Incoronata Galasso

Genomics of Phaseolus Beans, a Major Source of Dietary Protein and Micronutrients in the Tropics

Common bean is grown and consumed principally in developing countries in Latin America, Africa, and Asia. It is largely a subsistence crop eaten by its producers and, hence, is underestimated in production and commerce statistics. Common bean is a major source of dietary protein, which complements carbohydrate-rich sources such as rice, maize, and cassava. It is also a rich source of minerals, such as iron and zinc, and certain vitamins. Several large germplasm collections have been established, which contain large amounts of genetic diversity, including the five domesticated Phaseolus species and wild species, as well as an incipient stock collection. The genealogy and genetic diversity of P. vulgaris are among the best known in crop species through the systematic use of molecular markers, from seed proteins and isozymes to simple sequence repeats, and DNA sequences. Common bean exhibits a high level of genetic diversity, compared with other selfing species. A hierarchical organization into gene pools and ecogeographic races has been established. There are over 15 mapping populations that have been established to study the inheritance of agronomic traits in different locations. Most linkage maps have been correlated with the core map established in the BAT93 x Jalo EEP558 cross, which includes several hundreds of markers, including Restriction Fragment Length Polymorphisms, Random Amplified Polymorphic DNA, Amplified Fragment Length Polymorphisms, Short Sequence Repeats, Sequence Tagged Sites, and Target Region Amplification Polymorphisms. Over 30 individual genes for disease resistance and some 30 Quantitative Trait Loci for a broad range of agronomic traits have been tagged. Eleven BAC libraries have been developed in genotypes that represent key steps in the evolution before and after domestication of common bean, a unique resource among crops. Fluorescence in situ hybridization provides the first links between chromosomal and genetic maps. A gene index based on some P. vulgaris 21,000 expressed sequence tags (ESTs) has been developed. ESTs were developed from different genotypes, organs, and physiological conditions. They resolve currently in some 6,500–6,800 singletons and 2,900 contigs. An additional 20,000 embryonic P. coccineus ESTs provides an additional resource. Some 1,500 M2 Targeting Local Lesions In Genomes populations exist currently. Finally, transformation methods by biolistics and Agrobacterium have been developed, which can be applied for genetic engineering. Root transformation via A. rhizogenes is also possible. Thus, the Phaseomics community has laid a solid foundation towards its ultimate goal, namely the sequencing of the Phaseolus genome. These genomic resources are a much-needed source of additional markers of known map location for marker-assisted selection and the accelerated improvement of common bean cultivars.

The molecular cytogenetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18s-5.8s-25s rRNA genes, 5s rRNA genes, telomere-like sequences, and a family of centromeric repetitive DNA sequences

A knowledge of genome organization is important for understanding how genomes function and evolve, and provide information likely to be useful in plant breeding programmes involving hybridization and genetic manipulation. Molecular techniques, including in situ hybridization, molecular cloning and DNA sequencing, are proving valuable tools to investigate the structure, organization, and diversity of chromosomes in agricultural crops. Heterologous labelled 18 s-5.8 s-25 s (pTa71) and 5 s rDNAs (pTa794) were used for in situ hybridization on Vigna unguiculata (L.) Walp. chromosomes. Hybridization with 18 s-5.8 s-25 s rRNA gene probes occurred at the same chromosomal sites which were positive to the CMA fluorochrome. Silver staining of nucleolar-organizing regions indicated that all the rDNA sites detected using the 18 s-5.8 s-25 s rRNA gene probe possessed active genes. Degenerate telomeric repeats gave hybridization signals at the telomeres of most chromosomes and no intercalary sites were detected at metaphase; the sequences appear to have no preferential distribution in interphase nuclei. A repetitive DraI family from V. unguiculata was cloned (pVuKB1) and characterized. The DraI repeat is 488 nucleotides long, AT rich (74%), and hybridized on all chromosomes in the centromeric areas. The presence of this sequence family was investigated by Southern hybridization in different Vigna species and other Leguminoseae. It was only detected in V. unguiculata, and hence represents a species-specific DNA sequence.

Lectin-related resistance factors against bruchids evolved through a number of duplication events

Abstract. Abundant lectin-related proteins found in common beans (Phaseolus vulgaris L.) have been shown to confer resistance against the larvae of a number of bruchid species. Genes encoding for these proteins are members of the lectin multigene family, the most representative components being arcelins, phytohemagglutinins and α-amylase inhibitors. Arcelins have been described in seven variants, some of which are resistance factors against the Mexican bean weevil (Zabrotes subfasciatus), a major bean predator. In this study the isolation and sequencing of arcelin genes from wild P. vulgaris genotypes, containing Arc3 and Arc7 variants, is reported, and similarities and evolutionary relationships among the seven known arcelins are described. The evolutionary analysis shows that arcelins 3 and 4 cluster together and are the most-ancient variants. A duplication event gave rise to two additional clusters, one comprising arcelins 1, 2 and 6 and separated from the cluster of arcelins 5 and 7. A multiple number of arcelin genes were found in arcelin 3 and 4 genotypes indicating that more than one type of arcelin gene may be present in the same locus. Some of these sequences are reminiscent of ancient duplication events in arcelin evolution demonstrating that arcelins have evolved through multiple duplications. A further aim of this paper was to better understand and describe the evolution of the entire lectin multigene family. Beside arcelins, a number of other types of sequences, such as putative lectins and sequences not easily classifiable, were found in genotypes containing Arc3 and Arc4. These results, together with the evolutionary analysis, indicate that lectin loci are quite complex and confirm their origin by multiple duplication events.

Nuclear DNA content, chromatin organization and chromosome banding in brown and yellow seeds of Dasypyrum villosum (L.) P. Candargy

Banding patterns of metaphase chromosomes and nuclear DNA content in root meristematic cells of yellow and brown seeds of Dasypyrum villosum were determined. Microdensitometric evaluation of nuclear absorptions at different thresholds of optical density after Feulgen reaction indicated the organization of the chromatin in interphase nuclei, and allowed an evaluation of the amount of heterochromatin. These results were compared with those obtained after the application of banding techniques.

Characterisation of structural genes involved in phytic acid biosynthesis in common bean (Phaseolus vulgaris L.).

Phytate (myo-inositol hexakisphosphate), the major form of phosphorous storage in plant seeds, is an inositol phosphate compound poorly digested by humans and monogastric animals. A major goal for grain crop improvement is the reduction of its content in the seed to improve micronutrient bioavailability and phosphorus utilisation by humans and non-ruminant animals, respectively. We are interested in lowering phytic acid in common bean seed and to this goal we have undertaken a two-strategy approach: the isolation of mutants from an EMS mutagenised population (Campion et al. 2009) and the identification of genes coding for candidate enzymes involved in inositol phosphate metabolism for future targeted mutant isolation and/or study. In this paper we report data referred to the second approach and concerning the isolation and genomic organisation of Phaseolus vulgaris genes coding for myo-inositol 1-phosphate synthase (PvMIPSs and PvMIPSv), inositol monophosphatase (PvIMP), myo-inositol kinase (PvMIK), inositol 1,4,5-tris-phosphate kinase (PvIPK2), inositol 1,3,4-triphosphate 5/6-kinase (PvITPKα and PvITPKβ) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1). All these genes have been mapped on the common bean reference genetic map of McClean (NDSU) 2007 using a virtual mapping strategy. Bean markers, presumably associated to each gene of the phytic acid pathway, have also been identified. In addition, we provide a picture of the expression, during seed development, of the genes involved in phytic acid synthesis, including those such as MIK, IMP and IPK2, for which this information was lacking.

Identification of Lens culinaris ssp. culinaris chromosomes by physical mapping of repetitive DNA sequences.

We describe the characterisation and the chromosomal localisation of two repeated DNA sequences, named pLc30 (466 bp long, 64% AT residues) and pLc7 (408 bp long, 61% AT residues), isolated from lentil (Lens culinaris ssp. culinaris) genomic DNA. The pLc30 family is characterised by four internal repeats organised in a head-to-tail orientation, whereas the pLc7 contains many short direct subrepeats. The two families do not share significant sequence similarity. The distribution of these repetitive sequences in different Lens species and in other legumes was investigated. pLc30 is present in all Lens species investigated but absent from other genera examined. In contrast, pLc7 is present also in the genome of other legumes. As determined by FISH, the pLc30 sequence hybridises on six out of seven lentil chromosome pairs, while pLc7 hybridises on one only. The distribution of the nine different hybridisation sites of pLc30 allows the discrimination of all seven chromosome pairs and the construction of a karyotype of L. culinaris ssp. culinaris. Additionally, the combination of simultaneous and successive FISH with pLc7, 5S rRNA, 18S-5.8S-25S rRNA genes, and a telomeric sequence allowed the assembly of a physical map based on lentil karyotype.

Evaluation of Camelina sativa (L.) Crantz meal as an alternative protein source in ruminant rations

BACKGROUND Camelina sativa (CS) is an oilseed crop used for biofuel production. By-products from oil extraction are high in protein and can be used in ruminant rations; more information about their nutritive value is required also considering the antinutrional factor content of the by-products. The aim of this study was to evaluate the nutritive value of CS meal genotypes in comparison with canola. RESULTS Ten CS genotypes and one canola cultivar were evaluated. Meals were obtained from seeds after solvent oil extraction. CS average crude protein (CP) content (g kg⁻¹ dry matter) was 457. Numerical differences in lysine and sulfur amino acid content were observed among CS genotypes. Glucosinolate (mmol kg⁻¹) content was higher for CS (23.1) than canola (7.2). Sinapine content (g kg⁻¹) was lower for CS (2.79) than for canola (4.32). Differences were observed among CS genotypes for rumen undegraded protein (RUP). Average RUP (g kg⁻¹ CP) was 316 for CS and 275 for canola. CONCLUSIONS CS meal has potential for use in ruminant rations as a high-quality protein source. In vivo studies are needed to compare CS with other protein sources used in cattle rations. Implementation of breeding programs for improved meal quality is recommend.

h-TBP: an approach based on intron-length polymorphism for the rapid isolation and characterization of the multiple members of the β-tubulin gene family in Camelina sativa (L.) Crantz

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.

Lectin Gene Sequences and Species Relationships among Cultivated Legumes

Lectins are a class of defence proteins of non-immune origin that bind carbohydrate in a reversible fashion. In some cultivated legume species, lectin protein coding genes were PCR amplified using primers designed on the basis of conserved N- and C-terminal amino acid sequences of the common bean (one-chain) or pea (two-chains) lectins. Amplification products of the expected length were obtained in Lathyrus sativus L., Vicia faba L. var. faba, Phaseolus coccineus L., and Vigna unguiculata (L.) Walp. No amplification product or agglutinating activity against blood cells, and/or cross-reaction with specific antibodies were detected in Lupinus albus L. and Cicer arietinum L. Finally, the new isolated nucleotide sequences, together with other legume lectin sequences already present in the EMBL Database, were used for evolutionary analysis. This last indicated the existence of two main clusters; one grouping all the species belonging to the Phaseoleae tribe and the other one grouping Lens culinaris Medik., Pisum sativum L., L. sativus, and V. faba, members of the Vicieae tribe. Results were congruent with the taxonomic classification and suggested that the lectin genes divergence in legume followed species evolution.

Cytotaxonomic studies in Vigna. III. Chromosomal distribution and reacting properties of the heterochromatin in five wild species of the section Vigna

SUMMARYThe metaphase chromosomes of five wild species of the section Vigna of the genus Vigna, namely V. ambacensis, V. heterophylla, V. racemosa, V. marina, and V. gracilis were analysed in order to define the reacting properties to differential staining methods and chromosomal distribution of the different fractions of their heterochromatin, using C-banding and fluorochrome staining as discriminating techniques. Based on these characters it was possible to cluster the above species into three homogeneous groups: V. ambacensis and V. heterophylla belong to the first cluster, V. racemosa is the only member of the second group, and V. marina and V. gracilis form the third set. Species clustered into the first group show heterochromatin chromosomal distribution and reacting properties more similar to those of the cultivated species V. unguiculata than the other two sets. These findings are compared to those of earlier studies on related species.