A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.     

Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.     

Genetic and cytogenetic mapping of DMI1, DMI2, and DMI3 genes of Medicago truncatula involved in Nod factor transduction,nodulation, and mycorrhization

The DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal transduction. Here, we have used diverse approaches to pave the way for the map-based cloning of these genes. Molecular amplification fragment length polymorphism markers linked to the three genes were identified by bulked segregant analysis. Integration of these markers into the general genetic map of M. truncatula revealed that DMI1, DMI2, and DMI3 are located on linkage groups 2, 5, and 8, respectively. Cytogenetic studies using fluorescent in situ hybridization (FISH) on mitotic and pachytene chromosomes confirmed the location of DMI1, DMI2, and DMI3 on chromosomes 2, 5, and 8. FISH-pachytene studies revealed that the three genes are in euchromatic regions of the genome, with a ratio of genetic to cytogenetic distances between 0.8 and 1.6 cM per microm in the DMI1, DMI2, and DMI3 regions. Through grafting experiments, we showed that the genetic control of the dmi1, dmi2, and dmi3 nodulation phenotypes is determined at the root level. This means that mutants can be transformed by Agrobacterium rhizogenes to accelerate the complementation step of map-based cloning projects for DMI1, DMI2, and DMI3.     

Large-Scale Development of Cost-Effective Single-Nucleotide Polymorphism Marker Assays for Genetic Mapping in Pigeonpea and Comparative Mapping in Legumes

Single-nucleotide polymorphisms (SNPs, >2000) were discovered by using RNA-seq and allele-specific sequencing approaches in pigeonpea (Cajanus cajan). For making the SNP genotyping cost-effective, successful competitive allele-specific polymerase chain reaction (KASPar) assays were developed for 1616 SNPs and referred to as PKAMs (pigeonpea KASPar assay markers). Screening of PKAMs on 24 genotypes [23 from cultivated species and 1 wild species (Cajanus scarabaeoides)] defined a set of 1154 polymorphic markers (77.4%) with a polymorphism information content (PIC) value from 0.04 to 0.38. One thousand and ninety-four PKAMs showed polymorphisms between parental lines of the reference mapping population (C. cajan ICP 28 × C. scarabaeoides ICPW 94). By using high-quality marker genotyping data on 167 F₂ lines from the population, a comprehensive genetic map comprising 875 PKAMs with an average inter-marker distance of 1.11 cM was developed. Previously mapped 35 simple sequence repeat markers were integrated into the PKAM map and an integrated genetic map of 996.21 cM was constructed. Mapped PKAMs showed a higher degree of synteny with the genome of Glycine max followed by Medicago truncatula and Lotus japonicus and least with Vigna unguiculata. These PKAMs will be useful for genetics research and breeding applications in pigeonpea and for utilizing genome information from other legume species.     

Computed-tomography-based finite-element models of long bones can accurately capture strain response to bending and torsion

Finite element (FE) models of long bones constructed from computed-tomography (CT) data are emerging as an invaluable tool in the field of bone biomechanics. However, the performance of such FE models is highly dependent on the accurate capture of geometry and appropriate assignment of material properties. In this study, a combined numerical-experimental study is performed comparing FE-predicted surface strains with strain-gauge measurements. Thirty-six major, cadaveric, long bones (humerus, radius, femur and tibia), which cover a wide range of bone sizes, were tested under three-point bending and torsion. The FE models were constructed from trans-axial volumetric CT scans, and the segmented bone images were corrected for partial-volume effects. The material properties (Young's modulus for cortex, density-modulus relationship for trabecular bone and Poisson's ratio) were calibrated by minimizing the error between experiments and simulations among all bones. The R(2) values of the measured strains versus load under three-point bending and torsion were 0.96-0.99 and 0.61-0.99, respectively, for all bones in our dataset. The errors of the calculated FE strains in comparison to those measured using strain gauges in the mechanical tests ranged from -6% to 7% under bending and from -37% to 19% under torsion. The observation of comparatively low errors and high correlations between the FE-predicted strains and the experimental strains, across the various types of bones and loading conditions (bending and torsion), validates our approach to bone segmentation and our choice of material properties.     

Production and characterization of diverse developmental mutants of Medicago truncatula

The diploid annual legume Medicago truncatula has been developed as a tractable genetic system for studying biological questions that are unique to, or well suited for study in legume species. An efficient mutagenesis protocol using ethyl-methyl sulfonate and a polymorphic ecotype with properties appropriate for use as a mapping parent are described. Isolation and characterization of three developmental mutants are described. The mtapetala mutation results in homeotic conversions of floral organ whorls 2 and 3 into sepals and carpelloid structures, respectively, similar to mutations in the apetala3/pistillata genes of Arabidopsis. The palmyra mutation primarily affects seedling shoot meristem initiation, and thus phenocopies meristem function mutations identified in Arabidopsis such as the zwille locus. The phenotype of the palmyra and mtapetala double mutant is additive, with seedling shoot meristems and floral organs indistinguishable from those of the single palmyra and mtapetala mutants, respectively. These results are consistent with a lack of genetic interaction between these loci. A third mutant, speckle, is characterized by spontaneous necrotic lesion formation on leaves, root, and stems, similar to necrosis mutants identified in other plant species. In addition to documenting the efficient mutagenesis of M. truncatula, the availability of developmental mutants that phenocopy characterized Arabidopsis mutants will provide a basis for establishing orthologous gene function between M. truncatula and Arabidopsis, once the genes responsible are cloned. Moreover, the male-sterile, female-fertile nature of the mtapetala mutant provides a convenient tool for genetic analyses in M. truncatula.     

A high-throughput SNP marker system for parental polymorphism screening, and diversity analysis in common bean (Phaseolus vulgaris L.)

Single nucleotide polymorphism (SNP) detection has become a marker system of choice, because of the high abundance of source polymorphisms and the ease with which allele calls are automated. Various technologies exist for the evaluation of SNP loci and previously we validated two medium throughput technologies. In this study, our goal was to utilize a 768 feature, Illumina GoldenGate assay for common bean (Phaseolus vulgaris L.) developed from conserved legume gene sequences and to use the new technology for (1) the evaluation of parental polymorphisms in a mini-core set of common bean accessions and (2) the analysis of genetic diversity in the crop. A total of 736 SNPs were scored on 236 diverse common bean genotypes with the GoldenGate array. Missing data and heterozygosity levels were low and 94 % of the SNPs were scorable. With the evaluation of the parental polymorphism genotypes, we estimated the utility of the SNP markers in mapping for inter-genepool and intra-genepool populations, the latter being of lower polymorphism than the former. When we performed the diversity analysis with the diverse genotypes, we found Illumina GoldenGate SNPs to provide equivalent evaluations as previous gene-based SNP markers, but less fine-distinctions than with previous microsatellite marker analysis. We did find, however, that the gene-based SNPs in the GoldenGate array had some utility in race structure analysis despite the low polymorphism. Furthermore the SNPs detected high heterozygosity in wild accessions which was probably a reflection of ascertainment bias. The Illumina SNPs were shown to be effective in distinguishing between the genepools, and therefore were most useful in saturation of inter-genepool genetic maps. The implications of these results for breeding in common bean are discussed as well as the advantages and disadvantages of the GoldenGate system for SNP detection.     

Evaluation of TNF-α, IL-10 and IL-6 Cytokine Production and Their Correlation with Genotype Variants amongst Tuberculosis Patients and Their Household Contacts

Background

Household contacts of diagnostically established tuberculosis (TB) patients are highly susceptible to disease development. It is surmised that cytokines perhaps play a synergistic and a prognostic role in the activation of the otherwise latent infection in these house hold contacts. Evaluation of the cytokines and any of their inherent polymorphisms might provide a useful diagnostic tool in evaluating the immune regulation and the progression of the disease. The cytokines thus released in a paracrine manner in serum may also provide an indirect measure of the cytokine function.

Objective

The present study was aimed to evaluate the levels of TNF-α, IL-10 & IL-6 cytokines and their correlation with genotype variants amongst tuberculosis patients and their household contacts.

Methods

The cytokine levels were estimated in serum by enzyme-linked immunosorbent assay (ELISA) and their polymorphisms were studied by amplification refractory mutation system polymerase chain reaction (ARMs PCR) in active pulmonary tuberculosis patients (APTB = 150), household contacts (HHC = 190), and healthy controls (HC = 150).

Results

The median values of TNF-α cytokine were significantly high among APTB and HHC compared to HCs (P< 0.0001 and 0.0001). IL-6 levels also were elevated among APTB compared to HHC and HC, and a significant difference was observed between APTB and HHC at P<0.0001; APTB & HC at P< 0.04; HHC & HC at P< 0.01. The IL-10 levels were low in APTB compared to HHC and HCs and no significant difference was observed. TNF-α/IL-10 ratio was significant and indicated Th1 predominance in APTB and HHC. IL-6/IL-10 showed pronounced Th1 expression in APTB and Th2 in HHC and HC. The ROC analysis indicated that both IL-10 and IL-6 can be used to decide the risk of exposed individual to a disease. The results of multivariate analysis indicate that IL-10 (-1082) GA genotype was significantly associated with p<0.028 in APTB. No significant association was observed between genotypes, other serum cytokine levels and clinical characteristics between APTB, HHC and HCs.

Conclusion

Large sample size with follow-up at different time points may further illuminate the role of IL-10 and IL-6 cytokines as a prognostic marker in house hold contacts.