Eveline Teixeira Caixeta

Inheritance of angular leaf spot resistance in common bean line BAT 332 and identification of RAPD markers linked to the resistance gene

The existence of genetic variability for angular leaf spot (ALS) resistance in the common bean germplasm allows the development of breeding lines resistant to this disease. The BAT 332 line is an important resistance source to common bean ALS. In this work we determined the inheritance pattern and identified RAPD markers linked to a resistance gene present in BAT 332. Populations F1, F2,BCs and BCr derived from crosses between BAT 332 and cultivar Rudá were used. Rudá is a commercial cultivar with carioca type grains and susceptible to ALS. The resistance of BAT 332 to race 61.41 of the pathogen was confirmed. Segregation analysis of the plants indicated that a single dominant gene confers resistance. For identification of RAPD markers linked to the resistance gene, bulk segregant analysis (BSA) was used. Two RAPD markers,OPAA07950 and OPAO12950, linked in coupling phase at 5.10 and 5.83 cM of this gene, respectively, were identified. These molecular markers are important for common bean breeders and geneticists as source of genetic information and for marker assisted selection in breeding programs.

Allelic relationships for genes that confer resistance to angular leaf spot in common bean

Angular leaf spot is one of the major diseases of the common bean. The extensive genetic variability of this pathogen requires the constant development of new resistant cultivars. Different sources of resistance have been identified and characterized. For the State of Minas Gerais, Brazil, four main resistance sources were found: Mexico 54, AND 277, MAR 2 and Cornell 49-242. Independent characterization of these genotypes demonstrates that resistance in all four sources is dominant and monogenic. However, there are no studies on the relationship and independence of these genes. In the present work, allelism tests were carried out to understand the relationship among the resistance genes present in these four resistance sources. The data revealed a much higher complexity in the resistance inheritance of these genes than previously reported. It was demonstrated that Cornell 49-242 possesses a dominant gene (Phg-3); Mexico 54 possesses three genes, denominated Phg-2, Phg-5 and Phg-6. In MAR 2, two genes were found, one independent designated Phg-4 and the other, an allelic form of Phg-5, denominated of Phg-52. Allelic forms were also found in AND 277, Phg-22, Phg-32 and Phg-42. These results have special importance for breeding programs aiming to pyramid resistance genes.

Breeding potential and genetic diversity of "Híbrido do Timor" coffee evaluated by molecular markers

AFLP, RAPD and SSR molecular markers were used to study the genetic diversity and genetic structure of the Hibrido de Timor germplasm. The principal coordinate analysis, UPGMA cluster analysis based on genetic dissimilarity of Jaccard, Bayesian model-based cluster analysis, percentage of polymorphic loci, Shannons information index and Nei gene diversity were employed to assess the genetic diversity. The analyses demonstrated a high genetic diversity among Hibrido de Timor accessions. UPGMA and Bayesian cluster analyses grouped the accessions into three clusters. The genetic structure of Hibrido de Timor is reported. The management of Hibrido de Timor germplasm variability and its potential use in breeding programs are discussed.

Comparative study of different molecular markers for classifying and establishing genetic relationships in Coffea canephora

The genetic variability characterization of the accessions of the germplasm collection, using molecular markers, is being applied as a complementary strategy to the traditional approaches to redefine the plant genetic resources. In this study, we compared the informativeness and efficiency of the molecular markers RAPD, AFLP and SSR in the analysis of 94 accessions of Coffea canephora germplasm held by the breeding program of the Brazilian Agricultural Research Corporation (Embrapa), Rondônia State, Brazil. For this, we considered the marker’s discriminatory power and level of polymorphism detected and also the genetic relationships and clustering (dendrogram) analysis. The RAPD marker yielded low-quality data and problems in the discrimination of some accessions, being less recommended for genetic studies of C. canephora. The SSRs had a higher level of information content and yielded high-quality data, while AFLP was the most efficient marker system because of the simultaneous detection of abundant polymorphism markers per few reactions. Our results indicate that AFLP and SSR, allies to the intrinsic characteristics of each technique, are the most suitable molecular markers for genetic studies of C. canephora. However, the choice of AFLP or SSR in the species characterization should be made in agreement with some characteristics that are discussed in this work.

Molecular diversity in Coffea canephora germplasm conserved and cultivated in Brazil

This work aimed to characterize accessions that represent the C. canephora germplasm conserved and cultivated in Brazil. A total of 130 accessions from germplasm banks of IAC (Sao Paulo), UFV (Minas Gerais) and also collected in plantations of the State of Espirito Santo and Rondonia were evaluated with a set of 20 new microsatellite primers. Multivariate methods were used to estimate the relationship among the accessions. High level of polymorphism and two major diversity clusters were identified. First cluster was composed by the accessions conserved in the IAC and UFV collections and the second was formed by accessions collected in areas under cultivation. Accessions from Espirito Santo and Rondonia were clear separated, composing two subclusters. Despite the great polymorphism found in Brazilian plantations, the diversity may be increased, because a new threshold in the genetic gains is expected on breeding programs with the intensification of the use of conserved germplasm.

Mating System and Genetic Composition of the Macaw Palm (Acrocomia aculeata): Implications for Breeding and Genetic Conservation Programs

Acrocomia aculeata (Arecaceae), a palm endemic to South and Central America, is a potential oil crop. Knowledge of the mating system of this species is limited to its reproductive biology and to studies using molecular markers. The present study analyzed genetic diversity between its developmental stages and determined its prevailing mating system in order to support genetic conservation and breeding programs. We tested 9 microsatellite markers in 27 mother trees (adult plants) and 157 offspring (juvenile plants) from the southeastern region of Brazil. Heterozygosity levels differed between the 2 studied life stages, as indicated by the fixation index of adult and juvenile trees, suggesting that selection against homozygotes occurs during the plant life cycle. The mating system parameters analyzed indicate that A. aculeata is predominantly outcrossing (allogamous). However, its low levels of selfing suggest that there is individual variation with regard to self-incompatibility, which can be a survival strategy in isolated or fragmented habitats. Deviations in variance effective size were detected because of high mating rates among relatives and correlated matings. These findings indicate that the main source of inbreeding results from biparental inbreeding in the population and that the progenies are predominantly composed of full-sibs. The information provided by this study on the ecology and reproduction dynamics of A. aculeata should be useful to both breeding and genetic conservation programs, allowing the development of more precise mathematical models and the estimation of the appropriate number of mother trees for seed collection.

Inheritance study and linkage mapping of resistance loci to Hemileia vastatrix in Híbrido de Timor UFV 443-03

Coffee leaf rust (CLR) caused by Hemileia vastatrix Berk. et Br. is one of the major Coffea arabica diseases worldwide. CLR resistance has been attributed to at least nine dominant genes, as single or in combination. We present an inheritance study and mapping loci involved in the Híbrido de Timor (HDT) UFV 443-03 resistance to race I, race II, and pathotype 001 of H. vastatrix. Molecular markers were used to ascertain the phenotypic results and to map the putative resistance loci. For all tree isolates, the inheritance study indicated that the resistance of HDT UFV 443-03 is conditioned by two independent dominant loci or by three independent loci (two dominant and one recessive). Molecular marker analyses ascertained that the resistance of HDT UFV 443-03 to race II is conditioned by at least two independent dominant loci, while the resistance to race I and pathotype 001 is conditioned by at least four independent dominant loci. Gene pyramiding might result in a cultivar with durable resistance; however, it is difficult to distinguish between plants with one or more resistance genes due to epistatic effects. Molecular markers linked to these genes were indicated for marker-assisted selection, as it is an efficient breeding alternative for CLR resistance with no such epistatic effects.

Epigenetic regulation of a powdery mildew resistance gene in Medicago truncatula.

Dear Editor, Medicago truncatula is a model legume that is congeneric with alfalfa (Medicago sativa), a forage crop of global importance. Over the last decade, tremendous genetic and genomic tools have been developed for this model system, which has greatly facilitated the study of various aspects of legume genomics and biology. From an applied perspective, genomic information gained from M. truncatula is particularly useful for genetic improvement of cultivated alfalfa, a crop that is not amenable to genetic analysis because of its allogamous and autotetraploid nature (Yang et al., 2008). For instance, M. truncatula can be used to clone the orthologs of many economically important genes in alfalfa, because the two species share many common phenotypes. Owing to the high level of sequence homology and remarkably conserved gene function between the two species, the isolated genes from M. truncatula can be directly used for alfalfa improvement by transgenic approaches. We used M. truncatula as a model system to identify genes that confer resistance to powdery mildew caused by the biotrophic fungus Erysiphe pisi. For this purpose, the resistant genotype Jemalong A17 and the susceptible genotype F83005.5 were crossed to produce an F2 mapping population. The F2 plants were inoculated with an Erysiphe pisi race (we called race 1) originally obtained from naturally occurring infection on the leaves of F83005.5. Seven to 10 d after inoculation, the resistant plants grew normally and were symptomless, while the susceptible plants displayed typical powdery mildew symptoms presented by white patches of mycelium and conidia covering the leaf surface (Figure 1A). Phenotyping of 1107 F2 plants identified 822 resistant and 285 susceptible individuals. The segregation of resistance to susceptibility fits 3:1 ratio (χ2 = 0.31, df = 1, P = 0.58), indicating that a single dominant gene controls the powdery mildew resistance in Jemalong A17. This resistance gene was designated as MtREP1 (for resistance to Erysiphe pisi race 1). We used the susceptible F2 individuals for genetic mapping of the MtREP1 locus. Initial genotyping using simple sequence repeat (SSR) markers revealed that the MtREP1 locus is located between markers MtB330 and MtB38 on the M. truncatula chromosome 5 (Figure 1A), a position coincident with one of the two QTL loci that confer powdery mildew resistance against an E. pisi isolate from alfalfa (Ameline-Torregrosa et al., 2008). The availability of genomic sequence around this region allowed us to develop sequencebased CAPS (cleaved amplified polymorphic sequences) markers to finely map the locus. Through this process, we were able to delimit the MtREP1 locus between CAP52 and CAP23 that span ~194 kb, within which CAP8, CAP9, CAP20, and CAP22 were co-segregated with MtREP1 (Figure 1A). The 194-kb window contains more than 30 annotated genes (Medtr5g072060-Medtr5g072480), three of which encode a typical coiled-coil/nucleotide-binding site/leucinerich repeat (CC–NBS–LRR) plant R protein (Medtr5g072140, Medtr5g072250, and Medtr5g072340) (Young et al., 2011; version Mt3.5). These three CC–NBS–LRR genes were considered as candidates of MtREP1. We identified a single bacterial artificial chromosome (BAC) clone mth2-76g24 that contains all three candidate genes, from which genomic sequences for each candidate gene, including their native promoters and 3′ untranslated (UTR) regions, were obtained and cloned into the pCAMBIA2300 vector. Because M. truncatula is recalcitrant to efficient Agrobacterium tumefaciens-mediated transformation, we chose to use alfalfa as an alternative study system. For this purpose, we first screened the alfalfa cultivar Pioneer-53V08 to identify plants that were susceptible to the E. pisi race 1. The selected susceptible plants were then subjected to A. tumefaciens-mediated transformation using the genomic constructs described above. We obtained transgenic plants for each candidate gene, all of which were inoculated with the powdery mildew pathogen. Our results showed that independent transgenic plants containing Medtr5g072140 (n = 23), Medtr5g072250 (n = 11), or the empty vector pCAMBIA2300 (n = 19) were all susceptible. In contrast, independent transformants containing the Medtr5g072340 transgene (n = 15) were completely resistant to the pathogen (Figure 1B). Thus, we conclude that Medtr5g072340 is the MtREP1 gene. Alignment of genomic and cDNA sequences reveals that MtREP1 coding sequence is composed of a single exon that is predicted to encode a protein of 992 amino acids, consisting of an N-terminal CC domain, a centrally located NBS domain, and numerous degenerate LRRs C-terminal to the NBS domain. 3′-RACE (rapid amplification of cDNA ends) analysis identified 1486-bp transcribed sequence after the stop codon. Sequence alignment of the 1486-bp cDNA sequence with genomic DNA

Marker-assisted selection provides arabica coffee with genes from other Coffea species targeting on multiple resistance to rust and coffee berry disease

Selecting superior genotypes is facilitated by marker-assisted selection (MAS), which is particularly suitable for transferring disease resistance alleles because it nullifies environmental effects and allows selection of resistant individuals in the absence of the pathogen or race, enabling preventive breeding. Molecular markers linked to two major genes (SH3 and SH?), conferring resistance to coffee rust, and those linked to the Ck-1 gene, conferring resistance to coffee berry disease (CBD), have previously been identified. These markers were validated and used in a progeny of crosses between Indian selections with Coffea arabica cultivars. Eleven resistant individuals homozygous for SH3 were identified by MAS. Of these, seven carry SH? from Híbrido de Timor and the gene introduced from Coffea liberica (SH3). SH? was characterized as derived from Coffea canephora. Thus, it was possible to identify C. arabica genotypes carrying important genes for rust resistance introgressed from other coffee species. MAS also allowed identification of sources of CBD resistance for use in preventive breeding for resistance to this serious disease. Using two validated molecular markers, two coffee plants carrying Ck-1 were identified: the UFV 328-60 genotype (F2) was resistant and homozygous based on both molecular markers but exhibited no markers related to SH3 and SH?, and the UFV 317-12 genotype (F1) was resistant and homozygous but resistant and heterozygous based on CBD-Sat207 and CBD-Sat235, respectively. Along with possessing Ck-1, the latter carries SH?. Overall, plants carrying different genes for resistance to rust and CBD were identified. These plants are important sources for gene pyramiding in breeding programs aimed at multiple and durable resistance.

The effects of encoding data in diversity studies and the applicability of the weighting index approach for data analysis from different molecular markers

The use of molecular markers to study genetic diversity represents a breakthrough in this area, because of the increase in polymorphism levels and phenotypic neutrality. Codominant markers, such as microsatellites (SSR), are sensitive enough to distinguish the heterozygotes in genetic studies. Despite this advantage, there are some studies that ignore this feature and work with encoded data because of the simplicity of the evaluation, existence of polyploids and need for the combined analysis of different types of molecular markers. Thus, our study aims to investigate the consequences of these encodings on simulated and real data. In addition, we suggest an alternative analysis for genetic evaluations using different molecular markers. For the simulated data, we proposed the following two scenarios: the first uses SNP markers, and the second SSR markers. For real data, we used the SSR genotyping data from Coffea canephora accessions maintained in the Embrapa Germplasm Collection. The genetic diversity was studied using cluster analysis, the dissimilarity index, and the Bayesian approach implemented in the STRUCTURE software. For the simulated data, we observed a loss of genetic information to the encoded data in both scenarios. The same result was observed in the coffee studies. This loss of information was discussed in the context of a plant-breeding program, and the consequences were weighted to germplasm evaluations and the selection of parents for hybridization. In the studies that involved different types of markers, an alternative to the combined analysis is discussed, where the informativeness, coverage and quality of markers are weighted in the genetic diversity studies.