Ney Sussumu Sakiyama

Genetic and Molecular Characterization of the I Locus of Phaseolus vulgaris

The I locus of the common bean, Phaseolus vulgaris, controls the development of four different phenotypes in response to inoculation with Bean common mosaic virus, Bean common mosaic necrosis virus, several other related potyviruses, and one comovirus. We have generated a high-resolution linkage map around this locus and have aligned it with a physical map constructed with BAC clones. These clones were obtained from a library of the cultivar “Sprite,” which carries the dominant allele at the I locus. We have identified a large cluster of TIR–NBS–LRR sequences associated within this locus, which extends over a distance >425 kb. Bean cultivars from the Andean or Mesoamerican gene pool that contain the dominant allele share the same haplotypes as revealed by gel blot hybridizations with a TIR probe. In contrast, beans with a recessive allele display simpler and variable haplotypes. A survey of wild accessions from Argentina to Mexico showed that this multigene family has expanded significantly during evolution and domestication. RNA gel blot analysis indicated that the TIR family of genes plays a role in the response to inoculations with BCMV or BCMNV.

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.

ASSESSMENT OF EST-SSR MARKERS FOR GENETIC ANALYSIS ON COFFEE ( 1 )

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 Hibrido 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.

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.

Eficiência de absorção e utilização de boro, zinco, cobre e manganês em mudas enxertadas de cafeeiro

Efficiency of absorption and utilization of boron, zinc, copper and manganese in grafted coffee seedlings Studying nutritional efficiency of grafted coffee plants is important for the selection of graft/ rootstock combinations, aiming to achieve better plant development and yield. The objective of this work was to evaluate the genetic differences for B, Zn, Cu and Mn absorption and utilization efficiencies of grafted coffee seedlings. The experiment was conducted with seedlings planted in 20 L pots with a substrate consisting of soil, sand, and manure in the proportion of 3:1:1, in which they kept for 18 months, until harvest. Four genotypes of Coffea arabica L. were used as grafts: cultivars Catuai Vermelho IAC 15 (‘Catuai 15) and Oeiras MG 6851 (‘Oeiras’), and hybrids H419-10-3-4-4 (‘H419’) and H514-5-5-3 (‘H514’). All genotypes were obtained from the EPAMIG/UFV breeding program. Five half-sibling progenies of clones of Coffea canephora Pierre ex Froenher cv. Conilon were used as rootstocks: ‘ES 21’, ‘ES 36’, ‘ES 26’, ‘ES 23’ and ‘ES

Porta-enxertos afetando o desenvolvimento de mudas de coffea arabica l

The development of Coffea arabica L. young plants, influenced by the rootstock were evaluated under field condition, on the Jatoba farm, which is in Paula Cândido city, Minas Gerais State. Four genotypes of C. arabica L. were used as grafts: the varieties Catuai Vermelho IAC 15 and Oeiras MG 6851 and the lines H 419-10-3-1-5, H 514-5-5-3. As rootstocks were utilized three genotypes of Coffea canephora Pierre et Froenher were used as rootstocks: Apoata LC 2258, Conillon Muriae-1 and Robustao Capixaba (EMCAPA 8141) and one genotype of C. arabica L.: Mundo Novo IAC 376-4, plus four non-grafted plants. The grafting combination H419/EMCAPA promoted increase in the: plant height, number of nodes on the main stem, number of plagiotropic branches on the main stem, stem diameter, number of nodes on the medium plagiotropic branch, and yield, when compared to the respective non-grafted plant. The combinations Catuai/Apoata, H514/Apoata, H514/Conilon and H514/EMCAPA promoted yield decrease when compared to the respective non-grafted plant. The grafting in coffee trees may influence the plants development, when non-grafted plants and graft/rootstock combinations are compared. The use of rootstocks Apoata LC 2258 and EMCAPA 8141 enhanced the yield of the H419-10-3-1-5 line, and EMCAPA 8141 improved the plant growth.

In silico identification of coffee genome expressed sequences potentially associated with resistance to diseases

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. Fishers 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.

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.

Study of Combining Ability and Heterosis in Coffee

Combining ability analysis identifies the parents able to transfer their desirable traits to their descendants. It suggests the best hybrid combination and supplies data on the type of gene action, which controls the different agronomic traits. Combining ability in its most general form refers to the behaviour of lines, or cultivars when crossed in direct or reciprocal combinations to obtain the hybrids. The General Combining Ability (GCA) of a line refers to the behaviour of the line in a series of crosses based on the mean value of the resulting F1 hybrids. The performance of a particular cross may deviate from the mean of the GCA, and this deviation is known as the Specific Combining Ability (SCA) (Allard, 1971).

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.