Advances in BAC-Based Physical Mapping and Map Integration Strategies in Plants

In the advent of next-generation sequencing (NGS) platforms, map-based sequencing strategy has been recently suppressed being too expensive and laborious. The detailed studies on NGS drafts alone indicated these assemblies remain far from gold standard reference quality, especially when applied on complex genomes. In this context the conventional BAC-based physical mapping has been identified as an important intermediate layer in current hybrid sequencing strategy. BAC-based physical map construction and its integration with high-density genetic maps have benefited from NGS and high-throughput array platforms. This paper addresses the current advancements of BAC-based physical mapping and high-throughput map integration strategies to obtain densely anchored well-ordered physical maps. The resulted maps are of immediate utility while providing a template to harness the maximum benefits of the current NGS platforms

Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum

BACKGROUND: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8–10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome 1 orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40–50 million years ago. RESULTS: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome 1 in the wild species and provided evidence for at least one rearrangement on chromosome 1 of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa. CONCLUSION: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies

Homólogos de genes de resistencia en fríjol (Phaseolus vulgaris) y su aplicación en resistencia a colletotrichum lindemuthianum

ilustraciones, fotografías, gráficas, tablasEl cultivo de fríjol ( Phaseolus vulgaris) es afectado por diversos patógenos que limitan su producción, por esta razón la identificación de genes de resistencia funcionales es uno de los principales retos en investigación. Este proyecto tuvo por objetivo identificar homólogos de genes de resistencia (RGH) asociados con resistencia a Colletotrichum lindemuthianum agente causal de la antracnosis. Se evaluaron 544 combinaciones de primers degenerados, diseñados a partir de secuencias conservadas en el dominio NBS de genes de resistencia y genes RGH de la especie modelo para leguminosas Medicago truncatula. La amplificación, se realizó en ADN total del genotipo Andino G19833, originario de Perú y resistente a antracnosis y mancha angular. Cuatrocientas tres secuencias presentaron los motivos característicos de genes de resistencia tipo NBS, de las cuales 306 presentaron un ORF no interrumpido, por lo que se consideraron como genes RGH. A partir de estas secuencias se realizó el análisis filogenético, teniendo en cuenta un 90% de identidad nucleótidica, esto permitió confirmar la clasificación de los RGHs de fríjol, dentro de las familias TIR y no-TIR con valores de bootstrap altos. Las secuencias TIR se agruparon en 3 cluster discriminados en 14 clados y las secuencias no-TIR se agruparon en 3 cluster y 7 clados. Aunque la mayoría de comparaciónes pareadas mostró selección purificante aunque algunas secuencias pueden ser el resultado de selección diversificante, lo que muestra la diversidad de RGH en fríjol. Los RGHs específicos para frijol, se evaluaron en la librería BAC de G19833. En las secuencias terminales de los clones BAC (BES) positivos se identificaron 629 marcadores SSR y estos se evaluaron en los parentales G2333 (resistente) y G19839 (susceptible). 205 SSR polimórficos fueron evaluados en la población RIL derivada de la cruza entre G2333 x G19839. La evaluación fenotípica para la identificación de QTL se realizó en condiciones de campo e invernadero, donde se evaluaron en total 11 razas. La evaluación genotípica y fenotípica permitió identificar QTL asociados con resistencia a antracnosis, los cuales explicaron varianzas fenotípicas entre 9 y 89%. Estos marcadores constituyen una fuente importante de información, para el desarrollo de marcadores funcionales que puedan ser utilizados en selección asistida o para procesos de clonación posicional de genes de interés. (Texto tomado de la fuente).Common bean ( Phaseolus vulgaris) crop is constrained by different pathogens which affect its production. Thus, to identify functional resistance genes in one of the main goals in common bean research. One of the first goal to this project was to identify resistance gene homologues (RGH) associated with resistance to Colletotrichum lindemuthianum causal agent of anthracnose. 544 degenerate primers combinations designed from conserved sequences in the NBS domain of resistance genes and RGH genes of the model specie for legumes Medicago truncatula were evaluated. Amplification was carried out on G19833 total DNA this is an Andean genotype, came from Peru and shows resistant to anthracnose and angular leaf spot. Four hundred and three sequences showed the characteristic motifs of NBS type resistance genes, out of which 306 had an uninterrupted ORF, therefore were considered as RGH genes. An phylogenetic analysis was performed from these sequences, taking into account a 90% nucleotide identity, this confirmed the classification of common bean RGHs within TIR and non-TIR families with high bootstrap values. TIR sequences were grouped into 3 clusters discriminated in 14 clades and non-TIR sequences were grouped into 3 cluster and 7 clades. Most of pairwise comparisons showed purifying selection although some sequences may be the result of diversifying selection, which shows the diversity of RGH in beans. Common bean specific RGHs, were evaluated in G19833 BAC library. In BAC-end sequences (BES) of positive clones 629 SSR markers were identified and these were evaluated in the G2333 (resistant) and G19839 (susceptible) genotypes. 205 polymorphic SSR were evaluated in the RIL population derived from G2333 x G19839 cross. The phenotypic evaluation for the identification of QTL was carried out in greenhouse and field conditions, which evaluating 11 races. The genotypic and phenotypic evaluation allowed to identigy QTL associated with resistance to anthracnose, whith phenotypic variances between 9 and 89%. These markers are an important source of information for the development of functional markers that can be used in assisted selection or positional cloning.DoctoradoDoctor en Ciencias AgrariasMejoramiento de leguminosasCiencias Agronómica