Development Of Functional Markers And Transcript Map Of Chickpea (Cicer arietinum L)

To develop repertoire of genic molecular markers (GMMs) including single nucleotide polymorphism (SNP) and intron spanning region (ISR) based markers, in chickpea, the third food legume crop of the world and the first food legume crop of India. In first approach, Solexa 1 Gb was carried out on pooled RNA from all drought challenged root tissues of genotypes ICC 4958 and ICC 1882. 15.6 and 22.1 million reads were generated and aligned against chickpea transcriptome assembly. A total of 26,082 SNPs were identified between these two genotypes. In second approach for SNP discovery through allele re-sequencing, primer pairs were designed for 970 genes/ expressed sequence tags (ESTs) of chickpea and 657 genes/ESTs of heterologous (closely related to chickpea) species and 2,046 SNPs were identified in 84,073 bp sequence data. In the third approach, ISR markers were designed by aligning chickpea unigenes to Medicago truncatula. In the forth approach, KASPar assay was designed for 96-plex SNPs and 56 polymorphic markers were identified on the parental genotypes and genotyping was done by designing Veracode assay for BeadXpress reader. From all the approaches: 87-EST-SNPs, 1627 allele-specific sequencing from chickpea and heterologous species, 121- intron spanning region (CISR) and 56 CKAM from KASPar assay were designed. SNP2CAPS analysis of 87 and 264 sequence alignments from in silico mining of ESTs and allele specific primers, as mentioned above, provided a total of 311 CAPS candidates. 311 CAPS candidates provided scorable amplification in 205 (65.92%) cases of which predicted assays were validated in 152 (74.15%) cases (CGMM). Screening of easily assayable 295 markers including 152 CGMMs, 87 CISRs and 56 CKAM on 5 parental genotypes of three mapping populations identified 75 polymorphic markers on the intra-specific mapping population. 73 of these GMMs together with 241 earlier developed markers could be integrated into the intra-specific genetic map. The transcript map developed here, therefore, has a total of 285 maker loci including 50 GMMs loci and spans 595.73 cM with an average inter marker distance of 2.09 cM. Identification of QTL for drought related root traits resulted in 12 significant QTLs. The QTL analysis revealed the presence of a ―QTL hot-spot‖ region on LG04 that contained QTLs for several drought tolerance traits explaining upto 38.03% phenotypic variation. These resources will be useful not only for genome analysis and genetics and breeding applications of chickpea but also for comparative legume genomics. Moreover, markers and genes associated with QTLs for drought tolerance related traits will be useful for molecular breeding for drought tolerance in chickpea improvement

Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea (Cicer arietinum L.)

A transcript map has been constructed by the development and integration of genic molecular markers (GMMs) including single nucleotide polymorphism (SNP), genic microsatellite or simple sequence repeat (SSR) and intron spanning region (ISR)-based markers, on an inter-specific mapping population of chickpea, the third food legume crop of the world and the first food legume crop of India. For SNP discovery through allele re-sequencing, primer pairs were designed for 688 genes/expressed sequence tags (ESTs) of chickpea and 657 genes/ESTs of closely related species of chickpea. High-quality sequence data obtained for 220 candidate genic regions on 2–20 genotypes representing 9 Cicer species provided 1,893 SNPs with an average frequency of 1/35.83 bp and 0.34 PIC (polymorphism information content) value. On an average 2.9 haplotypes were present in 220 candidate genic regions with an average haplotype diversity of 0.6326. SNP2CAPS analysis of 220 sequence alignments, as mentioned above, provided a total of 192 CAPS candidates. Experimental analysis of these 192 CAPS candidates together with 87 CAPS candidates identified earlier through in silico mining of ESTs provided scorable amplification in 173 (62.01%) cases of which predicted assays were validated in 143 (82.66%) cases (CGMM). Alignments of chickpea unigenes with Medicago truncatula genome were used to develop 121 intron spanning region (CISR) markers of which 87 yielded scorable products. In addition, optimization of 77 EST-derived SSR (ICCeM) markers provided 51 scorable markers. Screening of easily assayable 281 markers including 143 CGMMs, 87 CISRs and 51 ICCeMs on 5 parental genotypes of three mapping populations identified 104 polymorphic markers including 90 markers on the inter-specific mapping population. Sixty-two of these GMMs together with 218 earlier published markers (including 64 GMM loci) and 20 other unpublished markers could be integrated into this genetic map. A genetic map developed here, therefore, has a total of 300 loci including 126 GMM loci and spans 766.56 cM, with an average inter-marker distance of 2.55 cM. In summary, this is the first report on the development of large-scale genic markers including development of easily assayable markers and a transcript map of chickpea. These resources should be useful not only for genome analysis and genetics and breeding applications of chickpea, but also for comparative legume genomics

Evaluate of action adaptogenica of roots the Pfaffia glomerata (Sprengel) Petersen - Amaranthaceae

Pesquisou-se a especie Pfaffia glomerata (Spreng.) Pedersen, cujas raizes sao referidas como um dos ginsengs brasileiros e empregadas para fortalecer a memoria e aumentar a resistencia fica. A planta foi coletada no municipio de Porto Rico (PR), montada em exsicatas, identificada por especialista botanico e avaliada quanto a qualidade de acordo com normas farmacopeicas. Obteve-se extratos hidroalcoolicos das raizes pelo processo da turbolise, que foram liofilizados e armazenados em dessecador a vacuo em freezer. Realizou-se testes agudos com camundongos jovens: screening farmacologico, teste de movimentacao espontanea, teste de coordenacao motora, teste de tempo de sono induzido pelo pentobarbital sodico e teste de esquiva passiva com escopolamina como agente amnesico. Realizou-se tambem testes com ratos jovens e idosos submetidos a administracao cronica: teste de discriminacao direita-esquerda (labirinto em T), teste de esquiva passiva, movimentacao espontanea e avaliacao da evolucao ponderal. O estudo toxicologico pre-clinico envolveu a toxicologia de uma dose (aguda), a toxicologia de doses repetidas (cronica) por 90 dias, a avaliacao da evolucao ponderal, a movimentacao espontanea e avaliacao sanguinea dos parametros bioquimicas e hematologicos bem como da anatomia-patologica do coracao, figado e rins dos animais. O estudo clinico envolveu 38 voluntarios normais, de idade entre 50-75 anos e de diferentes padroes de atividade fisica. Aplicou-se testes psicometricos basais (Teste de Atencao Concentrada, Wechsler Memory Scale e Teste de Matrizes Progressivas), bem como realizou-se um teste ergoespirometrico em bicicleta ergometrica com protocolo de aumento progressivo de cargas ate exaustao. Mediu-se o VO2 maximo, a carga maxima, a frequencia cardiaca submaxima e niveis de lactato no inicio e final do exercicio. Os voluntarios tomaram duas capsulas diarias de extrato seco da planta (300 mg por capsula) ou placebo (500 mg de acucar mascavo), durante seis meses. Nesse periodo, os mesmos foram acompanhados por consultas mensais. Ao final do tratamento, repetiram-se os testes psicometricos e a ergoespirometria. A especie utilizada neste trabalho foi identificada como Pfaffia glomerata (Sprengei) Pedersen - Amaranthaceae, cujos exemplares estao depositados no acervo do Herbarium Friburgense (FCAB) sob o numero 5426. Os dados da CLAE/HPLC evidenciaram os seguintes teores de b-ecdisona: O,76 por cento...(au)BV UNIFESP: Teses e dissertaçõe

Detailed steps performed by automated GBS pipeline.

<p>(A) De-multiplex samples: Raw paired-end Illumina reads are assigned to a sample using barcode sequences, which are subsequently trimmed. (B) Trim and filter reads: De-multiplexed paired-end reads are trimmed for base quality and Illumina adaptors. (C) Align reads to a reference genome. (D) Raw SNP calls: Every position in each sample’s alignment is scanned to determine the probability of a variant.</p

Classification and Characterization of Species within the Genus <i>Lens</i> Using Genotyping-by-Sequencing (GBS)

<div><p>Lentil (<i>Lens culinaris</i> ssp. <i>culinaris</i>) is a nutritious and affordable pulse with an ancient crop domestication history. The genus <b>Lens</b> consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely <i>L</i>. <i>culinaris/L</i>. <i>orientalis/L</i>. <i>tomentosus</i>, <i>L</i>. <i>lamottei/L</i>. <i>odemensis</i>, <i>L</i>. <i>ervoides</i> and <i>L</i>. <i>nigricans</i> which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.</p></div

Phylogenetic relationship within genus <i>Lens</i>.

<p>(A) Dendrogram generated using Neighbour-Joining model based on results from first GBS library. (B) A maximum-likelihood tree based on combined results from two GBS libraries.</p

Gene pool classification of lentil based on STRUCTURE results.

<p>(A) Graphical representation of STRUCTURE results indicates two clusters (K = 2) in genus <i>Lens</i> based on the highest delta K score. (B) Additional STRUCTURE analysis revealed substructures (K = 3) within individuals of <i>L</i>. <i>odemensis</i>, <i>L</i>. <i>lamottei</i>, <i>L</i>. <i>ervoides</i> and <i>L</i>. <i>nigricans</i>. (C) The new gene pool classification proposed in this study is shown next to a simplified maximum-likelihood tree of genus <i>Lens</i>. Accession IG 72815 is marked with asterisk.</p