Interval mapping of quantitative trait loci with selective DNA pooling data

Selective DNA pooling is an efficient method to identify chromosomal regions that harbor quantitative trait loci (QTL) by comparing marker allele frequencies in pooled DNA from phenotypically extreme individuals. Currently used single marker analysis methods can detect linkage of markers to a QTL but do not provide separate estimates of QTL position and effect, nor do they utilize the joint information from multiple markers. In this study, two interval mapping methods for analysis of selective DNA pooling data were developed and evaluated. One was based on least squares regression (LS-pool) and the other on approximate maximum likelihood (ML-pool). Both methods simultaneously utilize information from multiple markers and multiple families and can be applied to different family structures (half-sib, F2 cross and backcross). The results from these two interval mapping methods were compared with results from single marker analysis by simulation. The results indicate that both LS-pool and ML-pool provided greater power to detect the QTL than single marker analysis. They also provide separate estimates of QTL location and effect. With large family sizes, both LS-pool and ML-pool provided similar power and estimates of QTL location and effect as selective genotyping. With small family sizes, however, the LS-pool method resulted in severely biased estimates of QTL location for distal QTL but this bias was reduced with the ML-pool.     

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures

A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Partial desiccation of mature embryo-derived calli,a simple treatment that dramatically enhances the regeneration ability of indica rice

Regeneration of indica rice varieties remains a limiting factor for researchers undertaking rice Iransformation experiments. As reported for japonica rice and other crops, partial desiccation of indica rice calli dramatically promotes organogenesis and leads to high regeneration ability. We are now able to obtain 66.5%, 61.1% and 73.7% of calli that regenerate plants for the indica varieties TN1, IR72 and IR64 whereas in non desiccated controls only 30.0%, 15.5% and 18.7% of calli regenerated, respectively. Plants obtained were phenotypically normal and 50% were highly fertile. Partial desiccation is a reliable and simple method for improving indica rice regeneration. It also shortens the time of in vitro culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-Benzyl amino purine - DTT Dithiothreitol - EDTA Ethylene diamine tetra-acetic acid - MS Murashige and Skoog - NAA Naphtalene acetic acid - PAGE Polyacrylamide gel electrophoresis - PAR Photosynthetic active radiation - SDS Sodium dodecil sulfate     

Two new cytotypes reinforce that <Emphasis Type="Italic">Micronycteris hirsuta</Emphasis> Peters, 1869 does not represent a monotypic taxon

Background

The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n?=?26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data.

Results

We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n?=?26 and 25, NF?=?32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA.

Conclusions

The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

     

Male mate choice scales female ornament allometry in a cichlid fish

Background  

Studies addressing the adaptive significance of female ornamentation have gained ground recently. However, the expression of female ornaments in relation to body size, known as trait allometry, still remains unexplored. Here, we investigated the allometry of a conspicuous female ornament in Pelvicachromis taeniatus, a biparental cichlid that shows mutual mate choice and ornamentation. Females feature an eye-catching pelvic fin greatly differing from that of males.     

Coexpression of somatostatin-like immunoreactivity and catecholaminergic properties in neural crest derivatives: comodulation of peptidergic and adrenergic differentiation in cultured neural crest

In the avian embryo, somatostatin-like immunoreactivity (SLI) and adrenergic characteristics appear virtually simultaneously in the developing sympathetic nervous system and adrenal medulla. We have used double-labeling techniques to show that both properties coexist in the same cells. In the quail, not only do all somatostatin-containing cells in the adrenosympathetic system exhibit tyrosine hydroxylase immunoreactivity and possess catecholamines (CA), but this coexistence of the peptidergic and adrenergic phenotypes is already present very early in ontogeny. However, not all adrenergic cells express SLI. The development of sympathoadrenal precursors can be followed in vitro. Adrenergic precursor cells, obtained from the migrating neural crest, differentiate in culture into neuron-like cells that contain SLI and CA. This coexpression can be regulated by the same factors. For instance, corticosterone and progesterone increase SLI content and CA production in the neural crest cell cultures. The ontogeny of the autonomic lineage is discussed in the light of these results.     

Identification of Replication Specificity Determinants in Two Strains of Tomato Leaf Curl Virus from New Delhi

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.