X-ray imaging characterization of femoral bones in aging mice with osteopetrotic disorder.

Aging mice with a rare osteopetrotic disorder in which the entire space of femoral bones are filled with trabecular bones are used as our research platform. A complete study is conducted with a micro computed tomography (CT) system to characterize the bone abnormality. Technical assessment of femoral bones includes geometric structure, biomechanical strength, bone mineral density (BMD), and bone mineral content (BMC). Normal aging mice of similar ages are included for comparisons. In our imaging work, we model the trabecular bone as a cylindrical rod and new quantitative which are not previously discussed are developed for advanced analysis, including trabecular segment length, trabecular segment radius, connecting node number, and distribution of trabecular segment radius. We then identified a geometric characteristic in which there are local maximums (0.0049, 0.0119, and 0.0147 mm) in the structure of trabecular segment radius. Our calculations show 343% higher in percent trabecular bone volume at distal-metaphysis; 38% higher in cortical thickness at mid-diaphysis; 11% higher in cortical cross-sectional moment of inertia at mid-diaphysis; 42% higher in cortical thickness at femur neck; 26% higher in cortical cross-sectional moment of inertia at femur neck; 31% and 395% higher in trabecular BMD and BMC at distal-metaphysis; 17% and 27% higher in cortical BMD and BMC at distal-metaphysis; 9% and 53% higher in cortical BMD and BMC at mid-diaphysis; 25% and 64% higher in cortical BMD and BMC at femur neck. Our new quantitative parameters and findings may be extended to evaluate the treatment response for other similar bone disorders.

De novo sequencing, characterization, and comparison of inflorescence transcriptomes of Cornus canadensis and C. florida (Cornaceae).

BACKGROUND: Transcriptome sequencing analysis is a powerful tool in molecular genetics and evolutionary biology. Here we report the results of de novo 454 sequencing, characterization, and comparison of inflorescence transcriptomes of two closely related dogwood species, Cornus canadensis and C. florida (Cornaceae). Our goals were to build a preliminary source of genome sequence data, and to identify genes potentially expressed differentially between the inflorescence transcriptomes for these important horticultural species. RESULTS: The sequencing of cDNAs from inflorescence buds of C. canadensis (cc) and C. florida (cf), and normalized cDNAs from leaves of C. canadensis resulted in 251799 (ccBud), 96245 (ccLeaf) and 114648 (cfBud) raw reads, respectively. The de novo assembly of the high quality (HQ) reads resulted in 36088, 17802 and 21210 unigenes for ccBud, ccLeaf and cfBud. A reference transcriptome for C. canadensis was built by assembling HQ reads of ccBud and ccLeaf, containing 40884 unigenes. Reference mapping and comparative analyses found 10926 sequences were putatively specific to ccBud, and 6979 putatively specific to cfBud. Putative differentially expressed genes between ccBud and cfBud that are related to flower development and/or stress response were identified among 7718 shared sequences by ccBud and cfBud. Bi-directional BLAST found 87 (41.83% of 208) of Arabidopsis genes related to inflorescence development had putative orthologs in the dogwood transcriptomes. Comparisons of the shared sequences by ccBud and cfBud yielded 65931 high quality SNPs between two species. The twenty unigenes with the most SNPs are listed as potential genetic markers for evolutionary studies. CONCLUSIONS: The data provide an important, although preliminary, information platform for functional genomics and evolutionary developmental biology in Cornus. The study identified putative candidates potentially involved in the genetic regulation of inflorescence evolution and/or disease resistance in dogwoods for future analyses. Results of the study also provide markers useful for dogwood phylogenomic studies.

Chloroplast microsatellite markers in Liriodendron tulipifera (Magnoliaceae) and cross-species amplification in L. chinense.

PREMISE OF THE STUDY: A set of cpSSR markers were developed for the tree genus Liriodendron L. to investigate population genetic structure and phylogeographic history. METHODS AND RESULTS: Primers were designed directly from the chloroplast genome sequences of Liriodendron tulipifera. Among the 55 cpSSR markers tested, 11 polymorphic markers were identified in L. tulipifera. The number of alleles in the population tested ranged from two to five, and the unbiased haploid diversity per locus ranged from 0.074 to 0.644. Eighteen primer pairs generated polymorphic amplification in L. chinense. The number of alleles per locus ranged from two to seven, and the unbiased haploid diversity per locus was from 0.250 to 0.964. CONCLUSIONS: cpSSR markers developed here will be useful for phylogeography and population genetics studies of Liriodendron.

Mice with alopecia, osteoporosis, and systemic amyloidosis due to mutation in Zdhhc13, a gene coding for palmitoyl acyltransferase.

Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein-protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea-mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions). Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T) was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13), a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X), and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis.

Ten polymorphic microsatellite markers in Michelia maudiae (Magnoliaceae).

PREMISE OF THE STUDY: Michelia maudiae is a threatened species in the Magnoliaceae. Microsatellite markers were developed and characterized in M. maudiae for further investigation of its conservation genetics. • METHODS AND RESULTS: Microsatellite markers were developed in M. maudiae using the Fast Isolation by AFLP of Sequences Containing Repeats protocol. Ten polymorphic microsatellites were assessed in two populations of M. maudiae. The number of alleles detected per locus varied from 1 to 8. Observed and expected heterozygosities ranged from 0.000 to 0.792 and from 0.000 to 0.826, respectively. Six primer pairs showed transferability in the two related species Michelia foveolata and Michelia chapensis. • CONCLUSIONS: This set of microsatellite markers provides a useful tool for future studies of the conservation genetics of M. maudiae and other congeneric species.

Spontaneous interspecific hybridization and patterns of pollen dispersal in ex situ populations of a tree species (Sinojackia xylocarpa) that is extinct in the wild.

For endangered plants interspecific hybridization occurring in ex situ collections may lead to failure of reintroduction actions. We used Sinojackia xylocarpa, a well documented Chinese endemic species that is extinct in the wild, as a model case to address this concern. We used paternity analyses to assess the spontaneous hybridization and patterns of pollen flow between S. xylocarpa and its congener species, S. rehderiana, in conserved populations in Wuhan Botanic Garden. Interspecific hybridization events were detected in seven out of eight maternal trees of S. xylocarpa, and an average of 32.7% seeds collected from maternal trees of S. xylocarpa were hybrids. The paternity of 93 out of 249 seedlings from S. xylocarpa assigned to S. rehderiana provided convincing evidence that spontaneous interspecific hybridization occurred extensively in the living garden collection we studied. Different patterns of pollen dispersal (predominantly short-distance vs. long-distance pollination) were observed between intra- and interspecific hybridization events in the garden. Pollen dispersal within the ex situ populations was not restricted by distance, as evidenced by a lack of significant correlations between the average effective pollen dispersal distance (delta) and the geographic distances (d1 and d2) between maternal and paternal trees. The interspecific pollen-dispersal distance ranged from 10 to 620.1 m (mean 294.4 m). Such extensive hybridization in ex situ collections could jeopardize the genetic integrity of endangered species and irrevocably contaminate the gene pool if such hybrids are used for reintroduction and restoration. We recommend strongly that measures be taken to minimize the genetic risks of this kind of hybridization, including establishing buffer zones in ex situ collections, manipulating flowering phenology, testing seed lots before use in reintroduction programs, and controlling pollination for seed purity.

[Construction of cDNA library of 'Hongyang' kiwifruit and analysis of F3H expression].

The SMART switching mechanism at 5' end of the RNA transcript technique was used to construct a cDNA library from inner pericarp of the red flesh kiwifruit Actinidia chinesis cv 'Hongyang'. Construction of cDNA library facilitated cloning of the genes associated with the secondary metabolism, the specific genes in the course of anthocyanin biosynthesis. The titers of the primary library and the amplified library were 6.7x104 cfu/mL and 2.72x108 cfu/mL, respectively. The recom-bination rate was over 99.8%. The lengths of most cDNAs in the library ranged from 700 bp to 1 000 bp. A total of 1 014 clones randomly chosen from the cDNA library were sequenced and these expressed sequence tags (ESTs) were analyzed. A set of 963 sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 632 unigenes, includ-ing 92 contigs and 540 singletons. Among them, 441 EST unigenes were predicted to have known functions. Gene AcF3H, which participated in anthocyanin biosynthesis from sequencing, was obtained. The length of the AcF3H cDNA was 1 369 bp (GenBank accession No: FJ542819). Bioinformatic analysis showed that AcF3H ORF region was 1 101 bp, which en-coded a peptide with 366 amino acids. The amino acid sequences of this gene shared extensive homology to Arabidopsis, Vitis, and Eustoma. The expression of AcF3H was investigated in inner pericarp of 'Hongyang' at six stages during fruit development using RT-PCR. The expression level was high before colour-changed stage, and then decreased at the primary stage of pigmentation.

[Recent progress in plant molecular population genetics].

Molecular population genetics is not only one of the most important subjects of evolutionary biology, but also the basics subject of breeding, association mapping, and linkage analysis. Molecular population genetics has been developed from the classical population genetics aiming at studying population genetic structure and the factors that affect the population genetic structure by investigating the variation of DNA sequences. Therefore, population evolving history can be deduced accurately and quantitatively for evaluating the former conclusions about long-term evolution and the stability of genetic systems. Thus, molecular population genetics can avoid the shortcomings of classical population genetics, i.e. limiting to deduce the short evolving history of a population. Moreover, understanding of molecular variation patterns leads to further evaluation of the evolution theory, which is based on "Natural Selection" and introduced by Darwin. Molecular population genetics has made great progress and revealed many important scientific issues, such as the pattern of DNA polymorphism, the level of linkage disequilibrium, demographical history, and the genetic forces affecting gene evolvement. Furthermore, new research areas have been developed from molecular population genetics and become the hot fields, such as molecular phylogeography. In this review, we summarized studies and progresses of plant molecular population genetics.

Electrostatic funneling for precise nanoparticle placement: a route to wafer-scale integration.

We demonstrate a large-scale placement of nanoparticles through a scheme named "electrostatic funneling", in which charged nanoparticles are guided by an electrostatic potential energy gradient and placed on targeted locations with nanoscale precision. The guiding electrostatic structures are defined using current CMOS fabrication technology. The effectiveness of this scheme is demonstrated for a variety of geometries including one-dimensional and zero-dimensional patterns as well as three-dimensional step structures. Placement precision of 6 nm has been demonstrated using a one-dimensional guiding structure comprising alternatively charged lines with line width of approximately 100 nm. Detailed calculations using DLVO theory agree well with the observed long-range interactions and also estimate lateral forces as strong as (1-3) x 10(-7) dyn, which well explains the observed guided placement of Au nanoparticles.

An IL-7 splicing-defect lymphopenia mouse model revealed by genome-wide mutagenesis.

Homeostasis of the hematopoietic system is tightly regulated by an array of cytokines that control proliferation, differentiation and apoptosis of various cell lineages. To identify genes that are essential for hematopoietic homeostasis, we screened C57BL/6 mice that had been genome-wide mutagenized by N-ethyl-N-nitrosourea (ENU) to produce altered blood cell composition. We identified a mutant mouse line with a drastic reduction in the number of T and B cell lineages in lymphatic tissues and peripheral blood, as well as severe atrophy of the thymus and lymph nodes. Genotyping with a genome-wide single nucleotide polymorphism (SNP) marker set mapped the mutant phenotype to chromosome 3A and subsequent direct DNA sequencing revealed a G-to-A point mutation in the splicing donor site of the third exon of the candidate gene for IL-7, a lymphocyte survival cytokine. Such mutation resulted in skipping of exon 3 and production of an internally truncated IL-7 (DeltaE3-IL7). Furthermore, using recombinant proteins produced in a baculoviral system, we demonstrated that DeltaE3-IL7 had no detectable anti-apoptotic activity even at a dose that was 30 times more than that required for a wild-type protein to manifest a full activity in a naïve T cell survival assay. Our data suggest that this mutant mouse line provides an alternative animal model for the study of severe combined immunodeficiency (SCID) syndrome in humans.

[Genetic risks in plant ex situ conservation].

Conserving genetic diversity of rare and endangered species and their evolutionary potential is one of the long-term goals of ex-situ conservation. Some potential genetic risks in ex-situ conservation in botanical gardens are presented. The preserved species may lack genetic representativity because of poor sampling. Inappropriate plantations, inadequate records and unclear kinships jeopardize endangered species to genetic confusion, inbreeding depression or outbreeding depression. Artificial selection and habitat conversion also potentially result endangered plants in adapting to ex-situ conservation, which had been usually overlooked. All the genetic risks can decrease the success of reintroduction and recovery. Therefore, appropriate genetic management should be carried out in botanical gardens to decrease or avoid genetic risks in ex-situ conservation.

Genetic diversity and geographic differentiation in endangered Ammopiptanthus (Leguminosae) populations in desert regions of northwest China as revealed by ISSR analysis.

BACKGROUND AND AIMS: The desert legume genus Ammopiptanthus comprises two currently endangered species, A. mongolicus and A. nanus. Genetic variability and genetic differentiation between the two species and within each species were examined. METHODS: Inter-simple sequence repeat (ISSR) marker data were obtained and analysed with respect to genetic diversity, structure and gene flow. KEY RESULTS: Despite the morphological similarity between A. mongolicus and A. nanus, the two species are genetically distinct from each other, indicated by 63 % species-specific bands. Low genetic variability was detected for both population level (Shannon indices of diversity Hpop = 0.106, percentage of polymorphic loci P = 18.55 % for A. mongolicus; Hpop = 0.070, P = 12.24 % for A. nanus) and species level (Hsp = 0.1832, P = 39.39 % for A. mongolicus; Hsp = 0.1026, P = 25.89 % for A. nanus). Moderate genetic differentiation was found based on different measures (AMOVA PhiST and Hickory B) in both A. mongolicus (0.3743-0.3744) and A. nanus (0.2162-0.2369). CONCLUSIONS: The significant genetic difference between the two species might be due to a possible vicariant evolutionary event from a single common ancestor through the fragmentation of their common ancestor's range. Conservation strategies for these two endangered species are proposed.