Single strand conformation polymorphism based SNP and Indel markers for genetic mapping and synteny analysis of common bean (Phaseolus vulgaris L.)

Abstract

Background: Expressed sequence tags (ESTs) are an important source of gene-based markers such as those based on insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). Several gel based methods have been reported for the detection of sequence variants, however they have not been widely exploited in common bean, an important legume crop of the developing world. The objectives of this project were to develop and map EST based markers using analysis of single strand conformation polymorphisms (SSCPs), to create a transcript map for common bean and to compare synteny of the common bean map with sequenced chromosomes of other legumes.

Results: A set of 418 EST based amplicons were evaluated for parental polymorphisms using the SSCP technique and 26% of these presented a clear conformational or size polymorphism between Andean and Mesoamerican genotypes. The amplicon based markers were then used for genetic mapping with segregation analysis performed in the DOR364 x G19833 recombinant inbred line (RIL) population. A total of 118 new marker loci were placed into an integrated molecular map for common bean consisting of 288 markers. Of these, 218 were used for synteny analysis and 186 presented homology with segments of the soybean genome with an e-value lower than 7 x 10-12. The synteny analysis with soybean showed a mosaic pattern of syntenic blocks with most segments of any one common bean linkage group associated with two soybean chromosomes. The analysis with Medicago truncatula and Lotus japonicus presented fewer syntenic regions consistent with the more distant phylogenetic relationship between the galegoid and phaseoloid legumes.

Conclusion: The SSCP technique is a useful and inexpensive alternative to other SNP or Indel detection techniques for saturating the common bean genetic map with functional markers that may be useful in marker assisted selection. In addition, the genetic markers based on ESTs allowed the construction of a transcript map and given their high conservation between species allowed synteny comparisons to be made to sequenced genomes. This synteny analysis may support positional cloning of target genes in common bean through the use of genomic information from these other legumes.

Figure 1

Gel images for SNP and Indel based markers. Evaluations of the PCR products representing SNP markers developed in this study: a) gene based amplicons on agarose showing length polymorphisms (g755 and g762) or monomorphisms (g774 and g776) when comparing genotypes DOR364, G19833, BAT93 and JaloEEP558 (lanes 1 to 4, respectively). b) EST based amplicons for BNSP68, BNSP69 and BNSP70 on an agarose gel with the same four genotypes (lanes 1 to 4). c) gene based amplicons evaluated in a silver-stained SSCP gel showing three examples of polymorphisms with the same genotypes (lanes 1 to 4) for groups A, B and C, corresponding to molecular weights of 50-200 bp, 200-600 and 600 -1000 bp, respectively.

Figure 2

Genetic mapping of SSCP based markers. Segregation pattern of two BSNP markers and one "g" marker in the DOR364 × G19833 recombinant inbred line mapping population based on SSCP polymorphism and detection on silver stained gels.

Figure 3

Linkage analysis of SNP and Indel markers in the common bean genome. Common bean linkage map for the DOR364 × G19833 recombinant inbred line mapping population. Chromosome designations are indicated above the linkage groups (b01 to b11). The 118 newly mapped markers are shown in bold. The markers mapped by Galeano et al. [14] have asterisks while other genetic marker loci positions are as reported in Blair et al. [22,23].

Figure 4

Synteny relationships between common bean and soybean. Associations between common bean and soybean linkage groups through sequence based markers. The colored boxes represent the homologies with chromosome segments from the soybean genome with each chromosome from soybean assigned a given color. The boxes to the right side of the linkage group are the first similarity matches, while to the left side are the second similarity matches.

Figure 5

Synteny relationships between common bean and Lotus japonicus and Medicago truncatula. Associations between common bean, Lotus and Medicago linkage groups through sequence based markers. The number in the right and left hand boxes indicate the chromosomes numbers of the Medicago and Lotus hits, respectively. Each chromosome from each legume was assigned a different color with colors chosen to cover a large spectrum for ease of visualization.