QTL mapping of mandarin (<Emphasis Type="Italic">Citrus reticulata</Emphasis>) fruit characters using high-throughput SNP markers

Seedlessness, flavor, and color are top priorities for mandarin (Citrus reticulata Blanco) cultivar improvement. Given long juvenility, large tree size, and high breeding cost, marker-assisted selection (MAS) may be an expeditious and economical approach to these challenges. The objectives of this study were to construct high-density mandarin genetic maps and to identify single nucleotide polymorphism (SNP) markers associated with fruit quality traits. Two parental genetic maps were constructed from an F₁ population derived from ‘Fortune’ × ‘Murcott’, two mandarin cultivars with distinct fruit characters, using a 1536-SNP Illumina GoldenGate assay. The map for ‘Fortune’ (FOR) consisted of 189 SNPs spanning 681.07 cM and for ‘Murcott’ (MUR) consisted of 106 SNPs spanning 395.25 cM. Alignment of the SNP sequences to the Clementine (Citrus clementina) genome showed highly conserved synteny between the genetic maps and the genome. A total of 48 fruit quality quantitative trait loci (QTLs) were identified, and ten of them stable over two or more samplings were considered as major QTLs. A cluster of QTLs for flavedo color space values L, a, b, and a/b and juice color space values a and a/b were detected in a single genomic region on linkage group 4. Two carotenoid biosynthetic pathway genes, pds1 and ccd4, were found within this QTL interval. Several SNPs were potentially useful in MAS for these fruit characteristics. QTLs were validated in 13 citrus selections, which may be useful in further validation and tentative MAS in mandarin fruit quality improvement.     

TiO2 nanowire FET device: encapsulation of biomolecules by electro polymerized pyrrole propylic acid

Silane-based methods have become the standards for the conjugation of biomolecules, especially for the preparation of one-dimensional nanomaterial biosensors. However, the specific binding of those target molecules might raise problems with regard to the sensing and non-sensing regions, which may contaminate the sensing devices and decrease their sensitivity. This paper attempts to explore the encapsulation of biomolecules on a one-dimensional nanomaterial field effect transistor (FET) biosensor using polypyrrole propylic acid (PPa). Specifically, the encapsulation of biomolecules via the electropolymerization of pyrrole propylic acid (Pa), a self-made low-conductivity polymer, on TiO(2)-nanowire (NW)-based FETs is presented. The energy dispersive spectrum (EDS) was obtained and electrical analysis was conducted to investigate PPa entrapping anti-rabbit IgG (PPa/1°Ab) on a composite film. The specificity, selectivity and sensitivity of the sensor were analyzed in order to determine the immunoreaction of PPa/1°Ab immobilized NW biosensors. Our results show that PPa/1°Ab achieved high specificity immobilization on NWs under the EDS analysis. Furthermore, the TiO(2)-NW FET immunosensor developed in this work successfully achieved specificity, selectivity and sensitivity detection for the target protein rabbit IgG at the nano-gram level. The combination of PPa material and the electropolymerization method may provide an alternative method to immobilize biomolecules on a specific surface, such as NWs.     

Aberrant hippocampal subregion networks associated with the classifications of aMCI subjects: a longitudinal resting-state study

Background

Altered hippocampal structure and function is a valuable indicator of possible conversion from amnestic type mild cognitive impairment (aMCI) to Alzheimer''s disease (AD). However, little is known about the disrupted functional connectivity of hippocampus subregional networks in aMCI subjects.

Methodology/Principal Findings

aMCI group-1 (n = 26) and controls group-1 (n = 18) underwent baseline and after approximately 20 months follow up resting-state fMRI scans. Integrity of distributed functional connectivity networks incorporating six hippocampal subregions (i.e. cornu ammonis, dentate gyrus and subicular complex, bilaterally) was then explored over time and comparisons made between groups. The ability of these extent longitudinal changes to separate unrelated groups of 30 subjects (aMCI-converters, n = 6; aMCI group-2, n = 12; controls group-2, n = 12) were further assessed. Six longitudinal hippocampus subregional functional connectivity networks showed similar changes in aMCI subjects over time, which were mainly associated with medial frontal gyrus, lateral temporal cortex, insula, posterior cingulate cortex (PCC) and cerebellum. However, the disconnection of hippocampal subregions and PCC may be a key factor of impaired episodic memory in aMCI, and the functional index of these longitudinal changes allowed well classifying independent samples of aMCI converters from non-converters (sensitivity was 83.3%, specificity was 83.3%) and controls (sensitivity was 83.3%, specificity was 91.7%).

Conclusions/Significance

It demonstrated that the functional changes in resting-state hippocampus subregional networks could be an important and early indicator for dysfunction that may be particularly relevant to early stage changes and progression of aMCI subjects.     

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.     

Mining and characterizing microsatellites from citrus ESTs

Freely available computer programs were arranged in a pipeline to extract microsatellites from public citrus EST sequences, retrieved from the NCBI. In total, 3,278 bi- to hexa-type SSR-containing sequences were identified from 56,199 citrus ESTs. On an average, one SSR was found per 5.2 kb of EST sequence, with the tri-nucleotide motifs as the most abundant. Primer sequences flanking SSR motifs were successfully identified from 2,295 citrus ESTs. Among those, a subset (100 pairs) were synthesized and tested to determine polymorphism and heterozygosity between/within two genera, sweet orange (C. sinensis) and Poncirus (P. trifoliata), which are the parents of the citrus core mapping population selected for an international citrus genomics effort. Eighty-seven pairs of primers gave PCR amplification to the anticipated SSRs, of which 52 and 35 appear to be homozygous and heterozygous, respectively, in sweet orange, and 67 and 20, respectively, in Poncirus. By pairing the loci between the two intergeneric species, it was found that 40 are heterozygous in at least one species with two alleles (9), three alleles (28), or four alleles (3), and the remaining 47 are homozygous in both species with either one allele (31) or two alleles (16). These EST-derived SSRs can be a resource used for understanding of the citrus SSR distribution and frequency, and development of citrus EST-SSR genetic and physical maps. These SSR primer sequences are available upon request. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Direct electrochemistry of hemoglobin on carbonized titania nanotubes and its application in a sensitive reagentless hydrogen peroxide biosensor

Carbonized TiO(2) nanotubes (TNT/C) prepared by carbonization with organic polymers possess advantages combined from high conductivity of carbon and nanostructure of TiO(2) nanotubes. The material was used as a supporting matrix to immobilize a redox protein, hemoglobin (Hb), to explore its direct electron transfer ability. The apparent heterogeneous electron transfer rate constant (k(ET)) of Hb on TNT/C is 108s(-1), which is much higher than that in the reported works, demonstrating excellent direct electrochemistry behavior. The TNT/C-Hb modified glassy carbon electrode (GCE) demonstrates significant electrocatalytic activity for reduction of hydrogen peroxide with a small apparent Michaelis-Menten constant (87.5 microM). The TNT/C-Hb based H(2)O(2) sensor has a low detection limit (0.92 microM), fast response time (3s) and high dynamic response range (10(-6) to 10(-4)M), a much better performance than the reported works. These results demonstrate that a direct electrochemistry behavior can be significantly enhanced through simple carbon coating on a nanostructured material for higher reaction surface area and better conductivity. This work suggests that Hb-immobilized TNT/C has potential applications in a sensitive H(2)O(2) sensor.     

A Novel RUNX2 Mutation in Cleidocranial Dysplasia Patients

Cleidocranial dysplasia (CCD) is an autosomal-dominant heritable skeletal disease caused by heterozygous mutations in the RUNX2 gene. Here, the RUNX2 gene was analyzed within a CCD family from China, and a novel missense mutation (c. 475G --> C [p.G159R]) was identified. Normal and mutant RUNX2 expression vectors were then constructed and expressed transiently in NIH3T3 cells. Immunofluorescent staining and Western blotting showed that wild-type RUNX2 protein was localized exclusively in the nucleus; however, the mutant protein was found in both the nucleus and the cytoplasm, which demonstrated that transport of the RUNX2 mutant into the nucleus was disturbed by the G159R mutation. Therefore, we suggest that G159 is very important to promote RUNX2 nuclear localization. According to clinical analysis, the patient displays severe dysplasia of bones and relatively low-grade craniofacial abnormality, and we infer that G159 may be vital for normal skeletal development, other than control of tooth number. These findings confirm that mutations in the RUNX2 gene are associated with the pathogenesis of CCD across different ethnic backgrounds.     

Novel expression patterns of carotenoid pathway-related genes in citrus leaves and maturing fruits

Carotenoids are abundant in citrus fruits and vary among cultivars and species. In the present study, high performance liquid chromatography (HPLC) and real-time polymerase chain reaction (PCR) were used to investigate the expression patterns of 23 carotenoid biosynthesis gene family members and their possible relation with carotenoid accumulation in fresh flavedo, juice sacs and leaves of Valencia orange during fruit maturation. Violaxanthin and lutein mainly accumulated in fruit (flavedo and juice sacs) and leaves (young and mature), respectively, accounting for nearly 79 %, 57 %, 53 % and 70 % of corresponding total carotenoids in February. Violaxanthin content quickly began to increase in flavedo in December, but the increase in juice sacs began later in January. In mature leaves, lutein content was three times that in young leaves; α-carotene and β-carotene were also much higher in mature leaves than in flavedo or juice sacs. Generally most of the carotenoid biosynthesis gene members were expressed at higher levels in flavedo than in juice sacs, and the expression of some continued to increase in flavedo during fruit maturation. All CHYB members expressed at high levels and had similar patterns in juice sacs. Interestingly, the capsanthin capsorubin synthase (CCS) members had similar expression levels and patterns in flavedo and juice sacs. Differences in gene expression between leaf and fruit tissues were noted, pointing to some tissue specificity for certain members of the gene families associated with carotenogenesis. The expression patterns of these 23 citrus carotenoid biosynthesis gene members were also compared with their expression patterns in other plants. Taken together, these first-hand expression data will be useful to define the tissue-specific roles of each gene member in accumulation of different carotenoids in citrus leaves and maturing fruits.     

Mining and characterization of microsatellites from a genome of <Emphasis Type="Italic">Venturia carpophila</Emphasis>

A total of 4021 microsatellites were mined from a genome of Venturia carpophila, and 192 were selected to screen 39 isolates of the fungus collected from peach and nectarine in the southeastern USA. Of the 192 selected, 32 primers consistently and reliably produced polymorphic amplicons. Subsequently, the genotyping data from these 32 primers were used for preliminary analysis of the genetic diversity among the 39 isolates. The number of alleles identified ranged from 2 to 9, and the polymorphic information content from 0.097 to 0.792. Over all isolates, Shannon’s information index was 0.914, indicating genetic diversity. Stoddart and Taylor’s index of diversity and Simpson’s index also indicated high diversity (32.4 and 0.969, respectively). Evenness within the sample was high (0.955), but there was strong evidence for haploid linkage disequilibrium (3.799, P?=?0.001). Observations on diversity were supported by analysis of genetic distance, which showed little affinity for clustering based on isolate source population, location, or host. The microsatellites developed in this study should be useful in future research of the population genetic structure and dynamics of V. carpophila and evaluating the risks posed by the ability of the pathogen to adapt on peach and possibly other stone fruit hosts in the USA and elsewhere in the world.