Coexpression gene network analysis of cold-tolerant Solanum commersonii reveals new insights in response to low temperatures

Among abiotic stressors, cold is one of the most harmful for the cultivated potato (Solanum tuberosum L.), a frost-sensitive crop. RNA sequencing (RNA-seq) profiling of two different clones of wild potato (S. commersonii Dun.) contrasting in their capacity to withstand low temperatures revealed a higher number of differentially expressed genes (DEGs) under nonacclimated conditions (NAC) in tolerant clone cmm1T vs. the susceptible cmm6-6 (1,002 and 8,055 DEGs, respectively). By contrast, the number of DEGs was much more comparable when both genotypes were under acclimated conditions (AC). Indeed, a total of 5,650 and 8,936 DEGs were detected in the tolerant genotype vs. the susceptible. Gene ontology (GO) classification under NAC showed a significant role for transcription regulation, lignin catabolic genes, and regulation of plant type secondary cell wall in the cold-tolerant genotypes, suggesting an important role in conferring tolerance response. By contrast, response to stress and response to stimuli were enriched GO categories in both clones under AC. Unsigned weighted correlation networks analysis (WGCNA) allowed identification of coexpressed hub genes with possible main regulatory functions and major impacts on the phenotype. Among those identified, we clarified the role of CBF4. This gene showed contrasting expression profiles in the two clones under NAC, being induced in cold-tolerant cmm1T but suppressed in susceptible cmm6-6. By contrast, under AC, CBF4 was upregulated in both clones. Our study provides a global understanding of mechanisms involved following exposure to NAC and AC in S. commersonii. The mechanisms described here will inform future investigations for detailed validation in studies regarding cold tolerance in plants

Mapping a Novel Black Spot Resistance Locus in the Climbing Rose Brite Eyes™ (‘RADbrite’)

Rose black spot, caused by Diplocarpon rosae, is one of the most devastating foliar diseases of cultivated roses (Rosa spp.). The globally distributed pathogen has the potential to cause large economic losses in the outdoor cultivation of roses. Fungicides are the primary method to manage the disease, but are often viewed unfavorably by home gardeners due to potential environmental and health impacts. As such, rose cultivars with genetic resistance to black spot are highly desired. The tetraploid climbing rose Brite EyesTM (‘RADbrite’) is known for its resistance to black spot. To better characterize the resistance present in Brite EyesTM, phenotyping was conducted on a 94 individual F1 population developed by crossing Brite EyesTM to the susceptible tetraploid rose ‘Morden Blush’. Brite EyesTM was resistant to all D. rosae races evaluated except for race 12. The progeny were either resistant or susceptible to all races (2, 3, 8, 9, 10, 11, and 13) evaluated. The segregation ratio was 1:1 (χ2 = 0.3830, P = 0.5360) suggesting resistance is conferred by a single locus. The roses were genotyped with the WagRhSNP 68K Axiom array and the ‘polymapR’ package was used to construct a map. A single resistance locus (Rdr4) was identified on the long arm of chromosome 5 homoeolog 4. Three resistance loci have been previously identified (Rdr1, Rdr2, and Rdr3). Both Rdr1 and Rdr2 are located on a chromosome 1 homoeolog. The chromosomal location of Rdr3 is unknown, however, races 3 and 9 are virulent on Rdr3. Rdr4 is either a novel gene or an allele of Rdr3 as it provides resistance to races 3 and 9. Due to its broad resistance, Rdr4 is an excellent gene to introgress into new rose cultivars

Microsatellite isolation and marker development in carrot - genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae

<p>Abstract</p> <p>Background</p> <p>The Apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~21 million tons produced yearly worldwide. Despite its importance, molecular resources in this species are relatively underdeveloped. The availability of informative, polymorphic, and robust PCR-based markers, such as microsatellites (or SSRs), will facilitate genetics and breeding of carrot and other Apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. Thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for SSRs, and bioinformatic mining of SSRs in BAC-end sequence and EST sequence databases. This work reports on the development of 300 carrot SSR markers and their characterization at various levels.</p> <p>Results</p> <p>Evaluation of microsatellites isolated from both DNA sources in subsets of 7 carrot F₂mapping populations revealed that SSRs from the hybridization-based method were longer, had more repeat units and were more polymorphic than SSRs isolated by sequence search. Overall, 196 SSRs (65.1%) were polymorphic in at least one mapping population, and the percentage of polymophic SSRs across F₂populations ranged from 17.8 to 24.7. Polymorphic markers in one family were evaluated in the entire F₂, allowing the genetic mapping of 55 SSRs (38 codominant) onto the carrot reference map. The SSR loci were distributed throughout all 9 carrot linkage groups (LGs), with 2 to 9 SSRs/LG. In addition, SSR evaluations in carrot-related taxa indicated that a significant fraction of the carrot SSRs transfer successfully across Apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. SSR diversity evaluated in a collection of 65 <it>D. carota </it>accessions revealed a high level of polymorphism for these selected loci, with an average of 19 alleles/locus and 0.84 expected heterozygosity.</p> <p>Conclusions</p> <p>The addition of 55 SSRs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. The markers developed herein will be a valuable resource for assisting breeding, genetic, diversity, and genomic studies of carrot and other Apiaceae.</p

A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

<p>Abstract</p> <p>Background</p> <p><it>BCR-ABL </it>kinase domain (KD) mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI) in <it>BCR-ABL</it>-positive chronic myeloid leukemia (CML) patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage.</p> <p>Methods</p> <p>A single-tube allele specific-polymerase chain reaction (AS-PCR) method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC) and direct sequencing were performed as a comparison to AS-PCR.</p> <p>Results</p> <p>T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution.</p> <p>Conclusion</p> <p>A single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.</p

Characterization of the severe phenotype of pyruvate kinase deficiency

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162766/2/ajh25926.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162766/1/ajh25926_am.pd

Competition between Phytophthora infestans Effectors Leads to Increased Aggressiveness on Plants Containing Broad-Spectrum Late Blight Resistance

BACKGROUND: The destructive plant disease potato late blight is caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary. This disease has remained particularly problematic despite intensive breeding efforts to integrate resistance into cultivated potato, largely because of the pathogen's ability to quickly evolve to overcome major resistance genes. The RB gene, identified in the wild potato species S. bulbocastanum, encodes a protein that confers broad-spectrum resistance to most P. infestans isolates through its recognition of highly conserved members of the corresponding pathogen effector family IPI-O. IpiO is a multigene family of effectors and while the majority of IPI-O proteins are recognized by RB to elicit host resistance, some variants exist that are able to elude detection (e.g. IPI-O4). METHODS AND FINDINGS: In the present study, analysis of ipiO variants among 40 different P. infestans isolates collected from Guatemala, Thailand, and the United States revealed a high degree of complexity within this gene family. Isolate aggressiveness was correlated with increased ipiO diversity and especially the presence of the ipiO4 variant. Furthermore, isolates expressing IPI-O4 overcame RB-mediated resistance in transgenic potato plants even when the resistance-eliciting IPI-O1 variant was present. In support of this finding, we observed that expression of IPI-O4 via Agrobacterium blocked recognition of IPI-O1, leading to inactivation of RB-mediated programmed cell death in Nicotiana benthamiana. CONCLUSIONS: In this study we definitively demonstrate and provide the first evidence that P. infestans can defeat an R protein through inhibition of recognition of the corresponding effector protein

Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.)

<p>Abstract</p> <p>Background</p> <p>Cucumber, <it>Cucumis sativus </it>L. is an important vegetable crop worldwide. Until very recently, cucumber genetic and genomic resources, especially molecular markers, have been very limited, impeding progress of cucumber breeding efforts. Microsatellites are short tandemly repeated DNA sequences, which are frequently favored as genetic markers due to their high level of polymorphism and codominant inheritance. Data from previously characterized genomes has shown that these repeats vary in frequency, motif sequence, and genomic location across taxa. During the last year, the genomes of two cucumber genotypes were sequenced including the Chinese fresh market type inbred line '9930' and the North American pickling type inbred line 'Gy14'. These sequences provide a powerful tool for developing markers in a large scale. In this study, we surveyed and characterized the distribution and frequency of perfect microsatellites in 203 Mbp assembled Gy14 DNA sequences, representing 55% of its nuclear genome, and in cucumber EST sequences. Similar analyses were performed in genomic and EST data from seven other plant species, and the results were compared with those of cucumber.</p> <p>Results</p> <p>A total of 112,073 perfect repeats were detected in the Gy14 cucumber genome sequence, accounting for 0.9% of the assembled Gy14 genome, with an overall density of 551.9 SSRs/Mbp. While tetranucleotides were the most frequent microsatellites in genomic DNA sequence, dinucleotide repeats, which had more repeat units than any other SSR type, had the highest cumulative sequence length. Coding regions (ESTs) of the cucumber genome had fewer microsatellites compared to its genomic sequence, with trinucleotides predominating in EST sequences. AAG was the most frequent repeat in cucumber ESTs. Overall, AT-rich motifs prevailed in both genomic and EST data. Compared to the other species examined, cucumber genomic sequence had the highest density of SSRs (although comparable to the density of poplar, grapevine and rice), and was richest in AT dinucleotides. Using an electronic PCR strategy, we investigated the polymorphism between 9930 and Gy14 at 1,006 SSR loci, and found unexpectedly high degree of polymorphism (48.3%) between the two genotypes. The level of polymorphism seems to be positively associated with the number of repeat units in the microsatellite. The <it>in silico </it>PCR results were validated empirically in 660 of the 1,006 SSR loci. In addition, primer sequences for more than 83,000 newly-discovered cucumber microsatellites, and their exact positions in the Gy14 genome assembly were made publicly available.</p> <p>Conclusions</p> <p>The cucumber genome is rich in microsatellites; AT and AAG are the most abundant repeat motifs in genomic and EST sequences of cucumber, respectively. Considering all the species investigated, some commonalities were noted, especially within the monocot and dicot groups, although the distribution of motifs and the frequency of certain repeats were characteristic of the species examined. The large number of SSR markers developed from this study should be a significant contribution to the cucurbit research community.</p

Regulation of hTERT by BCR-ABL at multiple levels in K562 cells

<p>Abstract</p> <p>Background</p> <p>The cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells.</p> <p>Methods</p> <p>Molecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels.</p> <p>Results</p> <p>Our results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited <it>hTERT </it>at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in <it>hTERT </it>gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of <it>hTERT </it>mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec.</p> <p>Conclusions</p> <p>Our data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the <it>hTERT </it>gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients.</p

Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L.

Until recently, only a few microsatellites have been available for Cucurbita, thus their development is highly desirable. The Austrian oil-pumpkin variety Gleisdorfer Ölkürbis (C. pepo subsp. pepo) and the C. moschata cultivar Soler (Puerto Rico) were used for SSR development. SSR-enriched partial genomic libraries were established and 2,400 clones were sequenced. Of these 1,058 (44%) contained an SSR at least four repeats long. Primers were designed for 532 SSRs; 500 primer pairs produced fragments of expected size. Of these, 405 (81%) amplified polymorphic fragments in a set of 12 genotypes: three C. moschata, one C. ecuadorensis, and eight C. pepo representing all eight cultivar groups. On an average, C. pepo and C. moschata produced 3.3 alleles per primer pair, showing high inter-species transferability. There were 187 SSR markers detecting polymorphism between the USA oil-pumpkin variety “Lady Godiva” (O5) and the Italian crookneck variety “Bianco Friulano” (CN), which are the parents of our previous F2 mapping population. It has been used to construct the first published C. pepo map, containing mainly RAPD and AFLP markers. Now the updated map comprises 178 SSRs, 244 AFLPs, 230 RAPDs, five SCARs, and two morphological traits (h and B). It contains 20 linkage groups with a map density of 2.9 cM. The observed genome coverage (Co) is 86.8%