ABSTRACT: The biotrophic fungus Hemileia vastatrix causes coffee leaf rust (CLR), one of the most devastating diseases in Coffea arabica . Coffee, like other plants, has developed effective mechanisms to recognize and respond to infections caused by pathogens. Plant resistance gene analogs (RGAs) have been identified in certain plants as candidates for resistance ( R ) genes or membrane receptors that activate the R genes. The RGAs identified in different plants possess conserved domains that play specific roles in the fight against pathogens. Despite the importance of RGAs, in coffee plants these genes and other molecular mechanisms of disease resistance are still unknown. This study aimed to sequence and characterize candidate genes from coffee plants with the potential for involvement in resistance to H. vastatrix . Sequencing was performed based on a library of bacterial artificial chromosomes (BAC) of the coffee clone ‘Híbrido de Timor’ (HdT) CIFC 832/2 and screened using a functional marker. Two RGAs, HdT_LRR_RLK1 and HdT_LRR_RLK2, containing the motif of leucine-rich repeat-like kinase (LRR-RLK) were identified. Based on the presence or absence of the HdT_LRR_RLK2 RGA in a number of differential coffee clones containing different combinations of the rust resistance gene, these RGAs did not correspond to any resistance gene already characterized (SH1-9). These genes were also analyzed using qPCR and demonstrated a major expression peak at 24 h after inoculation in both the compatible and incompatible interactions between coffee and H. vastatrix . These results are valuable information for breeding programs aimed at developing CLR-resistant cultivars, in addition to enabling a better understanding of the interactions between coffee and H. vastatrix .

ABSTRACT Genomic selection (GS) emphasizes the simultaneous prediction of the genetic effects of thousands of scattered markers over the genome. Several statistical methodologies have been used in GS for the prediction of genetic merit. In general, such methodologies require certain assumptions about the data, such as the normality of the distribution of phenotypic values. To circumvent the non-normality of phenotypic values, the literature suggests the use of Bayesian Generalized Linear Regression (GBLASSO). Another alternative is the models based on machine learning, represented by methodologies such as Artificial Neural Networks (ANN), Decision Trees (DT) and related possible refinements such as Bagging, Random Forest and Boosting. This study aimed to use DT and its refinements for predicting resistance to orange rust in Arabica coffee. Additionally, DT and its refinements were used to identify the importance of markers related to the characteristic of interest. The results were compared with those from GBLASSO and ANN. Data on coffee rust resistance of 245 Arabica coffee plants genotyped for 137 markers were used. The DT refinements presented equal or inferior values of Apparent Error Rate compared to those obtained by DT, GBLASSO, and ANN. Moreover, DT refinements were able to identify important markers for the characteristic of interest. Out of 14 of the most important markers analyzed in each methodology, 9.3 markers on average were in regions of quantitative trait loci (QTLs) related to resistance to disease listed in the literature.

Abstract We evaluated the genetic variability of a collection of Jatropha curcas germplasm, represented by 93 accessions, using microsatellite markers. Among the 60 markers tested, five of them detected polymorphisms, with a total of 11 alleles and mean of 2.2 alleles per loci. These five markers enabled the quantification of genetic variability through estimates of expected (He=0.42) and observed (Ho=0.64) heterozygosity, Shannon-Weaver index (H'=0.62), coefficient of inbreeding (ƒ=-0.44) and the formation of 11 clusters. Simultaneously, 14 accessions randomly sampled among the 93 and represented by seven plants each, were analyzed with these same five markers to quantify the within and between variability. Most of the genetic variation (92.58%) was contained within the accessions. These analyses revealed, for the first time, expressive genetic variability to be explored in this collection. The accessions UFVJC 05, 07, 12, 18, and 53 presented expressive variability among them with potential for the constitution of a base population for the breeding program.

Abstract Coffee tree is a shrub with dimorphic branches. The orthotropic stem grows vertically with two near-horizontal plagiotropic branches, per node, in opposite phyllotaxy. The consecutive nodes of the orthotropic stem are organized in parastichies. We report here mutant plants of Coffea arabica with triple plagiotropic branch, resulted from the whorled phyllotaxy of the orthotropic stem. In mutant plants three plagiotropic branches in the same node are separated by angles of approximately 120o, while in normal plants two plagiotropic branches in the same node are separated by angles of approximately 180o. In mutant plants the angles of the parastichies increase approximately 50o clockwise between consecutive nodes, while in normal plants the angles increase approximately 87o. The plant architecture is changed. The triple plagiotropic branch is a putative mutant with potential to be explored in coffee breeding programs. The hypotheses for the origin of the mutation and the research perspectives are discussed.

Resumo: O objetivo deste trabalho foi avaliar o uso de redes neurais artificiais em comparação à modelagem por meio de modelos lineares generalizados na predição de resistência à ferrugem em café arábica (Coffea arabica). Foram utilizados 245 indivíduos provenientes de uma população F2, oriundos da autofecundação do híbrido F1 H511-1, resultante do cruzamento da cultivar suscetível Catuaí Amarelo IAC 64 (UFV 2148-57) e do genitor resistente Híbrido de Timor (UFV 443-03). Os 245 indivíduos foram genotipados com 137 marcadores. Realizaram-se análises com redes neurais artificiais e com modelos lineares generalizados sob o enfoque bayesiano. As redes neurais identificaram quatro marcadores importantes pertencentes a grupos de ligação que foram recentemente mapeados, enquanto o modelo generalizado bayesiano identificou somente dois marcadores pertencentes a esses grupos. Foram observadas taxas de erro de predição inferiores (1,60%) para predizer a resistência à ferrugem em café arábica, quando foram utilizadas as redes neurais artificiais em vez de modelos lineares generalizados sob o enfoque bayesiano (2,4%). Os resultados mostraram que as redes neurais artificiais são uma abordagem promissora para predizer a resistência à ferrugem em café arábica.

Abstract: The objective of this work was to evaluate the use of artificial neural networks in comparison with Bayesian generalized linear regression to predict leaf rust resistance in Arabica coffee (Coffea arabica). This study used 245 individuals of a F2 population derived from the self-fertilization of the F1 H511-1 hybrid, resulting from a crossing between the susceptible cultivar Catuaí Amarelo IAC 64 (UFV 2148-57) and the resistant parent Híbrido de Timor (UFV 443-03). The 245 individuals were genotyped with 137 markers. Artificial neural networks and Bayesian generalized linear regression analyses were performed. The artificial neural networks were able to identify four important markers belonging to linkage groups that have been recently mapped, while the Bayesian generalized model identified only two markers belonging to these groups. Lower prediction error rates (1.60%) were observed for predicting leaf rust resistance in Arabica coffee when artificial neural networks were used instead of Bayesian generalized linear regression (2.4%). The results showed that artificial neural networks are a promising approach for predicting leaf rust resistance in Arabica coffee.

The molecular characterization of ten genotypes of the Coffea arabica plants and of seven genotypes of C. canephora having interesting features for coffee breeding programs was carried to select the parents for breeding. A total of 40 SSR and 29 ISSR primers were used. The primers generated a total of 331 (307 polymorphic and 24 monomorphic) bands. Analysis of genetic diversity presented dissimilarity intervals ranging from 0.22 to 0.44 between the Conilon genotypes, from 0.02 to 0.28 between the Arabica genotypes, and from 0.49 to 0.60 between the genotypes of the two species in the joint analysis. Four groups were formed: I = genotypes of C. arabica, II = four progenies of C. canephora, Conilon group, and one non defined C. canephora (Conilon or Robusta), III = one progeny of un-defined C. canephora (Conilon or Robusta) and IV = one progeny of C. canephora of Robusta group. The grouping formed was consistent with the origins of each group. High stabilities of the bifurcations were found by bootstrap analysis. The use of molecular markers of the SSR and ISSR types in the diversity study was efficient in distinguishing genotypes between and within C. arabica and C. canephora.

Ao longo das três últimas décadas os estudos sobre os marcadores moleculares alcançaram avanços extraordinários, em especial nas técnicas de sequenciamento e bioinformática. A seleção assistida por marcadores tornou-se parte da rotina dos programas de melhoramento de importantes empresas de sementes, com o objetivo de acelerar e otimizar o processo de desenvolvimento de novas cultivares. Empresas privadas de sementes crescentemente adotam a seleção assistida por marcadores, especialmente para as espécies de grande importância para o mercado de sementes, como milho, soja e girassol. Nas instituições públicas brasileiras poucos programas de melhoramento a adotam eficientemente. As possíveis razões são: falta de "know-how", falta de laboratório adequado, poucos marcadores validados, alto custo e falta de urgência na obtenção de cultivares. Neste artigo são analisados o uso e as limitações da seleção assistida por marcadores nos programas de melhoramento de plantas dos institutos públicos brasileiros.

Over the past three decades, molecular marker studies reached extraordinary advances, especially for sequencing and bioinformatics techniques. Marker-assisted selection became part of the breeding program routines of important seed companies, in order to accelerate and optimize the cultivar developing processes. Private seed companies increasingly use marker-assisted selection, especially for the species of great importance to the seed market, e.g. corn, soybean, cotton, and sunflower. In the Brazilian public institutions few breeding programs use it efficiently. The possible reasons are: lack of know-how, lack of appropriate laboratories, few validated markers, high cost, and lack of urgency in obtaining cultivars. In this article we analyze the use and the constraints of marker-assisted selection in plant breeding programs of Brazilian public institutes.

Este trabalho objetivou caracterizar acessos de C. canephora oriundos de cultivos comerciais e bancos de germoplasma brasileiros. Um total de 130 acessos das coleções do IAC (São Paulo), UFV (Minas Gerais) e coletados em plantios comerciais no Espírito Santo e Rondônia foram genotipados com 20 novos microssatélites. Métodos multivariados foram utilizados para estimar a relação entre os acessos. Foi observado alto nível de polimorfismo e dois grupos foram identificados: o primeiro foi constituído pelos genótipos conservados nas coleções de germoplasma do IAC e UFV e o segundo foi composto pelos acessos coletados em plantios comerciais. Os acessos do Espírito Santo e de Rondônia formaram dois subgrupos distintos. Apesar do grande polimorfismo encontrado nas lavouras brasileiras de café canéfora, incrementar essa diversidade é necessário, pois um novo limiar de ganhos genéticos é esperado nos programas de melhoramento com a intensificação do uso do germoplasma conservado.

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 (São Paulo), UFV (Minas Gerais) and also collected in plantations of the State of Espírito Santo and Rondônia 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 Espírito Santo and Rondônia 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.

O objetivo deste trabalho foi identificar marcadores moleculares relacionados à resistência do cafeeiro (Coffea arabica) à ferrugem (Hemileia vastatrix). Foram identificadas sequências de DNA potencialmente envolvidas na resistência do cafeeiro a doenças, por meio de análise "in silico", a partir das informações geradas pelo Projeto Brasileiro do Genoma Café. A partir das sequências mineradas, foram desenhados 59 pares de iniciadores para amplificá-las. Os 59 iniciadores foram testados em 12 cafeeiros resistentes e 12 susceptíveis a H. vastatrix. Vinte e sete iniciadores resultaram em bandas únicas e bem definidas, enquanto um deles amplificou fragmento de DNA em todos os cafeeiros resistentes, mas não nos suscetíveis. Esse marcador molecular polimórfico amplificou uma região do DNA que corresponde a uma janela aberta de leitura parcial do genoma de C. arabica que codifica uma proteína de resistência a doenças. O marcador CARF 005 é capaz de diferenciar os cafeeiros analisados em resistentes e susceptíveis a H. vastatrix.

The objective of this work was to identify molecular markers related to the resistance of coffee (Coffea arabica) to rust (Hemileia vastatrix). DNA sequences potentially involved in coffee disease resistance were identified, using "in silico" analysis, from data obtained by the Brazilian coffee genome project. After data mining, 59 primer pairs were designed to amplify the sequences identified. The 59 primers were tested on 12 resistant and 12 susceptible coffee plants to H. vastatrix. Twenty-seven primers resulted in unique and well-defined bands, while one of these amplified a DNA fragment in all resistant plants, but not in the susceptible ones. This polymorphic molecular marker amplified a region of DNA that corresponds to a partial open reading frame of C. arabica genome that encodes a disease resistance protein. The marker CARF 005 can be used to differentiate between resistant and susceptible coffee plants to H. vastatrix.

Marcadores moleculares AFLP, RAPD e SSR foram usados para estudo da diversidade e estrutura genética do germoplasma de Híbrido de Timor. As análises de coordenadas principais, de agrupamento UPGMA baseado na dissimilaridade genética de Jaccard, de agrupamento baseado no modelo Bayesiano, a percentagem de locos polimórficos, o índice de Shannon e a diversidade genética de Nei foram utilizadas para acessar a diversidade genética. As análises demonstraram alta diversidade genética entre os acessos de Híbrido de Timor. A análise de agrupamento UPGMA e o modelo Bayesiano permitiram a alocação dos acessos em três grupos. A estrutura genética dos acessos de Híbrido de Timor foi descrita. A manutenção e manuseio da variabilidade do germoplasma de Híbrido de Timor e seu potencial uso nos programas de melhoramento de cafeeiro foram discutidos.

AFLP, RAPD and SSR molecular markers were used to study the genetic diversity and genetic structure of the Híbrido 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, Shannon's information index and Nei gene diversity were employed to assess the genetic diversity. The analyses demonstrated a high genetic diversity among Híbrido de Timor accessions. UPGMA and Bayesian cluster analyses grouped the accessions into three clusters. The genetic structure of Híbrido de Timor is reported. The management of Híbrido de Timor germplasm variability and its potential use in breeding programs are discussed.

Sequences potentially associated with coffee resistance to diseases were identified by in silico analyses using the database of the Brazilian Coffee Genome Project (BCGP). Keywords corresponding to plant resistance mechanisms to pathogens identified in the literature were used as baits for data mining. Expressed sequence tags (ESTs) related to each of these keywords were identified with tools available in the BCGP bioinformatics platform. A total of 11,300 ESTs were mined. These ESTs were clustered and formed 979 EST-contigs with similarities to chitinases, kinases, cytochrome P450 and nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, as well as with proteins related to disease resistance, pathogenesis, hypersensitivity response (HR) and plant defense responses to diseases. The 140 EST-contigs identified through the keyword NBS-LRR were classified according to function. This classification allowed association of the predicted products of EST-contigs with biological processes, including host defense and apoptosis, and with molecular functions such as nucleotide binding and signal transducer activity. Fisher's exact test was used to examine the significance of differences in contig expression between libraries representing the responses to biotic stress challenges and other libraries from the BCGP. This analysis revealed seven contigs highly similar to catalase, chitinase, protein with a BURP domain and unknown proteins. The involvement of these coffee proteins in plant responses to disease is discussed.

No estudo da diversidade genética entre e dentro de populações de café, foram usados marcadores EST-SSR, visando avaliar seu potencial para identificar cultivares comerciais e assistir programas de melhoramento. Os 17 marcadores utilizados foram desenvolvidos a partir das seqüências ESTs do Projeto Brasileiro do Genoma Café. Em todos os marcadores observou-se polimorfismo entre os genótipos avaliados, com um número médio de 5,1 alelos por primer. Os maiores polimorfismos foram constatados dentro de C. canephora (88,2%) e em variedades resistentes à ferrugem (35,3%). Dos marcadores analisados, 29,4% distinguiram C. arabica dos Híbridos de Timor (HDT), sendo possível identificar os mais próximos e os mais distantes de C. arabica . A análise dos genótipos agrupados por população revelou diversidade genética de 64% entre populações e 36% dentro delas, com um índice de diferenciação de 0,637. Em seis marcadores distinguiram-se quatro variedades resistentes à ferrugem, constando seu potencial para fingerprint. Esses resultados demonstram a utilidade dos EST-SSR para orientações dos cruzamentos e estudos de diversidade, introgressão e mapeamento genético.

EST-SSR markers were used to investigate the genetic diversity among and within coffee populations, to explore the possibility of their use for fingerprinting of cultivars and to assist breeding programs. Seventeen markers, developed from ESTs (Expressed Sequence Tags) from the Brazilian Coffee Genome Project, were used. All markers showed polymorphism among the genotypes assessed. The average number of allele per primer was 5.1. The highest polymorphisms were found within C. canephora (88.2%) and rust-resistant varieties (35.3%). About 29.4% of the markers differentiated C. arabica from Híbrido de Timor; it was also possible to identify those closest and farthest from C. arabica . The analysis of population-grouped genotypes revealed a 64.0% genetic diversity among and a 36.0% genetic diversity within populations. The differentiation index was 0.637. Six markers distinguished four rust-resistance varieties, showing their fingerprinting potential. These results demonstrate the usefulness of EST-SSR markers for cross orientation, in diversity and introgression studies, and in genetic mapping.

O objetivo deste trabalho foi caracterizar a herança da resistência do Híbrido de Timor UFV 443-03 à ferrugem-do-cafeeiro (Hemileia vastatrix). Para isso, a raça II e o patótipo 001 de ferrugem foram inoculados em 246 plantas da população F2, 115 plantas do retrocruzamento suscetível (RC S) e 87 plantas do retrocruzamento resistente (RC R), originadas do cruzamento entre o genótipo suscetível cv. Catuaí Amarelo IAC 64 e a fonte de resistência Híbrido de Timor UFV 443-03. Para ambos os inóculos, a cv. Catuaí Amarelo IAC 64 foi suscetível, enquanto o Híbrido de Timor UFV 443-03, a planta representante da geração F1 e as plantas do RC R foram resistentes. As plantas F2, quando inoculadas com a raça II, apresentaram dois padrões de segregação significativos: 15:1 e 61:3. A herança da resistência foi confirmada pela inoculação das plantas do RC S, que segregaram na proporção de 3:1, padrão esperado para herança condicionada por dois genes. A hipótese de segregação 7:1 para três genes foi rejeitada. Resultados semelhantes foram obtidos para o patótipo 001. Dois genes dominantes e independentes conferem a resistência genética do Híbrido de Timor UFV 443-03 à raça II e ao patótipo 001 de H. vastatrix.

The aim of this work was to characterize the resistance inheritance of the Timor Hybrid UFV 443-03 to coffee leaf rust (Hemileia vastatrix). For this, the race II and pathotype 001 of coffee leaf rust were inoculated in 246 F2 plants, 115 susceptible backcrossing (BCS) plants, and 87 resistant backcrossing (BC R) plants, derived from the crossing between the susceptible genotype 'Catuaí Amarelo' IAC 64 and the resistance source Timor Hybrid UFV 443-03. For both inoculums, the 'Catuaí Amarelo' IAC 64 was susceptible, while the Timor Hybrid, the plant representing F1 generation, and the BC R plants were resistant. The F2 plants inoculated with race II presented two significant segregation ratios: 15:1 and 61:3. The resistance inheritance was verified by the inoculation of the BCS plants, which segregated at a 3:1 ratio, an expected pattern for the inheritance controlled by two genes, whereas the segregation hypothesis of 7:1 for three genes was rejected. Two independent and dominant genes confer the genetic resistance of Timor Hybrid UFV 443-03 to race II and pathotype 001 of H. vastatrix.