Luciana Lasry Benchimol

Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies in tropical maize inbred lines

In order to compare their relative efficiencies as markers and to find the most suitable marker for maize diversity studies we evaluated 18 inbred tropical maize lines using a number of different loci as markers. The loci used were: 774 amplified fragment length polymorphisms (AFLPs); 262 random amplified polymorphic DNAs (RAPDs); 185 restriction fragment length polymorphisms (RFLPs); and 68 simple sequence repeats (SSR). For estimating genetic distance the AFLP and RFLP markers gave the most correlated results, with a correlation coefficient of r = 0.87. Bootstrap analysis were used to evaluate the number of loci for the markers and the coefficients of variation (CV) revealed a skewed distribution. The dominant markers (AFLP and RAPD) had small CV values indicating a skewed distribution while the codominant markers gave high CV values. The use of maximum values of genetic distance CVs within each sample size was efficient in determining the number of loci needed to obtain a maximum CV of 10%. The number of RFLP and AFLP loci used was enough to give CV values of below 5%, while the SSRs and RAPD loci gave higher CV values. Except for the RAPD markers, all the markers correlated genetic distance with single cross performance and heterosis which showed that they could be useful in predicting single cross performance and heterosis in intrapopulation crosses for broad-based populations. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationships among tropical maize inbred lines with high accuracy.

Relationship of intra- and interpopulation tropical maize single cross hybrid performance and genetic distances computed from AFLP and SSR markers

AbstractTwo sets of tropical maize inbred lines, one derived from the BR-105 population and another derived from the BR-106 population, were assayed for Amplified Fragment Length Polymorphisms (AFLP) and for Simple Sequence Repeat (SSR), in order to investigate genetic distances among lines and their relationship to heterotic group assignment and single cross yield performance. Genetic distances were on average greater for interpopulation than intrapopulation crosses for both AFLP and SSR. Cluster analysis was in agreement with the original assignment for heterotic groups. Inbred line 16, derived from BR-106, was assigned to the BR-105 set, in agreement with single cross yield performance from intra- and interpopulation crosses. However, the same pattern was not observed for SSR where another two lines from BR-106 were also assigned to the BR-105 set. Correlation coefficients of genetic distances (GD) with F1 grain yield and heterosis were high for BR-106 ×BR-106 crosses (0.91** and 0.82** for AFLP and SSR, respectively), moderate for BR-105 × BR-105 crosses (0.52* for AFLP and SSR) and low for BR-105 × BR-106 crosses (0.29 and 0.16 for AFLP and SSR, respectively). The lower correlation at interpopulation level was due to the smaller range of GD caused probably by a previous selection for combining ability. General results showed that the AFLP molecular marker is efficient in assigning maize lines to heterotic groups and that AFLP-based GD is suitable for predicting the maize single cross performance for intrapopulation crosses of broad-based populations. The efficiency of SSR in assigning lines to heterotic groups and for predicting single cross performance was smaller than AFLP.

Development, characterization, and comparative analysis of polymorphism at common bean SSR loci isolated from genic and genomic sources

Microsatellites or SSRs (single sequence repeats) have been used to construct and integrate genetic maps in crop species, including Phaseolus vulgaris. In the present study, 3 cDNA libraries generated by the Bean EST project (http://lgm.esalq.usp.br/BEST/), comprising a unigene collection of 3126 sequences and a genomic microsatellite-enriched library, were analyzed for the presence of SSRs. A total of 219 expressed sequence tags (ESTs) were found to carry 240 SSRs (named EST-SSR), whereas 714 genomic sequences contained 471 SSRs (named genomic-SSR). A subset of 80 SSRs, 40 EST-SSRs, and 40 genomic-SSRs were evaluated for molecular polymorphism in 23 genotypes of cultivated beans from the Mesoamerican and Andean genetic pools, including Brazilian cultivars and 2 related species. Of the common bean genotypes, 31 EST-SSR loci were polymorphic, yielding 2-12 alleles as compared with 26 polymorphic genomic-SSRs, accounting for 2-7 alleles. Cluster analysis from data using both genic and genomic-SSR revealed a clear separation between Andean and Mesoamerican beans. The usefulness of these loci for distinguishing bean genotypes and genetic mapping is discussed.

Structure of genetic diversity among common bean ( Phaseolus vulgaris L.) varieties of Mesoamerican and Andean origins using new developed microsatellite markers

A common bean genomic library was constructed using the ‘IAC-UNA’ variety enriched for (CT) and (GT) for microsatellite motifs. From 1,209 sequenced clones, 714 showed microsatellites distributed over 471 simple and 243 compound motifs. GA/CT and GT/CA were the most frequent motifs found among these sequences. A total of 123 microsatellites has been characterized. Out of these, 87 were polymorphic (73.7%), 33 monomorphic (26.8%), and 3 (2.4%) did not amplify at all. In a sample of 20 common bean materials selected from the Agronomic Institute Germplasm Bank, the number of alleles per locus varied 2–9, with an average of 2.82. The polymorphic information content (PIC) of each marker varied from 0.05 to 0.83, with a 0.45 average value. Cluster and principal coordinate analysis of the microsatellite data were consistent with the original assignment of the germplasm accessions into the Andean and Mesoamerican gene pools of common bean. Low polymorphism levels detected could be associated with the domestication process. These microsatellites could be a valuable resource for the bean community because of their use as new markers for genetic studies.

Microsatellite-assisted backcross selection in maize

Abstract A microsatellite marker (SSR) was chosen to simulate a target allele and three criteria (02, 04 and 06 markers perchromosome) were tested to evaluate the most efficient parameters for performing marker-assisted backcross(MAB) selection. We used 53 polymorphic SSRs to genotype 186 BC 1 maize (Zea mays L.) plants produced bycrossing the inbred maize lines L-08-05 (donor parent) and L-14-4B (recurrent parent). The second backcross (BC 2 )generation was produced with 180 plants and screened with markers which were not recovered from the firstbackcross (BC 1 ) generation. A total of 480 plants were evaluated in the third backcross (BC 3 ) generation from which48 plants were selected for parental genotype recovery. Recurrent genotype recovery averages in three backcrossgenerations were compatible with those expected in BC 4 or BC 5 , indicating genetic gain due to the marker-assistedbackcrossing. The target marker (polymorphic microsatellite PHI037) was efficiently transferred. Six markers perchromosome showed a high level of precision for parental estimates at different levels of maize genome saturationand donor alleles were not present in the selected recovered pure lines. Phenotypically, the plants chosen based onthis criterion (06 markers per chromosome) were closer to the recurrent parent than any other selected by othercriteria (02 or 04 markers per chromosome). This approach allowed the understanding that six microsatellites perchromosome is a more efficient parameter than 02 and 04 markers per chromosome for deriving a marker-assistedbackcross (MAB) experiment in three backcross generations.

Mapping of angular leaf spot resistance QTL in common bean (Phaseolus vulgaris L.) under different environments

BackgroundCommon bean (Phaseolus vulgaris L.) is the most important grain legume for human diet worldwide and the angular leaf spot (ALS) is one of the most devastating diseases of this crop, leading to yield losses as high as 80%. In an attempt to breed resistant cultivars, it is important to first understand the inheritance mode of resistance and to develop tools that could be used in assisted breeding. Therefore, the aim of this study was to identify quantitative trait loci (QTL) controlling resistance to ALS under natural infection conditions in the field and under inoculated conditions in the greenhouse.ResultsQTL analyses were made using phenotypic data from 346 recombinant inbreed lines from the IAC-UNA x CAL 143 cross, gathered in three experiments, two of which were conducted in the field in different seasons and one in the greenhouse. Joint composite interval mapping analysis of QTL x environment interaction was performed. In all, seven QTLs were mapped on five linkage groups. Most of them, with the exception of two, were significant in all experiments. Among these, ALS10.1DG,UC presented major effects (R2 between 16% - 22%). This QTL was found linked to the GATS11b marker of linkage group B10, which was consistently amplified across a set of common bean lines and was associated with the resistance. Four new QTLs were identified. Between them the ALS5.2 showed an important effect (9.4%) under inoculated conditions in the greenhouse. ALS4.2 was another major QTL, under natural infection in the field, explaining 10.8% of the variability for resistance reaction. The other QTLs showed minor effects on resistance.ConclusionsThe results indicated a quantitative inheritance pattern of ALS resistance in the common bean line CAL 143. QTL x environment interactions were observed. Moreover, the major QTL identified on linkage group B10 could be important for bean breeding, as it was stable in all the environments. Thereby, the GATS11b marker is a potential tool for marker assisted selection for ALS resistance.

Genetic diversity of cultivated accessions and wild species of rubber tree using EST‑SSR markers

O objetivo deste trabalho foi avaliar a eficiencia de marcadores EST‑SSR na determinacao da diversidade genetica de genotipos de seringueira e verificar a transferibilidade destes marcadores para especies silvestres de Hevea . Foram utilizados 45 acessos de seringueira ( H. brasiliensis ) do Instituto Agronomico e seis especies silvestres. As informacoes fornecidas pela distância genetica de Roger modificada foram usadas para analisar os dados de EST‑SSR. O agrupamento UPGMA dividiu as amostras em dois grandes grupos com alta diferenciacao genetica, enquanto o programa Structure distribuiu os 51 clones em oito grupos. Foi possivel tracar um paralelo entre ambos os metodos de agrupamento. Os 30 EST‑SSRs polimorficos mostraram de dois a dez alelos e foram eficientes em amplificar as seis especies silvestres. Microssatelites funcionais EST‑SSR sao eficientes na avaliacao da diversidade genetica entre clones de seringueira e podem ser usados para traduzir diferencas geneticas entre cultivares e para gerar perfis geneticos de materiais proximos. Os acessos do Instituto Agronomico apresentam elevada diversidade genetica. Os marcadores EST‑SSR, desenvolvidos para Hevea brasilensis , apresentam transferabilidade e sao capazes de amplificar outras especies de Hevea .