring them, using marker-assisted backcrossing, into a common genetic background. Most notable, one backcross population containing the most prevalent mutant haplotype, a 4.1kb retrotransposon, was found to begin and finish flowering significantly earlier than the recurrent parent and all other backcross populations. It is likely that determinate growth habit in association with earliness may have caused this haplotype to become the most agronomically desirable during or after domestication. More generations of backcrossing followed by recurrent parent background genetic selection will elucidate if identified trait associations are caused by pleiotropy or linkage. Common bean was domesticated independently in Mesoamerica and in the Andes. Within the Mesoamerican center, the domestication area has been narrowed to the Lerma-Santiago Basin of Mexico, while the Andean center still remains quite large. During Andean domestication, determinate growth habit was likely selected for as it led to a more compact growth habit and earliness. By analyzing shared haplotypes between wild and domesticated common bean, around the domestication locus PvTFL1y, it may be possible to narrow the Andean center of domestication. In chapter four we analyzed an 800 kb region around PvTFL1y, in 96 wild and domesticated common bean accessions. Preliminary findings have identified three unique haplotypes for growth habit, indicating that selection for determinacy, in the Andean gene pool, may have occurred several times since domestication. Two determinacy haplotypes appear most closely related to wild samples from Peru while the other clustered with Argentinian accessions. Complications in the analysis arose from high levels of admixture amongst wild accessions and inconsistent sequencing coverage across accessions sampled. Future studies should include important samples in replicate and reduce pooling to achieve maximum coverage across all accessions.