A Multiparent Advanced Generation Inter-Cross to Fine-Map Quantitative Traits in Arabidopsis thaliana

Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms.     

Genetic variation in South Indian castes: evidence from Y-chromosome, mitochondrial, and autosomal polymorphisms

Background

Major population movements, social structure, and caste endogamy have influenced the genetic structure of Indian populations. An understanding of these influences is increasingly important as gene mapping and case-control studies are initiated in South Indian populations.

Results

We report new data on 155 individuals from four Tamil caste populations of South India and perform comparative analyses with caste populations from the neighboring state of Andhra Pradesh. Genetic differentiation among Tamil castes is low (R_ST = 0.96% for 45 autosomal short tandem repeat (STR) markers), reflecting a largely common origin. Nonetheless, caste- and continent-specific patterns are evident. For 32 lineage-defining Y-chromosome SNPs, Tamil castes show higher affinity to Europeans than to eastern Asians, and genetic distance estimates to the Europeans are ordered by caste rank. For 32 lineage-defining mitochondrial SNPs and hypervariable sequence (HVS) 1, Tamil castes have higher affinity to eastern Asians than to Europeans. For 45 autosomal STRs, upper and middle rank castes show higher affinity to Europeans than do lower rank castes from either Tamil Nadu or Andhra Pradesh. Local between-caste variation (Tamil Nadu R_ST = 0.96%, Andhra Pradesh R_ST = 0.77%) exceeds the estimate of variation between these geographically separated groups (R_ST = 0.12%). Low, but statistically significant, correlations between caste rank distance and genetic distance are demonstrated for Tamil castes using Y-chromosome, mtDNA, and autosomal data.

Conclusion

Genetic data from Y-chromosome, mtDNA, and autosomal STRs are in accord with historical accounts of northwest to southeast population movements in India. The influence of ancient and historical population movements and caste social structure can be detected and replicated in South Indian caste populations from two different geographic regions.     

QTL fine-mapping with recombinant-inbred heterogeneous stocks and in vitro heterogeneous stocks

We compared strategies to fine-map Quantitative Trait Loci (QTL) in mice with heterogeneous stocks (HS). We showed that a panel of about 100 Recombinant Inbred Lines (RIL) derived from an HS, and which we called an RIHS, was ideally suited to fine-map QTL to very high resolution, without the cost of additional genotyping. We also investigated a strategy based on in vitro fertilization of large numbers of F₁ offspring of HS males crossed with an inbred line (IVHS). This method required some additional genotyping but avoided the breeding delays and costs associated with the construction of an RI panel. We showed that QTL detection was higher by using RIHS than with IVHS and that it was independent of the number of RI lines, provided the total number of animals phenotyped was constant. However, fine-mapping accuracy was slightly better with IVHS. We also investigated the effects of varying the number of HS generations and using multiallelic microsatellites instead of SNPs. We found that quite modest generation times of 10–20 generations were optimal. Microsatellites were superior to SNPs only when the generation time was 30 or more and when the markers were widely spaced.     

Hybrid origin of Japanese mice "Mus musculus molossinus": evidence from restriction analysis of mitochondrial DNA

The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction- site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.      

Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.     

Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.     

Management, presentation and interpretation of genome scans using GSCANDB

MOTIVATION: Advances in high-throughput genotyping have made it possible to carry out genome-wide association studies using very high densities of genetic markers. This has led to the problem of the storage, management, quality control, presentation and interpretation of results. In order to achieve a successful outcome, it may be necessary to analyse the data in different ways and compare the results with genome annotations and other genome scans. RESULTS: We created GSCANDB, a database for genome scan data, using a MySQL backend and Perl-CGI web interface. It displays genome scans of multiple phenotypes analysed in different ways and projected onto genome annotations derived from EnsMart. The current version is optimized for analysis of mouse data, but is customizable to other species. AVAILABILITY: Source code and example data are available under the GPL, in versions tailored to either human or mouse association studies, from http://gscan.well.ox.ac.uk/software.     

On the molecular discrimination between adenine and guanine by proteins

The molecular recognition and discrimination of adenine and guanine ligand moieties in complexes with proteins have been studied using empirical observations on carefully selected crystal structures. The distribution of protein folds that bind these purines has been found to differ significantly from that across the whole PDB, but the most populated architectures and folds are also the most common in three genomes from the three different domains of life. The protein environments around the two nucleic acid bases were significantly different, in terms of the propensities of amino acid residues to be in the binding site, as well as their propensities to form hydrogen bonds to the bases. Plots of the distribution of protein atoms around the two purines clearly show different clustering of hydrogen bond donors and acceptors opposite complimentary acceptors and donors in the rings, with hydrophobic areas below and above the rings. However, the clustering pattern is fuzzy, reflecting the variety of ways that proteins have evolved to recognise the same molecular moiety. Furthermore, an analysis of the conservation of residues in the protein chains binding guanine shows that residues in contact with the base are in general better conserved than the rest of the chain.     

Bayesian Modeling of Haplotype Effects in Multiparent Populations

A general Bayesian model, Diploffect, is described for estimating the effects of founder haplotypes at quantitative trait loci (QTL) detected in multiparental genetic populations; such populations include the Collaborative Cross (CC), Heterogeneous Socks (HS), and many others for which local genetic variation is well described by an underlying, usually probabilistically inferred, haplotype mosaic. Our aim is to provide a framework for coherent estimation of haplotype and diplotype (haplotype pair) effects that takes into account the following: uncertainty in haplotype composition for each individual; uncertainty arising from small sample sizes and infrequently observed haplotype combinations; possible effects of dominance (for noninbred subjects); genetic background; and that provides a means to incorporate data that may be incomplete or has a hierarchical structure. Using the results of a probabilistic haplotype reconstruction as prior information, we obtain posterior distributions at the QTL for both haplotype effects and haplotype composition. Two alternative computational approaches are supplied: a Markov chain Monte Carlo sampler and a procedure based on importance sampling of integrated nested Laplace approximations. Using simulations of QTL in the incipient CC (pre-CC) and Northport HS populations, we compare the accuracy of Diploffect, approximations to it, and more commonly used approaches based on Haley–Knott regression, describing trade-offs between these methods. We also estimate effects for three QTL previously identified in those populations, obtaining posterior intervals that describe how the phenotype might be affected by diplotype substitutions at the modeled locus.     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.