Evaluating <Emphasis Type="Italic">Theobroma grandiflorum</Emphasis> for comparative genomic studies with <Emphasis Type="Italic">Theobroma cacao</Emphasis>

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao’s two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches’ broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao’s most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences.     

Micropropagation of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. using somatic embryo-derived plants

Summary A micropropagation protocol was developed using cacao somatic embryo-derived plant as a source for nodal and apical stem explants, and apical microcuttings. Microcuttings were efficiently rooted and developed into plantlets. Axillary meristems within the remaining decapitated plantlets subsequently developed and were used for production of additional microcuttings, with an average 2.4 growing shoots per decapitated stem. The remaining plantelts were maintained as microcutting stock plants. When nodal stem explants were cultured on thidiazuron medium, axillary buds proliferated and developed into shoots, which were excised and rooted. However, the efficiency of this method is lower than rooting of apical microcuttings harvested directly from stock plants. During root induction, short treatment with indole-3-butyric acid (IBA) increased the total percentage of rooted microcuttings up to 89%. Longer exposures to IBA increased the average number of roots per microcutting (from 1.7 to 5.2). Plant acclimatization after rooting was achieved with an average success of 87%. During several months of growth in the greenhouse, the micropropagated plants developed functional taproots. Currently, cocoa plants produced by this micropropagation method have been successfully acclimated to field conditions in Ivory Coast, Ghana, and Saint Lucia.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Genome-wide association mapping of sexual incompatibility genes in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

Sexual compatibility limits the production of cacao plantations, being an important selection criterion in breeding programs. However, the current method for characterizing compatibility, based on the frequency of flower setting after controlled pollination, is time consuming, requiring a long time to identify self-compatible individuals. The identification of molecular markers in genomic regions can be an alternative to allow early selection of self-compatible plants. The present study aimed to identify SNP markers associated with sexual compatibility in cacao, by utilizing genome-wide association (GWAS) mapping. A population of 295 individuals mostly from third-generation breeding populations, but also founder clones, was used. This population was phenotypically characterized by hand pollinating 8199 flowers and evaluating the flower retention 15 days after pollination. In addition, leaf samples of each individual were collected and DNA extracted for genotyping by sequencing, generating 5301 SNP markers after cleaning. Genome-wide association mapping analysis was performed using Synbreed, GCTA, and TASSEL softwares. Significant markers associated to incompatibility, likely in strong linkage disequilibrium, were found within a region of 196 kb, in the proximal end of chromosome 4, suggesting the existence of a major gene in that region. However, this result should be validated in a larger population, considering that only 295 trees were used here. When the SNP effects were treated as random in the estimation process, many other regions in the genome appears to be involved with sexual incompatibility in cacao. Candidate genes were found not only in the proximal end of chromosome 4 but also spread in several other regions of the genome.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Development and characterization of novel tetra-, tri- and di-nucleotide microsatellite markers in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">polyfermenticus</Emphasis> isolated from <Emphasis Type="Italic">Meju,</Emphasis> Korean soybean fermentation starter

Bacteria of the Bacillus species have been reported as an important microorganism in fermented soybean products. In the present study, thirty Bacillus isolates were screened from Meju, a Korean soybean fermentation starter. The comparative analysis of 16S rDNA sequences, 16S-23S internal transcribed spacer sequences, phenotypic, and biochemical characterizations revealed three phylogenetically distinct groups namely Bacillus atrophaeus, Bacillus polyfermenticus and Bacillus subtilis. The isolates were assayed for poly-γ-glutamate production and fibrinolytic activity. Among the isolates, B. polyfermenticus exhibited maximum poly-γ-glutamate production and fibrinolytic activity. Moreover, the soybean products fermented by B. polyfermenticus have increased the time taken for coagulation and hemorrhage in mice. The results of the present study clearly indicate the functional role of B. polyfermenticus in fermented soybean products.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Photosynthesis,chloroplast ultrastructure,chemical composition and oxidative stress in <Emphasis Type="Italic">Theobroma cacao</Emphasis> hybrids with the lethal gene <Emphasis Type="Italic">Luteus-Pa</Emphasis> mutant

The Theobroma lethal character Luteus-Pa segregates in a 3:1 ratio, expresses in recessive homozygosis, initially inducing leaf chlorosis and finally provoking seedlings death. The objective of this work was to evaluate gas exchange, chlorophyll fluorescence emission, chemical composition and oxidative stress of wild and mutant seedlings resulting from the crosses Pa 30 × Pa 169 and its reciprocal, aiming to elucidate the seedlings death induced by Luteus-Pa. At 15 day after emergence (DAE) differences began to appear between the wild type and mutant. Mutant seedlings showed: (1) lack of photosynthesis and alterations in chloroplast morphology; (2) lower level of three abundant groups of proteins in leaves; (3) decrease in the content of chloroplastidic pigments (4) decrease in peroxidases activities and increase in leaf polyphenol oxidase activity; (5) decrease in carbohydrate and concentration of some nutrients and low dry mass in all plant parts. In leaves of mutant seedlings of both crosses damages occurred in the system responsible for the photochemical phase of photosynthesis. Variations in growth parameters and subsequent seedling death up to 60 DAE were related to exhaustion of cotyledonary reserves, inactive photosynthetic apparatus and oxidative stress.     

Selection of international molecular standards for DNA fingerprinting of <Emphasis Type="Italic">Theobroma cacao</Emphasis>

A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.