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Abstract Background The 1RS arm of wheat-rye 1BL.1RS translocations contains several subtelomeric tandem repeat families. To study the effect of the difference in the composition of these tandem repeats on the meiotic recombination of 1RS arms can help to enrich the genetic diversity of 1BL.1RS translocation chromosomes. Results Five wheat-rye 1BL.1RS translocation cultivars/lines were used to build two cross combinations including group 1 (20T401 × Zhou 8425B, 20T401 × Lovrin 10 and 20T401 × Chuannong 17) and group 2 (20T360-2 × Zhou 8425B, 20T360-2 × Lovrin 10 and 20T360-2 × Chuannong 17). Oligonucleotide (oligo) probes Oligo-s120.3, Oligo-TR72, and Oligo-119.2-2 produced the same signal pattern on the 1RS arms in lines 20T401 and 20T360-2, and another signal pattern in the three cultivars Zhou 8425B, Lovrin 10 and Chuannong 17. The Oligo-pSc200 signal disappeared from the 1RS arms of the line 20T401, and the signal intensity of this probe on the 1RS arms of the line 20T360-2 was weaker than that of the three cultivars. The five cultivars/lines had the same signal pattern of the probe Oligo-pSc250. The recombination rate of 1RS arms in group 1 was significantly lower than that in group 2. In the progenies from group 1, unequal meiotic recombination in the subtelomeric pSc119.2 and pSc250 tandem repeat regions, and a 1BL.1RS with inversion of 1RS segment between the pSc200 and the nucleolar organizer region were found. Conclusions This study provides a visual tool to detect the meiotic recombination of 1RS arms. The meiotic recombination rate of 1RS arms was affected by the variation of pSc200 tandem repeat, indicating the similar composition of subtelomeric tandem repeats on these arms could increase their recombination rate. These results indicate that the 1RS subtelomeric structure will affect its recombination, and thus the localization of genes on 1RS by means of meiotic recombination might also be affected.

Abstract Background The wheat-rye 1BL.1RS translocations have played an important role in common wheat breeding programs. Subtelomeric tandem repeats have been often used to investigate polymorphisms of 1RS arms, but further research about their organizations on the 1RS chromosome is needed. Results 162 1RS arms from a wild rye species (Secale strictum) and six cultivated rye accessions (Secale cereale L.) (81 plants), 102 1BL.1RS and one 1AL.1RS translocations were investigated using oligo probes Oligo-TaiI, Oligo-pSc119.2–1, Oligo-pTa71A-2, Oligo-pSc200 and Oligo-pSc250, which were derived from tandem repeats TaiI, pSc119.2, pTa71, pSc200 and pSc250, respectively. The variations of 1RS arms were revealed by signal intensity of probes Oligo-pSc119.2–1, Oligo-pTa71A-2, Oligo-pSc200 and Oligo-pSc250. Proliferation of rDNA sequences on the 1RS chromosomes was observed. According to the presence of probe signals, 34, 127 and 144 of the 162 1RS arms contained TaiI, pSc200 and pSc250, respectively, and all of them contained pSc119.2 and pTa71. Most of the 1RS arms in rye contained three kinds of subtelomeric tandem repeats, the combination of pSc119.2, pSc200 and pSc250 was most common, and only eight of them contained TaiI, pSc119.2, pSc200 and pSc250. All of the 1RS arms in 1BL.1RS and 1AL.1RS translocations contained pSc119.2, pTa71, pSc200 and pSc250, but the presence of the TaiI family was not observed. Conclusion New organizations of subtelomeric tandem repeats on 1RS were found, and they reflected new genetic variations of 1RS arms. These 1RS arms might contain abundant allelic diversity for agricultural traits. The narrow genetic base of 1RS arms in 1BL.1RS and 1AL.1RS translocations currently used in agriculture is seriously restricting their use in wheat breeding programs. This research has found new 1RS sources for the future restructuring of 1BL.1RS translocations. The allelic variations of these 1RS arms should be studied more intensely as they may enrich the genetic diversity of 1BL.1RS translocations.

Abstract Irritable bowel syndrome (IBS) is the most common gastrointestinal disorder significantly decreasing patients’ lives of quality and placing huge economic burden on our society. Existing studies indicated that the therapeutic effects maintained for a period of time after the treatments were discontinued. It is clinically important to assess these post-treatment therapeutic effects (PTTE), which prevent IBS from relapsing. To assess the PTTE in pinaverium treatment and obtain high-quality evidence to justify the use of PTTE for long-term IBS management, we performed this controlled, double blind study on patients with IBS who were randomized to pinaverium 50 mg (n = 132) or placebo (n = 132), three times daily, for 4 weeks, and were followed up for 57 weeks after the treatments. The primary endpoints were abdominal pain and stool consistency. The secondary endpoints were pain frequency and stool frequency. The tertiary endpoints were global overall symptom and adverse events. Three days after pinaverium was discontinued, endpoints rebounded only 23.2–42.8% (P 0.05)], during which an average of 51.5–56.4% of patients (pool pinaverium and placebo data together) had IBS symptoms. These results provide clinical insights into efficient and cost-effective management of refractory IBS, and lend support to the IBS management that the selection of a therapy should consider both its effectiveness during treatment and its PTTE after the treatment. Trial registration number: NCT02330029 (16/08/2016).

Diarrhea predominant irritable bowel syndrome (IBS) is a highly relapsing gastrointestinal disorder decreasing the quality of life. Existing studies indicated that the therapeutic effects maintained for a period of time after the treatments were discontinued (post-treatment therapeutic effects or PTTE). In this study, we aim to assess the PTTE of tongxie. We performed a multiple center, controlled, double blind study of patients with IBS randomized to tongxie (n = 120) or placebo (n = 120) for 4 weeks and followed up for 57 weeks. The primary outcomes were abdominal pains and stool consistency. The secondary outcomes were pain frequency and stool frequency. Tertiary outcomes were adverse effects and global overall symptom. The outcome data were collected at days 1, 2, 3, weeks 1 and 4 during the treatment and at days 1, 2, 3, until week 57 during the post-treatment. Significantly more patients receiving tongxie were clinical responders to the primary and secondary endpoints from day 1 until the end of the treatment. The positive effects of tongxie were maintained until 17–25 weeks after tongxie was discontinued. The relapse-free probabilities in the tongxie group were significantly higher than those in the placebo group (P

To study the effects of structural alterations of chromosomes caused by tandem repeats on the meiotic recombination, the wheat (Triticum aestivum L.) 5A chromosomes with different structure from ten wheat cultivars were used to investigate their meiotic recombination using non-denaturing fluorescence in situ hybridization (ND-FISH) technology. Fifteen cross combinations were carried out and they were divided into seven F1 categories. The structural difference between the intercalary regions of the long arms of the two 5A chromosomes (5AL) in the F1 categories III, VI, and VII was greater than that in the categories I and II, subsequently, the recombination frequencies in the distal regions of the 5AL arm in the progenies from the three categories were significantly lower than that from the categories I and II. For the two 5A chromosomes in the F1 categories VI and VII, the structural differences in the distal regions of both of the two arms were greater than that in the categories IV and V. So, the recombination frequencies in the intercalary region of the 5AL arm in the progeny from the categories IV and V were higher than that in the progeny from the categories VI and VII. The breakage of 5A chromosome together with the 5A translocations and the breakage of some other chromosomes were observed in the progeny from the F1 categories V, VI, and VII. These chromosomal variations were not observed in the progenies from the other four F1 categories. In conclusion, the smaller structural difference between the 5A chromosomes in distal regions of the two arms resulted in a higher recombination frequency in interstitial region and vice versa. The 5A chromosome with complex cytological structure can be used to induce genetic variations of wheat genome.

Abstract Background DNA sequence composition affects meiotic recombination. However, the correlation between tandem repeat composition and meiotic recombination in common wheat (Triticum aestivum L.) is unclear. Results Non-denaturing fluorescent in situ hybridization (ND-FISH) with oligonucleotide (oligo) probes derived from tandem repeats and single-copy FISH were used to investigate recombination in three kinds of the long arm of wheat 5A chromosome (5AL). 5AL535–18/275 arm carries the tandem repeats pTa-535, Oligo-18, and pTa-275, 5AL119.2–18/275 arm carries the tandem repeats pSc119.2, Oligo-18 and pTa-275, and 5AL119.2 arm carries the tandem repeats pSc119.2. In the progeny of 5AL535–18/275 × 5AL119.2, double recombination occurred between pSc119.2 and pTa-535 clusters (119–535 interval), and between pTa-535 and Oligo-18/pTa-275 clusters (535–18 interval). The recombination rate in the 119–535 interval in the progeny of 5AL535–18/275 × 5AL119.2–18/275 was higher than that in the progeny of 5AL535–18/275 × 5AL119.2. Recombination in the 119–535 interval produced 5AL119 + 535 segments with pTa-535 and pSc119.2 tandem repeats and 5ALNo segments without these repeats. The 5AL119 + 535 and 5ALNo segments were localized between the signal sites of the single-copy probes SC5A-479 and SC5A-527. The segment between SC5A-479 and SC5A-527 in the metaphase 5ALNo was significantly longer than that in the metaphase 5AL119 + 535. Conclusion The structural variations caused by tandem repeats might be one of the factors affecting meiotic recombination in wheat. Meiotic recombination aggregated two kinds of tandemly repeated clusters into the same chromosome, making the metaphase chromosome more condensed. To conclude, our study provides a robust tool to measure meiotic recombination and select parents for wheat breeding programs.

Cancer induced bone pain (CIBP) occurs in patients with advanced osteosarcoma or metastasized bone tumors that can negatively affects the patient's quality of life. However, motor impairment in CIBP is still understudied. To improve the quality of life of patients with CIBP, the study of CIBP induced movement impairment is of particular importance. Here, we presented a model of metastatic cancer induced bone pain caused by an allograft of Lewis lung cancer cells. In this method, we injected Lewis lung cancer cells into the femoral medulla cavity and recorded the pain behavior and motor behavior after CIBP surgery. We observed enhanced pain after the initial surgery. Interestingly, we found the latency on rotarod was significantly reduced concomitant with tumor growth and pain. This result indicated that the motor coordination and balance were severely impaired in CIBP. We also found the pain and motor behavioral differences in models that severed the patellar ligament vs. maintaining the patellar ligament. These findings provide a novel clue for further investigating the mechanisms responsible for the generation and development of CIBP.

It was reported that the chromosome 6R of rye (Secale cereale L.) carries stripe rust resistance gene Yr83, and the region with the candidate resistance gene(s) still needs to be narrowed down. This study confirmed that the chromosome 6RLAr derived from rye AR106BONE contains stripe rust resistance gene(s). A wheat-rye T6BS.6RLAr translocation chromosome, a wheat-rye small-segment translocation T6RLAr-6AS.6AL, and three kinds of deleted T6BS.6RLAr translocations, T6BS.6RLAr-1, T6BS.6RLAr-2, and T6BS.6RLAr-3, were identified. Translocations T6BS.6RLAr, T6BS.6RLAr-2, and T6RLAr-6AS.6AL were highly resistant to stripe rust and T6BS.6RLAr-1 and T6BS.6RLAr-3 were highly susceptible. The molecular markers specific to 6RL determined that the three regions of the 6RLAr arm from 732,999,830 bp to the telomere, from 735,010,030 to 848,010,414 bp, and from 848,011,262 bp to the telomere were deleted from T6BS.6RLAr-1, T6BS.6RLAr-2, and T6BS.6RLAr-3, respectively. T6BS.6RLAr-2 and T6RLAr-6AS.6AL contained the segment that was deleted in T6BS.6RLAr-3. Therefore, it can be concluded that about 37 Mb segment from 848,011,262 bp to the telomere carried stripe rust resistance gene(s), and it was smaller than that with the Yr83 gene. Gene annotation indicated that about 37 Mb region contains 43 potential resistance genes, and 42 of them are nucleotide-binding site and leucine-rich repeat (NBS-LRR)-like resistance protein genes. The results in this study narrowed down the size of the region with candidate stripe rust resistance gene(s) on the 6RL arm, and the T6RLAr-6AS.6AL is a promising small-segment translocation for improvement of wheat cultivars.

Rye (Secale cereale L.) has been widely used to improve wheat (Triticum aestivum L.) cultivars. Oligo probes combined with non-denaturing fluorescence in situ hybridization (ND-FISH) technology provide a convenient and efficient way to identify individual rye chromosomes. However, suitable ND-FISH-positive oligo probes for recognizing specific segments of rye chromosomes are lacking. Five new ND-FISH-positive oligo probes: Oligo-5BL.46, Oligo-5A8080, Oligo-5A8080.1, Oligo-1AL.73, and Oligo-0R3, combined with two previously reported oligo probes, Oligo-44 and Oligo-45, were used in this study. Probes Oligo-5BL.46, Oligo-5A8080 and Oligo-44 produced signals only in intercalary regions of arms 1RS, 5RS, and 5RL, respectively. Probe Oligo-5A8080.1 combined with probe Oligo-45 distinguished the intercalary regions of arms 1RS, 5RS, and 6RS simultaneously. Oligo-5A8080 and Oligo-5A8080.1 revealed variation in the distribution of 5S rDNA sequences and polymorphism among 5R chromosomes. Probe Oligo-1AL.73 produced signals only on chromosomes 4R and 7R and contributed to the construction of an improved FISH map of chromosome 4RKu and to the confirmation of 4RLKu breakpoints in wheat-rye 4RLKu translocation chromosomes. Oligo-0R3 produced signals in the telomeric and subtelomeric regions of 14 rye chromosomes. These oligo probes also revealed five new tandem repeats in rye. Using the oligo probes reported in this study, the short arms of 1R, 5R, and 6R and the long arms of 4R and 7R can be easily discriminated when these chromosomes are broken. Keywords: Wheat, Rye, ND-FISH, Oligo probe, Tandem repeats

Abstract Background Introgression of chromatin from Secale species into common wheat has for decades been a successful strategy for controlling the wheat diseases. The wild Secale species, Secale africanum Stapf., is a valuable source for resistance to foliar disease of wheat. A wheat-S. africanum chromosome 6Rafr substitution line displayed resistance to both powdery mildew and stripe rust at the adult-plant stage. Results Wheat-S. africanum chromosome 6Rafr deletion and translocation lines were produced and identified by sequential non-denaturing fluorescence in situ hybridization (ND-FISH) using multiple Oligo-based probes. Different ND-FISH patterns were observed between S. cereale 6R and S. africanum 6Rafr. With reference to the physical map of the draft genome sequence of rye inbred line Lo7, a comprehensive PCR marker analysis indicated that insertions and deletions had occurred by random exchange between chromosomes 6R and 6Rafr. A survey of the wheat- S. africanum 6Rafr lines for disease resistance indicated that a powdery mildew resistance gene(s) was present on the long arm of 6Rafr at FL0.85–1.00, and that a stripe rust resistance gene(s) was located in the terminal region of 6RafrS at FL0.95–1.00. The wheat-S. africanum 6Rafr introgression lines also displayed superior agronomic traits, indicating that the chromosome 6Rafr may have little linkage drag in the wheat background. Conclusions The combination of molecular and cytogenetic methods allowed to precisely identify the chromosome rearrangements in wheat- S. africanum 6Rafr substitution, deletion and translocation lines, and compare the structural difference between chromosomes 6R and 6Rafr. The wheat- S. africanum 6Rafr lines containing gene(s) for powdery mildew and stripe rust resistance could be used as novel germplasm for wheat breeding by chromosome engineering.

Quercetin has numerous functions including antioxidant and anti-inflammatory effects. The beneficial effect of quercetin against microcystin-LR (MC-LR)-induced testicular tight junctions (TJs) defects in vitro and in vivo were investigated. Significant reductions in transepithelial electrical resistance, occludin, and zonula occludens-1(ZO-1) levels were detected in the MC-LR-treated TM4 cells, and quercetin attenuated these effects. Interestingly, quercetin suppressed MC-LR-induced phosphorylation of protein kinase B (AKT). It effectively inhibited the accumulation of reactive oxygen species (ROS) in cells stimulated by MC-LR. In addition, ROS inhibitors blocked the TJ damage that is dependent on the AKT signaling pathway induced by MC-LR. In conclusion, our results suggest that alleviates MC-LR-impaired TJs by suppressing the ROS-regulated activation of the AKT pathway.

Abstract Background Hexaploid bread wheat (Triticum aestivum L) arose by two polyploidisation events from three diploid species with homoeologous genomes. Nullisomic-tetrasomic (nulli-tetra or NT) lines are aneuploid wheat plants lacking two and adding two of six homoeologous chromosomes. These plants can grow normally, but with significantly morphological variations because the adding two chromosomes or the remaining four chromosomes compensate for those absent. Despite these interesting phenomena, detailed molecular mechanisms underlying dosage deletion and compensation in these useful genetic materials have not been determined. Results By sequencing the transcriptomes of leaves in two-week-old seedlings, we showed that the profiles of differentially expressed genes between NT stocks for homoeologous group 7 and the parent hexaploid Chinese Spring (CS) occurred throughout the whole genome with a subgenome and chromosome preference. The deletion effect of nulli-chromosomes was compensated partly by the tetra-chromosomes via the dose level of expressed genes, according to the types of homoeologous genes. The functions of differentially regulated genes primarily focused on carbon metabolic process, photosynthesis process, hormone metabolism, and responding to stimulus, and etc., which might be related to the defective phenotypes that included reductions in plant height, flag leaf length, spikelet number, and kernels per spike. Conclusions The perturbation of the expression levels of transcriptional genes among the NT stocks for homoeologous group 7 demonstrated the gene dosage effect of the subgenome at the genome-wide level. The gene dosage deletion and compensation can be used as a model to elucidate the functions of the subgenomes in modern polyploid plants.

Wheat (Triticum aestivum L.) is rich in tandem repeats, and this is helpful in studying its karyotypic evolution. Some tandem repeats have not been assembled into the wheat genome sequence. Alignment using the blastn tool in the B2DSC web server indicated that the genomic sequence of 5B chromosome (IWGSC RefSeq v2.1) does not contain the tandem repeat pTa-275, and the tandem repeat (GA)26 distributed throughout the whole 5B chromosome. The nondenaturing fluorescence in situ hybridization (ND-FISH) using the oligonucleotide (oligo) probes derived from pTa-275 and (GA)26 indicated that one signal band of pTa-275 and two signal bands of (GA)26 appeared on the 5B chromosome of Chinese Spring wheat, indicating the aggregative distribution patterns of the two kinds of tandem repeats. Single-copy FISH indicated that the clustering region of pTa-275 and the two clustering regions of (GA)26 were located in ~160–201 Mb, ~153–157 Mb, and ~201–234 Mb intervals, respectively. Using ND-FISH and single-copy FISH technologies, the translocation breakpoint on the 5BS portion of the translocation T7BS.7BL-5BS, which exists widely in north-western European wheat cultivars, was located in the region from 157,749,421 bp to 158,555,080 bp (~0.8 Mb), and this region mainly contains retrotransposons, and no gene was found. The clustering regions of two kinds of tandem repeats on wheat chromosome 5B were determined and this will be helpful to improve the future sequence assembly of this chromosome. The sequence characteristics of the translocation breakpoint on the translocation T7BS.7BL-5BS obtained in this study are helpful to understand the mechanism of wheat chromosome translocation.

Single-copy FISH analysis is a useful tool to physically locate a given sequence on chromosome. Centromeric single-copy sequences can be used to locate the position of centromere and disclose the subtle differences among different centromeres. Nine centromeric single-copy sequences 1R1, 3R1, 4R1, 4R2, 5R1, 5R2, 6R2, 6R3, and 7R1 were cloned from Kustro (Secale cereale L.). FISH analysis using these sequences as probes indicated that the signals of 1R1, 3R1, 4R1, 4R2, 5R1, 5R2, 6R1, 6R2, and 7R1 were located in the centromeric regions of rye 1R, 3R, 4R, 4R, 5R, 5R, 6R, 6R, and 7R chromosomes, respectively. In addition, for each of the centromeric single-copy sequences, high sequence similarity was observed among different Secale species. Combined with rye genomic sequence, single-copy FISH analysis indicated that the 1BL.1RS translocations in wheat cultivar CN17 and wheat line 20T363-4 contained the centromeric segment of 1R chromosome from 349,498,361 to 349,501,266 bp, and the 1BL.1RS translocations in the other two wheat cultivars did not contain this segment. The nine sequences are useful in determining the centromere location on rye chromosomes, and they have the potential to disclose the accurate structural differences of centromeres among the wheat-rye centric fusion translocation chromosomes; therefore, more centromeric single-copy sequences are needed.

Background/Aims: Opiates are potent analgesics but their clinical use is limited by sex-associated side effects, such as drug tolerance, opioid-induced hyperalgesia and withdrawal reaction. OPRM1, as the main receptor of opioids, plays an important role in the pharmacological process of opioids in rodents and human. We have previously investigated OPRM1, the μ opioid receptor gene, which have dozens of alternatively spliced variants probably correlating with opioid-induced effects in brain regions of four inbred mouse strains and demonstrated the strain-specific expressions of these splice variants. Also, within a strain, the regional expression patterns of some of the variants were similar while others were opposite. Thus, we are aiming to seek out the relationship between sex differences and these alternatively spliced variants. Methods: The present studies follow a SYBR green quantitative PCR (qPCR) which we had used before to examine the expression of OPRM1 splice variant mRNAs in selected brain regions of male and female C57BL/6 mice. Sex-associated differences in baseline latency, opioid-induced tolerance, analgesia and addiction were examined and determined by Tail-flick test, jumps and statistical analysis. Results: The mRNA levels of opioid receptor gene splice variants in male and female mice showed significant differences among the brain regions, implying region-specific alternative splicing of the OPRM1 gene, which was consistent with our previous study. More importantly, the complete mRNA expression profiles of the OPRM1 splice variants was also gender-specific, suggesting a sexual influence on OPRM1 alternative splicing. Conclusion: In brief, we put forward that the distinctions among baseline latency, opioid-induced tolerance, analgesia and physical dependence in male and female mice might correlate with sex associated differential expressions of OPRM1 gene.

Non-denaturing FISH (ND-FISH) technology has been widely used to study the chromosomes of Triticeae species because of its convenience. The oligo probes for ND-FISH analysis of wheat (Triticum aestivum L.) chromosomes are still limited. In this study, the whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of Chinese Spring (T. aestivum L.) were used to find new tandem repeats. One hundred and twenty oligo probes were designed according to the new tandem repeats and used for ND-FISH analysis of chromosomes of wheat Chinese Spring. Twenty nine of the 120 oligo probes produce clear or strong signals on wheat chromosomes. Two of the 29 oligo probes can be used to conveniently distinguish wheat A-, B-, and D-genome chromosomes. Sixteen of the 29 oligo probes only produce clear or strong signals on the subtelomeric regions of 1AS, 5AS, 7AL, 4BS, 5BS, and 3DS arms, on the telomeric regions of 1AL, 5AL, 2BS, 3BL, 6DS, and 7DL arms, on the intercalary regions of 4AL and 2DL arms, and on the pericentromeric regions of 3DL and 6DS arms. Eleven of the 29 oligo probes generate distinct signal bands on several chromosomes and they are different from those previously reported. In addition, the short and long arms of 6D chromosome have been confirmed. The new oligo probes developed in this study are useful and convenient for distinguishing wheat chromosomes or specific segments of wheat chromosomes.

Maximum tiller number and productive tiller number are important traits for wheat grain yield, but research involving the temporal expression of tiller number at different quantitative trait loci (QTL) levels is limited. In the present study, a population set of 371 recombined inbred lines derived from a cross between Chuan-Nong18 and T1208 was used to construct a high-density genetic map using a Wheat55K SNP Array and to perform dynamic QTL analysis of the tiller number at four growth stages. A high-density genetic map containing 11,583 SNP markers and 59 SSR markers that spanned 4,513.95 cM and was distributed across 21 wheat chromosomes was constructed. A total of 28 single environmental QTL were identified in the recombined inbred lines population, and among these, seven QTL were stable and used for multi-environmental and dynamic analysis. These QTL were mapped to chromosomes 2D, 4A, 4D, 5A, 5D, and 7D, respectively. Each QTL explained 1.63–21.22% of the observed phenotypic variation, with an additive effect from -20.51 to 11.59. Dynamic analysis showed that cqTN-2D.2 can be detected at four growth stages of tillering, explaining 4.92–17.16% of the observed phenotypic variations and spanning 13.71 Mb (AX-109283238-AX-110544009: 82189047-95895626) according to the physical location of the flanking markers. The effects of the stable QTL were validated in the recombined inbred lines population, and the beneficial alleles could be utilized in future marker-assisted selection. Several candidate genes for MTN and PTN were predicted. The results provide a better understanding of the QTL selectively expressing the control of tiller number and will facilitate future map-based cloning. 9.17% SNP markers showed best hits to the Chinese Spring contigs. It was indicated that Wheat55K Array was efficient and valid to construct a high-density wheat genetic map.

Neuropathic pain is a chronic pain and reduces the life quality of patients substantially. Transient receptor potential vanilloid channel 1 (TRPV1), a nonselective cation channel, has been shown to play a crucial role in neuropathic pain. Although TRPV1 plays an important role in neuropathic pain, the mechanism of how TRPV1 was regulated in neuropathic pain remains unclear. Pirt is a membrane protein and binds to TRPV1 to enhance its activity. It was suggested that Pirt should also be involved in neuropathic pain. In this study, we investigated the role of Pirt in neuropathic pain (CCI model); the results show that mechanical allodynia and thermal hyperalgesia were alleviated in Pirt−/− mice in CCI models. TRPV1 expression was increased by immunofluorescence and real-time PCR experiments. The increase in TRPV1 expression was less in Pirt knockout mice in CCI models. Moreover, the number of capsaicin-responding neurons and the magnitude of evoked calcium response were attenuated in DRG neurons from Pirt−/− mice in CCI models. Finally, we found that the pain behavior attenuated in dysfunction of both Pirt and TRPV1 was much stronger than in dysfunction of Pirt or TRPV1 only in a CCI model in vitro study. Taken together, Pirt together with TRPV1 is involved in CCI-induced neuropathic pain.

Rye has been used worldwide as a source for the genetic improvement of wheat. In this study, two stable wheat-rye primary T1RS.1BL translocation lines were selected from the progeny of the crossing of the wheat cultivar Mianyang11-1 and a Chinese local rye variety, Weining. These two novel translocation lines were identified by molecular cytogenetic analysis. PCR results, multi-color fluorescence in situ hybridization (MC-FISH), and acid polyacrylamide gel electrophoresis (A-PAGE) indicated that both new translocation lines harbor a pair of T1RS.1BL translocation chromosomes, and have been named RT828-10 and RT828-11, respectively. The cytogenetic results also indicated that the pSc119.2 signals of 5AL were absent in both lines along with the pSc119.2 signals of 4AL of RT828-11. When inoculated with different stripe rust and powdery mildew isolates, both lines expressed high resistance to Puccinia striiformis f. sp. tritici and Blumeria graminis f. sp. tritici pathotypes, which are prevalent in China and are virulent on Yr9 and Pm8. The line RT828-11 also exhibited excellent agronomic traits in the field. The present study indicates that this rye variety may carry untapped variations that could potentially be used for wheat improvement.

Frankincense and myrrh are widely used in clinics as a pair of herbs to obtain a synergistic effect for relieving pain. To illuminate the analgesia mechanism of frankincense and myrrh, we assessed its effect in a neuropathic pain mouse model. Transient receptor potential vanilloid 1 (TRPV1) plays a crucial role in neuropathic pain and influences the plasticity of neuronal connectivity. We hypothesized that the water extraction of frankincense and myrrh (WFM) exerted its analgesia effect by modulating the neuronal function of TRPV1. In our study, WFM was verified by UHPLC-TQ/MS assay. In vivo study showed that nociceptive response in mouse by heat and capsaicin induced were relieved by WFM treatment. Furthermore, thermal hypersensitivity and mechanical allodynia were also alleviated by WFM treatment in a chronic constriction injury (CCI) mouse model. CCI resulted in increased TRPV1 expression at both the mRNA and protein levels in predominantly small-to-medium neurons. However, after WFM treatment, TRPV1 expression was reverted in real-time PCR, Western blot, and immunofluorescence experiments. Calcium response to capsaicin was also decreased in cultured DRG neurons from CCI model mouse after WFM treatment. In conclusion, WFM alleviated CCI-induced mechanical allodynia and thermal hypersensitivity via modulating TRPV1.

The rye (Secale cereale L.) 5R chromosome contains some elite genes that can be used to improve wheat cultivars. In this study, a set of 5RKu dissection lines was obtained, and 111 new PCR-based and 5RKu-specific markers were developed using the specific length amplified fragment sequencing (SLAF-seq) method. The 111 markers were combined with the 52 5RKu-specific markers previously reported, and 65 S. cereale Lo7 scaffolds were physically mapped to six regions of the 5RKu chromosome using the 5RKu dissection lines. Additionally, the 5RLKu arm carried stripe rust resistance gene(s) and it was located to the region L2, the same region where 22 5RKu-specific markers and 11 S. cereale Lo7 scaffolds were mapped. The stripe rust resistance gene(s) located in the 5RLKu arm might be new one(s) because its source and location are different from the previously reported ones, and it enriches the resistance source of stripe rust for wheat breeding programs. The markers and the S. cereale Lo7 scaffolds that were mapped to the six regions of the 5RKu chromosome can facilitate the utilization of elite genes on the 5R chromosome in the improvement of wheat cultivars.

Non-denaturing fluorescence in situ hybridization (ND-FISH) has been used to distinguish wheat chromosomes and to detect alien chromosomes in the wheat genome. In this study, five different oligonucleotide probes were used with ND-FISH to examine 21 wheat cultivars and lines. These oligonucleotide probes distinguished 42 wheat chromosomes and also detected rye chromatin in the wheat genome. Moreover, the signal patterns of the oligonucleotide probes Oligo-pTa535-1 and Oligo-pSc119.2-1 showed high polymorphism in the wheat chromosomes. A total of 17.6% of the A group chromosomes, 25.9% of the B group chromosomes and 8.9% of the D group chromosomes showed obvious mutations when they were compared to the standard ND-FISH signal patterns, and most of them were Oligo-pSc119.2-1 mutants. The results suggested that these polymorphisms could be induced by the crossing of wheat cultivars. The results provided more information for the further application of oligonucleotide probes and ND-FISH.

Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG) neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip)—a histamine H4 receptor special agonist under cutaneous injection—obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3–50 μM) could also induce a dose-dependent increase in intracellular Ca2+ (Ca2+i) of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca2+ responses. In addition, immepip-induced Ca2+i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons’ responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation.

Secale cereale L. has been used worldwide as a source of genes for agronomic and resistance improvement. In this study, a stable wheat-rye substitution line and 3 primary 1RS.1BL translocation lines were selected from the progeny of the crossing of the Chinese local rye Aigan variety and wheat cultivar Mianyang11. The substitution and translocation lines were identified by molecular cytogenetic analysis. PCR results, fluorescence in situ hybridization and acid polyacrylamide gel electrophoresis indicated that there were a pair of 1R chromosomes in the substitution line which have been named RS1200-3, and a pair of 1RS.1BL translocation chromosomes in the other 3 translocation lines, which have been named RT1163-4, RT1217-1, and RT1249. When inoculated with stripe rust isolates, these 4 lines expressed high resistance to several Puccinia striiformis f. sp Tritici pathotypes that are virulent on Yr9. Moreover, the different response pattern of resistance among them suggested that the diversity of resistance genes for wheat stripe rust exists in the rye. These 4 lines also showed better agronomic performances than their wheat parent. The GS indices also showed the genetic diversity of the 1RS which derived from same rye variety. The present study indicates that rye cultivars may carry untapped variations that could potentially be used for wheat improvement.

Oligonucleotide probes and the non-denaturing fluorescence in situ hybridization (ND-FISH) technique are widely used to analyze plant chromosomes because they are convenient tools. New oligonucleotide probes, Oligo-Ku, Oligo-3B117.1, Oligo-3B117.2, Oligo-3B117.2.1, Oligo-3B117.3, Oligo-3B117.4, Oligo-3B117.5, Oligo-3B117.6, Oligo-pTa71A-1, Oligo-pTa71A-2, Oligo-pTa71B-1, Oligo-pTa71B-2, Oligo-pTa71C-1, Oligo-pTa71C-2, Oligo-pTa71C-3 and Oligo-pTa71D were designed based on the repetitive sequences KU.D15.15, pSc119.2-like sequence 3B117 and pTa71. Oligonucleotide probe (GT)7 was also used. Oligo-Ku and (GT)7 can be together used to identify Dasypyrum villosum from wheat chromosomes and to distinguish individual D. villosum chromosomes. The oligonucleotide probes that were derived from the same repeat sequence displayed different signal intensity and hybridization sites on the same chromosomes. Both the length and the nucleotide composition of oligonucleotide probes determined their signal intensity. For example, Oligo-3B117.2 (25 bp) and Oligo-pTa71A-2 (46 bp) produced the strongest signals on chromosomes of wheat (Triticum aestivum L.), rye (Secale cereale L.), barley (Hordeum vulgare ssp. vulgare) or D. villosum, the signal of Oligo-3B117.4 (18 bp) on the short arm of 7B chromosome was weaker than that of Oligo-3B117.2.1 (15 bp) and Oligo-3B117.3 (16 bp), and Oligo-pTa71A-1 (38 bp) produced the same strong signals as Oligo-pTa71A-2 did on 1B and 6B chromosomes, but its signals on 1R and 1V chromosomes were weaker than the ones of Oligo-pTa71A-2. Oligonucleotide probes and ND-FISH analysis can reflect the distribution and structural statues of different segments of tandem repeats on chromosomes. The possible reasons why different segments derived from the same repeat sequence produced different signal patterns are discussed.

Chromosomal rearrangements induced by wheat-rye hybridization is a very well investigated research topic. However, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported.Octoploid triticale lines were derived from common wheat Triticum. aestivum L. 'Mianyang11'×rye Secale cereale L. 'Kustro'. Some progeny were obtained by the controlled backcrossing of triticale with 'Mianyang11' and common wheat T. aestivum L. 'Chuannong27' followed by self-fertilization. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and rye genomic DNA as probes were used to analyze the mitotic chromosomes of these progeny. Alterations of wheat chromosomes including 5A, 6A, 1B, 2B, 6B, 7B, 1D, 3D and 7D were observed. 5AL arm carrying intercalary Oligo-pSc119.2-1, Oligo-pTa535-1 or both Oligo-pSc119.2-1 and Oligo-pTa535-1 signals, 6AS, 1BS and 1DL arms containing terminal Oligo-pSc119.2-1 signal, 6BS and 3DS arms without terminal Oligo-pSc119.2-1 signal, 7BS without subtelomeric Oligo-pSc119.2-1 signal and 7DL with intercalary Oligo-pSc119.2-1 signal have been observed. However, these changed wheat chromosomes have not been detected in 'Mianyang11' and Chuannong 27. The altered 5A, 6A, 7B and 7D chromosomes in this study have not been reported and represent several new karyotype structures of common wheat chromosomes.These rearranged wheat chromosomes in the present study afford some new genetic variations for wheat breeding program and are valuable materials for studying the biological function of tandem repetitive DNA sequences.

BACKGROUND: Wheat-rye addition lines are an old topic. However, the alterations and abnormal mitotic behaviours of wheat chromosomes caused by wheat-rye monosomic addition lines are seldom reported. METHODOLOGY/PRINCIPAL FINDINGS: Octoploid triticale was derived from common wheat T. aestivum L. 'Mianyang11'×rye S. cereale L. 'Kustro' and some progeny were obtained by the controlled backcrossing of triticale with 'Mianyang11' followed by self-fertilization. Genomic in situ hybridization (GISH) using rye genomic DNA and fluorescence in situ hybridization (FISH) using repetitive sequences pAs1 and pSc119.2 as probes were used to analyze the mitotic chromosomes of these progeny. Strong pSc119.2 FISH signals could be observed at the telomeric regions of 3DS arms in 'Mianyang11'. However, the pSc119.2 FISH signals were disappeared from the selfed progeny of 4R monosomic addition line and the changed 3D chromosomes could be transmitted to next generation stably. In one of the selfed progeny of 7R monosomic addition line, one 2D chromosome was broken and three 4A chromosomes were observed. In the selfed progeny of 6R monosomic addition line, structural variation and abnormal mitotic behaviour of 3D chromosome were detected. Additionally, 1A and 4B chromosomes were eliminated from some of the progeny of 6R monosomic addition line. CONCLUSIONS/SIGNIFICANCE: These results indicated that single rye chromosome added to wheat might cause alterations and abnormal mitotic behaviours of wheat chromosomes and it is possible that the stress caused by single alien chromosome might be one of the factors that induced karyotype alteration of wheat.

BACKGROUND: Monosomic alien addition lines (MAALs) can easily induce structural variation of chromosomes and have been used in crop breeding; however, it is unclear whether MAALs will induce drastic genetic and epigenetic alterations. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, wheat-rye 2R and 5R MAALs together with their selfed progeny and parental common wheat were investigated through amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) analyses. The MAALs in different generations displayed different genetic variations. Some progeny that only contained 42 wheat chromosomes showed great genetic/epigenetic alterations. Cryptic rye chromatin has introgressed into the wheat genome. However, one of the progeny that contained cryptic rye chromatin did not display outstanding genetic/epigenetic variation. 78 and 49 sequences were cloned from changed AFLP and MSAP bands, respectively. Blastn search indicated that almost half of them showed no significant similarity to known sequences. Retrotransposons were mainly involved in genetic and epigenetic variations. Genetic variations basically affected Gypsy-like retrotransposons, whereas epigenetic alterations affected Copia-like and Gypsy-like retrotransposons equally. Genetic and epigenetic variations seldom affected low-copy coding DNA sequences. CONCLUSIONS/SIGNIFICANCE: The results in the present study provided direct evidence to illustrate that monosomic wheat-rye addition lines could induce different and drastic genetic/epigenetic variations and these variations might not be caused by introgression of rye chromatins into wheat. Therefore, MAALs may be directly used as an effective means to broaden the genetic diversity of common wheat.