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Poster7: Development and diversity of gene-based microsatellites for common bean (Phaseolus vulgaris L.)
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Poster7: Development and diversity of gene-based microsatellites for common bean (Phaseolus vulgaris L.)

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  • 1. Development and diversity of gene-based microsatellites for common bean (Phaseolus vulgaris L.). M. W. Blair, M. Muñoz, M. C. Giraldo, F. Pedraza, L. M. Díaz, C. Chavarro CIAT - International Center for Tropical Agriculture, A. A. 6713, Cali, Colombia, Introduction Figures and Tables Gene-based (genic) microsatellites are useful tools for plant genetics and simple sequence repeat loci can often be found in coding regions of the genome. While EST sequencing can be used to discover genic microsatellites, direct screening of cDNA libraries for A) BMc315 B) BMc241 repeat motifs can also provide a source of new loci. Andean The objective of this research was to screen a large cDNA library from an Andean common bean genotype for six di-nucleotide and tri-nucleotide repeat motifs 155 through a filter hybridization approach and to develop 112 145 Mesoamerican microsatellite markers from positive clones. Materials and Methods G4825 G3513 G11360 G11350 G21657 G21078 G21242 G14519 G4825 G19833 DOR364 BAT477 G3513 BAT881 G21212 G24404 G24390 DOR390 G19892 G19833 G11360 G11350 G21657 G21078 G21242 G14519 G19833 DOR364 BAT477 BAT881 G21212 G24404 RADICAL G24390 DOR390 G19892 G19833 RADICAL Library construction: A cDNA library was made from total RNA Figure 2. Neighbor joining dendogram of relationships between wild (G19892, prepared from common bean leaves of three week old plants of the G24390 and G24404) and cultivated (all other) accessions of common bean based Andean cultivar ‘G19833', grown at CIAT. The poly(A) RNA was purified C) BMc180 D) BMc316 on 248 cDNA derived SSR markers with Andean and Mesoamerican genepools and reverse transcribed. cDNA was synthesized and cloned into the indicated. Among the cultivated genotypes, 4 were Mesoamerican advanced breeding lines from CIAT (BAT477, BAT881, DOR364 and DOR390), one was a plasmid vector, pCMVSport6.0 (Invitrogen). The library was transformed locally bred Andean large red seeded variety (Radical Cerinza) and the remainder into E. coli EMDH12S cells which were plated on Q-plates. A Q-Bot were germplasm accessions (G lines) 158 robot and automatic blue/white screening was used to pick colonies and array onto double replicate 4 x 4 Hybond filters. 88 Table 1. SSR repeats identified in the sequenced cDNA clones. 82 Filter hybridization and library screening: Six simple sequence repeat SSR type1 5’ 3’ total Percentage Proportion motif oligonucleotide probes were used to screen the cDNA library filters 128 sequences sequences of total 5’ end (two for dinucleotides repeats, CA and GA; four for trinucleotide repeats, Di-nucleotides 181 28 209 42.1 86.6 AAT, CAG, CAA and ACG). Probes were end-labeled and hybridized to ac/gt/ca/tg 44 4 48 9.7 91.7 ag/ct/ga/tc 124 13 137 27.6 90.5 library fiilters with standard protocols (Edwards et al., 1996). Filters were at/ta 13 11 24 4.8 54.2 re-used for sequential screening of different oligonucleotide repeats. G4825 G3513 G4825 DOR364 G3513 DOR390 G11360 G11350 G21657 G21078 G21242 G14519 G19833 DOR364 BAT477 BAT881 G21212 G24404 RADICAL G24390 DOR390 G19892 G11360 G11350 G21657 G21078 G21242 G14519 G19833 BAT477 BAT881 G21212 G24404 RADICAL G24390 G19892 G19833 gc/cg 0 0 0 0.0 -- Tri-nucleotides 194 30 224 45.1 86.6 Clone identification and sequencing: Positive clones were identified by aag/aga/gaa/ttc/tct/ctt 24 1 25 5.0 96.0 which filter they were on; which field within the filter they were in, and aat/ata/taa/tta/tat/att 2 7 9 1.8 22.2 what address they had within the field. Only double-spotted clones were aac/aca/caa/ttg/tgt/gtt 34 2 36 7.2 94.4 acc/cac/cca/tgg/gtg/ggt 19 2 21 4.2 90.5 selected. DNA was extracted by standard alkaline lysis and used for Figure 1. Representative cDNA based microsatellite markers showing four agc/cag/gca/tcg/gtc/cgt 68 15 83 16.7 81.9 sequencing from the 5’ and 3’ ends using SP6 and T7 primers. patterns of diversity. A) BMc315, a monomorphic microsatellite. B) BMc241, a agg/gag/gga/tcc/ctc/cct 30 2 32 6.4 93.8 polymorphic microsatellite that detected different alleles in the wild accessions, atc/cat/tca/tag/gta/agt 4 1 5 1.0 80.0 Primer design: SSRs were found in the sequenced clones using the but not within the cultivated accessions. C) BMc180, a bi-allelic microsatellite that ccg/gcc/cgc/ggc/cgg/gcg 8 0 8 1.6 100.0 detected different alleles for Andean versus Mesoamerican accessions. D) SSR identification tool (SSRIT). Primer 3.0 was then used to design BMc316, a more polymorphic microsatellite that detected different alleles across gac/cga/acg/ctg/gct/tgc 5 0 5 1.0 100.0 Tetra-nucleotides 31 20 51 10.3 60.8 primer pairs which would produce PCR amplification fragments of aprox. most of the accessions, especially those of the Andean genepool. Penta-nucleotide 11 2 13 2.6 84.6 150 bp. The newly-designed microsatellites were tested against a Total 417 80 497 100.0 - standard survey of 18 genotypes from Blair et al. (2006) and run on silver stain gels. A matrix of allele values was used to construct a simple- 1Complementary sequences for a given motif are given and were the basis for grouping of matching neighbor joining dendogram which was subjected to 1000 Discussion di-nucleotide and tri-nucleotide motif SSR. bootstraps using program DARwin 5.0 (Perrier et al., 2003). The approach taken here for gene-based microsatellite development was based on the targeted Results identification of SSRs in high-density filter arrayed Conclusions Robotics were used for high-throughput colony picking clones from a large cDNA library. This was facilitated The present study provides a set of validated gene- and to create a high-density filter of 18,432 double by the robotic picking of a large number of clones and based markers for common bean that are derived from spotted cDNA clones which was followed by the automated library duplication and filter preparation G19833, an Andean landrace that is an important hybridization with repeat motif containing probes based features of the Q-bot. Meanwhile, the cDNA library source of disease and abiotic stress tolerance which on GA, CA, AAT, CAG, CAA and ACG repeats. A total we used was made to represent a wide range of has been used for physical map development and as a of 1203 positive clones were identified by their vegetative tissue from three week old, greenhouse- mapping parent. Gene-based markers appear to be addresses and sequenced from 5’ ends and if required grown plants including young and old leaves, stem very efficient at separating divergent wild and cultivated from 3’ ends to confirm repeat motif and length. Out of and vascular tissues, as well as vegetative meristems accessions as well as Andean and Mesoamerican 886 high quality sequences, 497 had complete and therefore as a multi-tissue library was appropriate genepools and therefore will be useful for diversity microsatellite loci that were not truncated by the to upscale with this technology. To picture the scale analyses and for comparative and transcript mapping in sequencing reaction. Different motifs were found in of the library, a total of nearly 24 MB of the common bean. different frequencies in the 5’ and 3’ ends of the cDNAs transcriptome was represented in the filter set based (Table 1). Interestingly, while the AG and AC motif on the number of clones screened and the average References microsatellites were much more likely to be in the 5’ cDNA insert size of 1.3 kb. The use of di-nucleotide 1. Blair et al. (20^6) Microsatellite marker diversity in common bean sequences, about half of the AT microsatellites were and tri-nucleotide SSR probes in the filter (Phaseolus vulgaris L.). Theor Appl Genet. 113: 100–109. found in the 3’ sequences. In a microsatellite hybridization and the sequencing confirmation of 2. Edwards et al (1994) Microsatellite libraries enriched for several development program, primers were designed for 248 microsatellite sequences in plants. Biotechniques 20: 759-760. clones allowed us to calculate the frequencies of 3. Perrier et al. (2003) Darwin software. In: Hamon, P., Seguin, M., Perrier, SSR loci which were tested on a panel of 18 common various types of motifs in the cDNA library. The X., Glaszmann, J. C. (Eds.), Genetic diversity of cultivated tropical plants. bean genotypes (Figure 1) finding higher average results showed that di-nucleotide and tri-nucleotide Enfield, Science Publishers. Montpellier. 43-76. polymorphism information content for di-nucleotide clones were about equal in the cDNA library although repeat markers than for tri-nucleotide repeat markers the frequency of the tri-nucleotide positive clones was Acknowledgements Clemson University Genome Institute for technical assistance and the but very accurate genetic differentiation of cultivars, somewhat higher than that of the di-nucleotide Generation Challenge Program and USAID for funding. This poster wild accessions and genepools (Figure 2). positive clones. was based on a publication in BMC Plant Biology (2009).