Notas Científicas New microsatellite markers developed from an enriched microsatellite common bean library

The objective of this work was to develop new microsatellite markers in common bean. Ninety nine new microsatelitte loci were developed from a microsatellite enriched library for (CT)8 and (GT)8 motifs, from CAL-143 line. The majority of microsatellite sequences (51%) was related to cellular metabolism. The remaining sequences were associated to transcription functions. Only 17.2% of the sequences presented some level of similarity with other plant species genes.

Microsatellites have to be continuously isolated for new species, as cross-species amplification is not always possible.Once developed, microsatellites are ideal markers, as they are stable and easy to assay by polymerase chain reaction.Several important genes, such as resistance ones, may be linked to microsatellite motifs and, therefore, they are quite relevant for studies such as germplasm characterization, mapping and markerassisted selection.
When compared to commodity crops, the number of available microsatellites for common bean is insufficient, and success of mapping with limited number of markers is reduced (McClean et al., 2004).
Polymorphisms showed by microsatellite markers have been restricted to the narrow genetic based elite population.More microsatellites for common bean represent a step beyond the constraint of polymorphism and better tools to identify useful genetic variation (Acosta-Gallegos et al., 2007).Campos et al. (2007) identified and characterized twenty microsatellite loci in common bean, from an 'IAC-UNA' microsatellite enriched library.These new informative microsatellite loci can be used for selecting promising common bean segregant populations (Pereira et al., 2007).
Nevertheless, the number of microsatellites available for common bean is still insufficient, and requires the development of additional markers, so as to accelerate the analytical capacity of genetic studies in this crop.
The objective of this work was to develop new microsatellites from a 'CAL-143' enriched library.
A microsatellite enriched library (Billotte et al., 1999) was developed for common bean line CAL-143.This library was developed together with other 'IAC-UNA' libraries (Benchimol et al., 2007;Campos et al., 2007), as part of a broadened mapping project.CAL-143 is an Andean line with red stringed seeds (cream coat color) and calima type.It is susceptible to anthracnose (Colletotrichum lindemuthianum) and resistant to some races of angular leaf spot (Phaeoisariopsis griseola).
Genomic DNA was extracted from leaf tissue using CTAB method (Hoisington et al., 1994).The genomic libraries were screened by picking 2 µL of frozen white colonies and their amplification in a PCR reaction.Each amplification reaction contained 25 µL of 1x reaction buffer, 2 mM MgCl 2 , 0.5 µM of RsaI primer, 200 µM of total dNTP mixture, and 0.5U Taq DNA polymerase.Amplifications were performed in a PTC-100 MJ Research thermocycler, programmed with a hot start of 4 min at 95ºC; followed by 30 cycles of 30 s at 94ºC, 45 s at 52ºC, 1 min 30 s at 72ºC, followed by 8 min at 72ºC.PCR products were separated onto 3% agarose gels.Plasmid DNA was isolated according to Maniatis et al. (1982).
Sequencing of the inserts was performed using the ABI 377 Big Dye Terminator.Microsat software CIRAD (Risterrucci et al., 2005) was used for excising adaptation sequences and finding possible RsaI sites.Reads were processed by Phred version 0.000925.cbase calling program (Ewing et al., 1998) and vector sequences, poly-A tail, and adaptators were trimmed after cross-match analysis.Clustering was performed using CAP3 software, with default parameters (Huang & Madan, 1999).
BLASTN search utility program (available in NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify similarities between 'CAL-143' new sequences and 'IAC-UNA' sequences (Benchimol et al., 2007;Campos et al., 2007), in order to avoid redundancies among the microsatellites.BLASTX search utility program was used to identify similarities of 'CAL-143' sequences within known genes represented in the GenBank non-redundant database (Altschul et al., 1990).
Only sequences containing five or more repeated units were considered, regardless if the sequences were perfect or not.The free software SSRIT -simple sequence repeat identification tool (Temnykh et al., 2001), available at http://www.gramene.org/db/searches/ssrtool -was used to identify, count, and localize the microsatellite motifs inside the sequences.Complementary primers to the single sequences flanking the microsatellites were designed with Primer Select software from Lasergene program, with the following conditions: amplification DNA size from 150 to 350 pb; GC content between 40 and 60%; annealing temperature (Ta) between 45 and 60 o C; primer length between 18 and 22 pb; no hairpins or dimmers.
This analysis showed that 45 sequences, containing compound microsatellite motifs of 'CAL-143', were redundant to a unique 'IAC-UNA''s library contig.A total of 1,440 clones were identified and sequenced.Seventy percent (1,002) of these clones were putative positive (one unique insert cloned, having neither contamination of other clones, nor showing double bands).
Among all sequences, 256 microsatellites were found: 176 (68.8%) presented dinucleotide compound motifs, 74 (28.9%) presented dinucleotide simple repetitions and six (2.3%) presented trinucleotides.Therefore, dinucleotide compound (perfect and imperfect) motifs were the most frequent kind of microsatellite found in this Andean variety enriched library.The same was also reported for 'IAC-UNA' studies (Benchimol et al., 2007;Campos et al., 2007).The AC/TG motifs were the most frequent, followed by the GT/CA ones.This result is different from the one reported for 'IAC-UNA' (Mesoamerican cultivar), in which most of microsatellites found contained GT/CA motifs followed by GA/CT motifs (Benchimol et al., 2007).Gaitán-Solís et al. (2002) reported that GA repeat was the most common dinucleotide detected, accounting for 62.7% of the microsatellites found among three Andean accession libraries.Yu et al. (1999) have searched GenBank database for microsatellites sequences and found a higher frequency for AT dinucleotide repeats in P. vulgaris and Vigna sp., followed by GA repeats.
Only ninety-nine primer pairs were designed for the 256 microsatellites found (Table 1).It was not possible to develop primer pairs for all the microsatellites, because: it was impossible to draw primers with an amplification product over 350 pb, as there was no available ladder (the one of 10 pb has the largest fragment at 330 pb); there were complementarity and hairpin formation among some of the primers; and, in some cases, many were found at the initial or at the end of the sequenced sequences.These ninety-nine microsatellites are being used to accomplish the mapping of resistance loci associated to anthracnose and angular leaf spot, and for evaluating genetic diversity among 'Carioca' beans.
The results of BLASTX revealed that only 17.2% of the microsatellite sequences presented some level of similarity with some other plant gene, especially of Arabidopsis thaliana and Oryza sativa.From these, four sequences (SSR-IAC129, SSR-IAC179, SSR-IAC204 and SSR-IAC205) presented similarities with proteins with unknown function (E-value < 10 -12 ), and two sequences (SSR-IAC166 and contig10 -SSR-IAC209 and SSR-210) showed similarities with the retrotransposon gag protein family (E-value < 10 -31 ).
This protein is present in some virus capsids and in plants; it may be involved with pathogenic resistance to virus, bacteria and fungi (Grandbastien et al., 1997;Benko-Iseppon et al., 2003).These results suggest that the new microsatellites reported here could be used for synteny studies, establishing the conservation of genes between other species of plants.Around 51% of sequences were involved in the cellular metabolism, such as lipid and kinase metabolism and photosynthesis (E-value < 10 -49 ).
The remaining sequences (SSR-IAC174 and SSR-IAC175) were associated to transcription functions (E-value < 10 -12 ) related, in several aspects, to the developmental processes, which might be interesting for manipulating important agronomic traits.

Table 1 .
Primer sequences of microsatellite markers obtained from line CAL-143.