Whole-genome sequencing enables molecular dissection and candidate gene identification of the rust resistance gene R12 in sunflower (Helianthus annuus L.).

KEY MESSAGE: We finely mapped the rust resistance gene R12 to a 0.1248-cM region, identified a potential R12 candidate gene in the XRQ reference genome, and developed three diagnostic SNP markers for R12. Rust is a devastating disease in sunflower that is damaging to the sunflower production globally. Identification and utilization of host-plant resistance are proven to be preferable means for disease control. The rust resistance gene R12 with broad-spectrum specificity to rust was previously localized to a 2.4 Mb region on sunflower chromosome 11. To understand the molecular mechanism of resistance, we conducted whole-genome sequencing of RHA 464 (R12 donor line) and reference genome-based fine mapping of the gene R12. Overall, the 213 markers including 186 SNPs and 27 SSRs' were identified from RHA 464 sequences and used to survey polymorphisms between the parents HA 89 and RHA 464. Saturation mapping identified 26 new markers positioned in the R12 region, and fine mapping with a large population of 2004 individuals positioned R12 at a genetic distance of 0.1248 cM flanked by SNP markers C11_150451336 and S11_189205190. One gene, HanXRQChr11g0348661, with a defense-related NB-ARC-LRR domain, was identified in the XRQr1.0 genome assembly in the R12 region; it is predicted to be a potential R12 candidate gene. Comparative analysis clearly distinguished R12 from the rust R14 gene located in the vicinity of the R12 gene on chromosome 11. Three diagnostic SNP markers, C11_147181749, C11_147312085, and C11_149085167, specific for R12 were developed in the current study, facilitating more accurate and efficient selection in sunflower rust resistance breeding. The current study provides a new genetic resource and starting point for cloning R12 in the future.

[Combined application of immunohistochemical markers to identify pathologic subtypes of ampullary carcinoma and its clinical significance].

Objective: To investigate the expression of immunomarkers CK7, CK20, CK17, CDX2, MUC1 and MUC2 in primary adenocarcinoma of the ampulla of Vater, to explore the role of these markers in the histopathologic subclassification of ampullary carcinoma; and to provide biologic basis for precision treatment of patients with different types of ampullary carcinoma. Methods: Forty-two cases of primary ampullary carcinoma were collected at Peking University People's Hospital, from 2012 to 2018 year. There were 22 males and 20 females. Aged range 42 to 88 years old, with mean aged (62±11) years. Among the patients, 6 was high differentiation, 19 median differentiation, and 17 low differentiation. Immunohistochemical studies on the expression of CK7, CK20, CK17, CDX2, MUC1 and MUC2 were performed in 42 cases of primary ampullary carcinoma. The relationship between different ampullary carcinoma subtypes and clinicopathologic survival data was analyzed using SPSS 16.0 statistical software. Results: Three histopathologic subtypes were observed. Among 42 cases, 8(19.0%)were classified as intestinal subtype, which showed a positive expression rate of 8/8 for both CK20 and CDX2, and 5/8 for MUC2. Both CK7 and CK17 were weakly expressed in one case (1/8). No expression was observed for MUC1 in this subtype. Twenty-two (52.4%,22/42) cases were classified as pancreaticobiliary subtype, which showed a positive expression rate of 100.0%(22/22) for both CK7 and MUC1, and 90.9% (20/22) for CK17. No expression was observed for CK20, CDX2 and MUC2.The remaining 12 (28.6%) cases were classified as mixed subtype, which showed variable expression patterns. The expression frequencies of these 6 immunomarkers in different subtypes of ampullary carcinoma did not correlate with various clinicopathologic factors such as patient gender and age, tumor size, histologic differentiation, pancreatic and bile duct invasion, or the depth of duodenal invasion. However, stage Ⅲ+Ⅳ diseases were more commonly seen in pancreaticobiliary type (63.6%,14/22) than intestinal type (2/8) and mixed type (3/9; χ(2)=6.508, P=0.039). Follow-up data showed a trend of better survival rate for patients with intestinal subtype than those with mixed and pancreaticobiliary subtypes. Conclusions: Ampullary carcinoma can be subclassified into three different subtypes using a panel of six immunomarkers, especially for the identification of subtypes of poorly differentiated carcinoma. CK7, CK17 and MUC1 are major markers of pancreaticobiliary subtype, whereas CK20, CDX2 and MUC2 are useful markers for intestinal subtype. The mixed subtype variably expresses these markers. The prognosis of patients with intestinal subtype appears better than that of pancreaticobiliary and mixed subtypes. Accurate subtyping of ampullary carcinoma is clinically important to patient management and prognosis assessment.

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, R 15 , in sunflower (Helianthus annuus L.).

KEY MESSAGE: A novel rust resistance gene, R 15 , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R 15 were identified, facilitating marker-assisted selection of resistance genes. The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F2 individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R 15 ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R 15 was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

[Analysis of proliferative lesions of haematopoietic and lymphoid tissue in the female productive tract].

Objective: To study the clinicopathologic features, diagnosis and differential diagnosis of tumors of haematopoietic and lymphoid tissue in the female productive tract. Methods: Eleven cases of myeloid sarcoma and leukemia, 9 of non Hodgkin lymphoma (NHL) , 13 of cervical lymphoma-like lesions were selected from Peking University People's Hospital from January 2006 to August 2017. According to WHO classification of tumors of haematopoietic and lymphoid tissues (2008) and updated classification(2016), the cases were studied by microscopy, immunohistochemistry and in situ hybridization. Results: In 20 cases of tumors of haematopoietic and lymphoid tissue, the mean and median age was 48.5 and 56 years old (range: 16-77 years old) . In cases of lymphoma-like lesion of uterine cervix, the mean and median age was 45.9 and 48 years old (range: 23-62 years old) . The patients with neoplasm present as fever, fatigue, hypogastralgia, colporrhagia and mass etc. Eight cases had history of acute myeloid leukemia, and 3 had myeloid leukemia while pregnancy. One case of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) had history of ovary small cell carcinoma and high grade serous carcinoma resected with chemotherapy. One case of diffuse large B cell lymphoma (DLBCL) had history of renal transplantation. Lactic dehydrogenase (LDH) was elevated in 9 cases (9/18) . The cases of lymphoma-like lesion present as contact bleeding in most cases and all located in cervix. Four cases of neoplasm located in vulva, 1 in vagina, 4 in cervix, 4 in uterine corpus, 8 in ovary and 2 in placenta. Clinical staging of NHL: 4 case was stageⅠ, 1 case of stageⅢ, and 4 cases of stage Ⅳ. Pathological morphology: 9 cases were myeloid sarcoma, 2 cases were placenta invaded by myeloid leukemia. Six cases were DLBCL, and 1 case was CLL/SLL, 1 case was mucosa associated lymphoid tissuse lymphoma (MALToma) , and 1 case was anaplastic large cell lymphoma. Resected mass, chemotherapy was performed in tumors of haematopoietic and lymphoid tissue. Five cases of myeloid sarcoma and 2 of NHL died. In 13 cases of lymphoma-like lesion of uterine cervix, the general condition was good as following up. Conclusions: The clinical history, pathological morphology and immunohistochemistry are very important for diagnosing tumors of haematopoietic and lymphoid tissue in the female productive tract. Resection with chemotherapy is recommended in treatment. The prognosis of lymphoma-like lesion of uterine cervix is good, and should be differentiated from lymphoma.

Genotyping-by-sequencing targeting of a novel downy mildew resistance gene Pl 20 from wild Helianthus argophyllus for sunflower (Helianthus annuus L.).

KEY MESSAGE: Genotyping-by-sequencing revealed a new downy mildew resistance gene, Pl 20 , from wild Helianthus argophyllus located on linkage group 8 of the sunflower genome and closely linked to SNP markers that facilitate the marker-assisted selection of resistance genes. Downy mildew (DM), caused by Plasmopara halstedii, is one of the most devastating and yield-limiting diseases of sunflower. Downy mildew resistance identified in wild Helianthus argophyllus accession PI 494578 was determined to be effective against the predominant and virulent races of P. halstedii occurring in the United States. The evaluation of 114 BC1F2:3 families derived from the cross between HA 89 and PI 494578 against P. halstedii race 734 revealed that single dominant gene controls downy mildew resistance in the population. Genotyping-by-sequencing analysis conducted in the BC1F2 population indicated that the DM resistance gene derived from wild H. argophyllus PI 494578 is located on the upper end of the linkage group (LG) 8 of the sunflower genome, as was determined single nucleotide polymorphism (SNP) markers associated with DM resistance. Analysis of 11 additional SNP markers previously mapped to this region revealed that the resistance gene, named Pl 20 , co-segregated with four markers, SFW02745, SFW09076, S8_11272025, and S8_11272046, and is flanked by SFW04358 and S8_100385559 at an interval of 1.8 cM. The newly discovered P. halstedii resistance gene has been introgressed from wild species into cultivated sunflower to provide a novel gene with DM resistance. The homozygous resistant individuals were selected from BC2F2 progenies with the use of markers linked to the Pl 20 gene, and these lines should benefit the sunflower community for Helianthus improvement.

[Tumors of lymphoid and hematopoietic tissue of spleen: a clinicopathologic analysis of 53 cases].

Objective: To study the clinicopathologic features, diagnosis and differential diagnosis of the tumors of lymphoidand hematopoietic tissue of the spleen(TLTS). Methods: Fifty-three cases of TLTS were selected from the pathologic files from Peking University People's Hospital from April 2002 to April 2017. According to WHO classification of tumors of hematopoietic and lymphoid tissues (2008) and its updated classification (2016), the cases were studied by microscopy, immunohistochemistry and in situ hybridization, combined with the bone marrow biopsy and clinical examination. Results: In 53 cases of TLTS, the male to female ratio was 3.4∶1.0; the mean age was 55.4 years (range 21-76 years), and all patients presented with variable degree of splenomegaly. Laboratory examination showed increased percentage of lymphocyte in peripheral blood in 22 cases, and elevated serum LDH level in 24 cases. Abnormal blood counts were seen in 26 cases pre-operatively, in which 22 cases showed complete or partial correction of these abnormalities post-operatively (84.6%, 22/26). The clinical symptoms included abdominal pain or distension, fatigue, fever, and weight loss, etc. Seventeen cases presented with lymphadenopathy of abdomen or other sites. Bone marrow biopsy was performed in 30 cases, and 19 cases were involved by tumor (63.3%). Of all 53 cases, 43 were diagnosed as primary splenic lymphoma (PSL), and the remaining 10 cases as secondary TLTS. According to Ann Arbor staging, 14 cases were stages Ⅰ or Ⅱ, 6 were stage Ⅲ and 28 were stage Ⅳ. By histopathologic classification, 43 cases of PSL were splenic B-cell marginal zone lymphoma (SMZL; 48.8%, 21/43), diffuse large B cell lymphoma (DLBCL; 23.3%, 10/43), splenic diffuse red pulp small B-cell lymphoma (11.6%, 5/43), mantle cell lymphoma (9.3%, 4/43), follicular lymphoma (4.7%, 2/43), and composite lymphoma (CL, DLBCL and classical Hodgkin lymphoma; 2.3%, 1/43). The remaining 10 cases were chronic lymphocytic leukaemia/small lymphocytic lymphoma (4 cases), hairy cell leukaemia (1 case), hepatosplenic T-cell lymphoma (HSTL; 5 cases), with lesions in other sites. Of the 53 cases of TLTS, 47 were B cell neoplasm (88.7%, 47/53), and the T cell neoplasms were all HSTL(5 cases, 9.4%, 5/53), 1 case was composite lymphoma. In 11 cases of TLTS, EBER in situ hybridization was performed and all cases were negative. Forty eight cases had follow-up data, and the median survival period was 17.0 months(range: 1-96 months). The survival of patients with SMZL and DLBCL were 25.7 and 18.6 months respectively. Thirteen patients died (27.1%, 13/48). The prognosis of those with elevated LDH level, high clinical stage, B symptoms and older than 60 years of age was worse. And the prognosis of DLBCL was worse than that of SMZL. There was no statistically significant difference between these factors and prognosis (P>0.05). Conclusions: Most TLTS cases present with splenomegaly and abnormal blood counts, and complete or partial remission of blood counts isseen after splenectomy. The most common pathologic types of TLTS are SMZL and DLBCL. Definite diagnosis of TLTS could be made by combining clinical features, histopathology, immunophenotype, genetics, bone marrow biopsy and laboratory examination.

New approaches in the diagnosis and prognosis of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is the most common pregnancy metabolic disorder in which a person with no history of hyperglycemia exhibits any degree of impaired glucose tolerance during gestation. GDM can be resolved on its own after birth, but mothers with GDM are more at risk for future problems, such as type 2 diabetes, obesity, and cardiovascular disease. In addition, GDM can cause macrosomia in infants and obesity or even the risk of diabetes in childhood. Standard diagnostic tests for GDM are the oral glucose tolerance test (OGTT) and glucose challenge test (GCT), which is a mandatory test at 28-28 weeks of pregnancy in most countries. Disorders in various molecular mechanisms, such as hepatocyte growth factor (HGF), mechanistic target of rapamycin (mTOR), and nuclear factor-kappaB (NF-κB) signaling pathways are involved in GDM. Therefore, a better understanding of these mechanisms can help find new therapeutic and diagnostic strategies accordingly. In this review, we first deal with molecular mechanisms involved in GDM occurrence and then summarized the studies that hired this knowledge for early diagnosis and prognosis of GDM. Finally, we present the latest achievements in the diagnosis of GDM based on exosomes, microRNAs, glycosylated hemoglobin, and inflammatory factors detection in maternal circulation.

A New Biotype of Phaeosphaeria sp. of Uncertain Affinity Causing Stagonospora Leaf Blotch Disease in Cereals in Poland.

A new Phaeosphaeria sp. biotype was isolated from winter ryes in Poland during 1995. Two isolates, Sn23-1 and Sn48-1, were obtained from diseased leaves of cvs. Motto and Dankowskie, respectively. The rye Phaeosphaeria sp. represented by isolate Sn48-1 has similar pycnidiospore morphology and induces disease symptoms in cereals similar to Phaeosphaeria nodorum, the causal agent of Stagonospora nodorum blotch disease (4). The pathogen (Sn48-1) produces hyaline, cylindrical pycnidiospores that are mostly three-septate and measure 12.8 to 23.7 × 2.1 to 3.2 µm (average size = 16 × 2.6 µm) on water agar. A molecular comparison of several genes in isolates Sn23-1 and Sn48-1 revealed that the rye Phaeosphaeria sp. was different from P. nodorum. In the conserved alpha-box sequence (1,93 bp) of the MAT1-1 gene, a four nucleotide difference occurred between the wheat-biotype P. nodorum and isolates Sn23-1 and Sn48-1 (GenBank Accession Nos. AY072933 and AF322008). In addition, the length of the internal transcribed spacer (ITS) region of the nuclear rDNA was the same for the wheat-biotype P. nodorum and the two rye Phaeosphaeria sp. isolates. However, a six nucleotide discrepancy was found in the ITS region (GenBank Accession Nos. U77362 and AF321323). The beta-glucosidase (bgl1) and beta-tubulin (tubA) genes differ in length between the wheat-biotype P. nodorum and two rye Phaeosphaeria sp. isolates (2,3). The main difference was due to the intron sizes of these two genes. One extra nucleotide was found in the intron2 of the bgl1 gene (GenBank Accession Nos. AY683619 and AY683620) and the intron1 of the tubA gene (GenBank Accession Nos. AY786337 and AY786331), respectively, in these two rye Phaeosphaeria sp. isolates. Disease severity on the fifth leaf (GS15) of Polish wheat (Alba, Begra, and Liwilla), triticale (Bogo and Pinokio), and rye (Zduno) cultivars was assessed with one (resistant) to nine (susceptible) scales 14 days after inoculation. Aggressiveness of wheat-biotype P. nodorum isolate Sn26-1 and rye Phaeosphaeria sp. isolate Sn48-1 was significant (P < 0.01) in five cultivars except in the moderately resistant wheat cv. Liwilla. The rye Phaeosphaeria sp. isolate Sn48-1 severely affected Polish rye Zduno (8.3) and two triticale cultivars (6.5), while the infection by isolate Sn26-1 was moderate (3-4). On the contrary, the wheat-biotype P. nodorum isolate Sn26-1 was more aggressive on wheat (4.1 on moderately resistant Alba and 6.2 on highly susceptible Begra) than the rye Phaeosphaeria sp. isolate Sn48-1, which had a scale of 2.2 and 4.3, respectively. Under laboratory conditions, the rye isolate Sn48-1 was able to cross with the wheat-biotype P. nodorum isolate Sn26-1 that has an opposite mating-type (MAT1-2) gene, but few viable ascospores were produced (1). References: (1) P. C. Czembor and E. Arseniuk. Mycol. Res. 104:919, 2000. (2) A. Malkus et al. FEMS (Fed. Eur. Microbiol. Soc.) Lett. 249:49, 2005. (3) E. Reszka et al. Can. J. Bot. 83:1001, 2005. (4) M. J. Richardson and M. Noble. Plant Pathol. 19:159, 1970.

Transformation of microcystins to 2-methyl-3-methoxy-4-phenylbutyric acid by room temperature ozone oxidation for rapid quantification of total microcystins.

Microcystins (MCs) are cyanobacterial hepatotoxins capable of accumulation into animal tissues. To determine the total microcystins in water, a novel analytical method, including ozonolysis, methylation of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) with methylchloroformate (MCF) and gas chromatography mass spectrometry (GC-MS) detection was developed. The results show that MCs can be oxidized by ozone to produce MMPB at ambient temperature, proving ozonation is an effective, rapid and green method for the transformation of MCs to MMPB without secondary pollution. The oxidation conditions as well as the esterification process were optimized and, subsequently applied to analysis of environmental samples. The method shows wide linear range and high sensitivity with a detection limit of 0.34 µg L(-1). The established method was successfully applied to the analysis of microcystins in water samples.

Single-nucleotide polymorphisms in soybean.

Single-nucleotide polymorphisms (SNPs) provide an abundant source of DNA polymorphisms in a number of eukaryotic species. Information on the frequency, nature, and distribution of SNPs in plant genomes is limited. Thus, our objectives were (1) to determine SNP frequency in coding and noncoding soybean (Glycine max L. Merr.) DNA sequence amplified from genomic DNA using PCR primers designed to complete genes, cDNAs, and random genomic sequence; (2) to characterize haplotype variation in these sequences; and (3) to provide initial estimates of linkage disequilibrium (LD) in soybean. Approximately 28.7 kbp of coding sequence, 37.9 kbp of noncoding perigenic DNA, and 9.7 kbp of random noncoding genomic DNA were sequenced in each of 25 diverse soybean genotypes. Over the >76 kbp, mean nucleotide diversity expressed as Watterson's theta was 0.00097. Nucleotide diversity was 0.00053 and 0.00111 in coding and in noncoding perigenic DNA, respectively, lower than estimates in the autogamous model species Arabidopsis thaliana. Haplotype analysis of SNP-containing fragments revealed a deficiency of haplotypes vs. the number that would be anticipated at linkage equilibrium. In 49 fragments with three or more SNPs, five haplotypes were present in one fragment while four or less were present in the remaining 48, thereby supporting the suggestion of relatively limited genetic variation in cultivated soybean. Squared allele-frequency correlations (r(2)) among haplotypes at 54 loci with two or more SNPs indicated low genome-wide LD. The low level of LD and the limited haplotype diversity suggested that the genome of any given soybean accession is a mosaic of three or four haplotypes. To facilitate SNP discovery and the development of a transcript map, subsets of four to six diverse genotypes, whose sequence analysis would permit the discovery of at least 75% of all SNPs present in the 25 genotypes as well as 90% of the common (frequency >0.10) SNPs, were identified.

Characterization of trinucleotide SSR motifs in wheat.

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer "stutter bands" as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat ( Triticum aestivumL.). Four genomic libraries of cultivar 'Chinese Spring' were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) (n), (GGA/CCT) (n), (TAA/ATT) (n), (CAA/GTT) (n), (GGT/CCA) (n), (CAT/GTA) (n), (CGA/GCT) (n), (CTA/GAT) (n), and (CGT/GCA) (n) repeats were estimated to be 5.4x10(4), 3.5x10(4), 3.2x10(4), 1.2x10(4), 6.3x10(3), 4.9x10(3), 4.5x10(3), 4.5x10(3) and 3.6x10(3), i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) (n), 30 (43%) (CTT/GAA) (n), 16 (59%) (CAA/GTT) (n), 3 (27%) (CAT/GTA) (n) and 2 (4%) (GGA/CCT) (n) clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) (n), (CTT/GAA) (n) and (CAA/GTT) (n) microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) (n), four (14.8%) to (CTT/GAA) (n), and two (12.5%) to (CAA/GTT) (n) resulted in polymorphic markers. The results indicated that (TAA/ATT) (n) microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat.

Improvement of preservation with cardioplegia induced by heat stress is mediated by calcitonin gene-related peptide.

Previous studies have shown improvement of preservation with cardioplegia by calcitonin gene-related peptide (CGRP)-induced preconditioning. Therefore we examined the hypothesis that endogenous CGRP may be involved in the protection of heat stress against myocardial damages after prolonged cardioplegic arrest in isolated rat heart. Reperfusion after 4 h of hypothermic ischemia caused a decline of cardiac function and an increase of creatine kinase (CK) release. Heat stress induced by pretreatment with whole body hyperthermia (rectal 42 degrees C) for 15 min produced a significant increase in the plasma content of CGRP, an improvement of cardiac function and a decrease in the release of CK. However, after pretreatment with capsaicin (50 mg/kg, s.c.) to deplete CGRP in cardiac sensory nerves, the plasma concentration of CGRP was no longer increased and the cardioprotection afforded by heat stress was abolished. These findings suggest that improvement of preservation with cardioplegia by heat stress may be mediated by endogenous CGRP in the rat.

Early and delayed cardioprotection by heat stress is mediated by calcitonin gene-related peptide.

Brief ischaemia or heat stress protects the myocardium against ischaemia-reperfusion injury. Heat stimulus evokes release of sensory nerve transmitters, including calcitonin gene-related peptide (CGRP). Since CGRP has been shown to play an important role in the mediation of ischaemic preconditioning, the present study examined whether early or delayed preconditioning induced by retrograde hyperthermic perfusion in vitro or by whole-body hyperthemia in vivo also involves endogenous CGRP. Isolated rat hearts were perfused in the Langendorff mode and subjected to 30 min global ischaemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure and its first derivatives (+/-dp/dt) were recorded and the CGRP-like immunoreactivity (CGRP-LI) content and the release of creatine kinase (CK) during reperfusion were measured. Retrograde hyperthermic perfusion (42 degrees C) for 5 min improved the recovery of cardiac function, decreased the release of CK and elevated the content of CGRP-LI in the coronary effluent. CGRP(8-37) (10(-7 mol/l), a selective CGRP receptor antagonist, abolished the cardioprotection by heat stress. Pretreatment with capsaicin (50 mg/kg s.c.), which specifically depletes sensory nerve transmitter content, abolished both the cardioprotection and the increased release of CGRP-LI. Whole-body hyperthermia (42 degrees C for 15 min) caused an increase in the plasma concentration of CGRP-LI. Early or delayed protection was shown in the hearts obtained from the animals subjected to whole-body hyperthermia 10 min or 48 h before the experiments. The early or delayed protection by heat stress was also abolished by pretreatment with capsaicin. The present study suggests that, in the rat, the early and delayed cardioprotection induced by heat stress involves endogenous CGRP.

Protective effects of calcitonin gene-related peptide on guinea-pig cardiac anaphylaxis.

Anaphylactic events occurring in cardiac tissues can result in cardiac dysfunction via vasoconstriction and arrhythmias. Calcitonin gene-related peptide (CGRP) is the most potent vasodilator and possesses anti-arrhythmic action. We examined the influence of CGRP on cardiac anaphylaxis in guinea-pigs. In the Langendorff-perfused heart of passively sensitized guinea-pigs, antigen challenge evoked a decrease in coronary flow, left ventricular pressure and its maximum first derivatives (+/-dP/dtmax) and an increased heart rate. Antigen challenge also induced atrioventricular conduction block. Treatment with CGRP (1 or 3 nM) significantly improved the recovery of cardiac function and reduced the incidence and duration of atrioventricular block without influencing the increased heart rate. Pretreatment with capsaicin caused effects similar to those of CGRP and markedly elevated the content of CGRP in coronary effluent. Ischaemic preconditioning, induced by two cycles each of 5 min global ischaemia and 5 min reperfusion, also improved cardiac function and raised the level of CGRP in coronary effluent. The protective effects of ischaemic preconditioning were abolished in the presence of the CGRP receptor antagonist CGRP8-37. Histamine release did not differ significantly during any of the interventions. The findings of the present study indicate that, in guinea-pig hearts, CGRP protects against cardiac anaphylaxis and that the cardioprotection by CGRP is independent of histamine release.

BARCSoySNP23: a panel of 23 selected SNPs for soybean cultivar identification.

This report describes a set of 23 informative SNPs (BARCSoySNP23) distributed on 19 of the 20 soybean linkage groups that can be used for soybean cultivar identification. Selection of the SNPs to include in this set was made based upon the information provided by each SNP for distinguishing a diverse set of soybean genotypes as well as the linkage map position of each SNP. The genotypes included the ancestors of North American cultivars, modern North American cultivars and a group of Korean cultivars. The procedure used to identify this subset of highly informative SNP markers resulted in a significant increase in the power of identification versus any other randomly selected set of equal number. This conclusion was supported by a simulation which indicated that the 23-SNP panel can uniquely distinguish 2,200 soybean cultivars, whereas sets of randomly selected 23-SNP panels allowed the unique identification of only about 50 cultivars. The 23-SNP panel can efficiently distinguish each of the genotypes within four maturity group sets of additional cultivars/lines that have identical classical pigmentation and morphological traits. Comparatively, the 13 trinucleotide SSR set published earlier (BARCSoySSR13) has more power on a per locus basis because of the multi-allelic nature of SSRs. However, the assay of bi-allelic SNP loci can be multi-plexed using non-gel based techniques allowing for rapid determination of the SNP alleles present in soybean genotypes, thereby compensating for their relatively low information content. Both BARCSoySNP23 and BARCSoySSR13 were highly congruent relative to identifying genotypes and for estimating population genetic differences.

Development and mapping of microsatellite (SSR) markers in wheat.

Microsatellite DNA markers are consistently found to be more informative than other classes of markers in hexaploid wheat. The objectives of this research were to develop new primers flanking wheat microsatellites and to position the associated loci on the wheat genome map by genetic linkage mapping in the ITMI W7984 x Opata85 recombinant inbred line (RIL) population and/or by physical mapping with cytogenetic stocks. We observed that the efficiency of marker development could be increased in wheat by creating libraries from sheared rather than enzyme-digested DNA fragments for microsatellite screening, by focusing on microsatellites with the [ATT/TAA]n motif, and by adding an untemplated G-C clamp to the 5'-end of primers. A total of 540 microsatellite-flanking primer pairs were developed, tested, and annotated from random genomic libraries. Primer pairs and associated loci were assigned identifiers prefixed with BARC (the acronym for the USDA-ARS Beltsville Agricultural Research Center) or Xbarc, respectively. A subset of 315 primer sets was used to map 347 loci. One hundred and twenty-five loci were localized by physical mapping alone. Of the 222 loci mapped with the ITMI population, 126 were also physically mapped. Considering all mapped loci, 126, 125, and 96 mapped to the A, B, and D genomes, respectively. Twenty-three of the new loci were positioned in gaps larger than 10 cM in the map based on pre-existing markers, and 14 mapped to the ends of chromosomes. The length of the linkage map was extended by 80.7 cM. Map positions were consistent for 111 of the 126 loci positioned by both genetic and physical mapping. The majority of the 15 discrepancies between genetic and physical mapping involved chromosome group 5.

Calcitonin gene-related peptide: an endogenous mediator of preconditioning.

Preconditioning of the heart induced by a brief ischemia or hyperthermia is exerted in two phases, early and delayed protection. The cardioprotection of ischemic preconditioning is related to the release of endogenous mediators. Calcitonin gene-related peptide (CGRP), a principal transmitter of capsaicin-sensitive sensory nerves, is involved in the mediation of ischemic preconditioning, and CGRP-mediated ischemic preconditioning has been shown to protect the endothelial cells. The cardioprotection mediated by endogenous CGRP is also found in heat stress reaction. Drug-induced preconditioning, such as by nitroglycerin, may be related to stimulated release of CGRP. These findings suggest that CGRP may be an endogenous myocardial protective substance and plays an important role in the mediation of preconditioning.

Cardioprotective effect of bradykinin-induced preconditioning mediated by calcitonin gene-related peptide in isolated rat heart.

AIM: To study the mediation of calcitonin gene-related peptide (CGRP) in the cardioprotective effect of bradykinin-induced preconditioning in heart. METHODS: The isolated rat hearts were perfused in a Langendorff mode. The cardiac function and creatine kinase (CK) were measured. RESULTS: Pretreatment with bradykinin for 5 min caused an improvement of heart function and a decrease of CK release during reperfusion [CK was (0.18 +/- 0.06), (1.07 +/- 0.14), and (0.37 +/- 0.15) U.min-1.g-1/(wet wt) for control, ischemia-reperfusion, and bradykinin, respectively, P < 0.01], and the effect of bradykinin was abolished in the presence of icatibant acetate (Hoe140 1 mumol.L-1) or CGRP8-37 (0.1 mumol.L-1) [CK was (0.37 +/- 0.15), (1.01 +/- 0.23), and (1.07 +/- 0.23) U.min-1.g-1 (wet wt) for bradykinin, Hoe140, and CGRP8-37, respectively, P < 0.01]. Pretreatment with capsaicin also abolished the protection of bradykinin [CK was (0.30 +/- 0.04) and (1.14 +/- 0.12) U.min-1.g-1 (wet wt) for vehicle and capsaicin, respectively, P < 0.01]. CONCLUSION: The cardioprotective effect of bradykinin-induced preconditioning was related to stimulation of CGRP release in the rat.

Role of calcitonin gene-related peptide in prostaglandins-mediated ischemic preconditioning in guinea pig hearts.

AIM: To examine the role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning induced by prostaglandins in isolated guinea pig hearts. METHODS: The isolated guinea pig hearts were perfused in a Langendorff model. The heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dp/dt(max)) were recorded and the calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and 6-keto-PGF(1 alpha) were measured. RESULTS: Endothelin-1 (200 pmol in 1 mL K-H buffer) reduced the left ventricular developed pressure and its first derivatives (+/-dp/dt(max)), heart rate, and coronary flow. Preconditioning with two cycles of 5-min global ischemia and 5-min reperfusion attenuated endothelin-1 induced myocardial injury, and concentrations of both CGRP and 6-keto-PGF(1alpha) in the coronary effluent were markedly raised in the preconditioning periods. Pretreatment with capsaicin, which depletes endogenous CGRP, abolished the elevated level of CGRP concomitantly with loss of the cardioprotection induced by ischemic preconditioning. CGRP(8-37) (100 nmol/L), a selective CGRP1 receptor antagonist, also abolished the protective effects of ischemic preconditioning. After pretreatment with indometacin (10 micromol/L), an inhibitor of cyclooxygenase, the protective effects of ischemic preconditioning were abolished and the release of 6-keto-PGF1alpha was no longer elevated. Pretreatment with indometacin abolished the elevated level of CGRP in the coronary effluent. CONCLUSION: Endogenous prostaglandins are involved in the protective effects of ischemic preconditioning, and the beneficial effects of prostaglandins are mediated by CGRP in the guinea pig heart.

A new integrated genetic linkage map of the soybean.

A total of 391 simple sequence repeat (SSR) markers designed from genomic DNA libraries, 24 derived from existing GenBank genes or ESTs, and five derived from bacterial artificial chromosome (BAC) end sequences were developed. In contrast to SSRs derived from EST sequences, those derived from genomic libraries were a superior source of polymorphic markers, given that the mean number of tandem repeats in the former was significantly less than that of the latter ( P<0.01). The 420 newly developed SSRs were mapped in one or more of five soybean mapping populations: "Minsoy" x "Noir 1", "Minsoy" x "Archer", "Archer" x "Noir 1", "Clark" x "Harosoy", and A81-356022 x PI468916. The JoinMap software package was used to combine the five maps into an integrated genetic map spanning 2,523.6 cM of Kosambi map distance across 20 linkage groups that contained 1,849 markers, including 1,015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, six AFLPs, ten isozymes, and 12 others. The number of new SSR markers added to each linkage group ranged from 12 to 29. In the integrated map, the ratio of SSR marker number to linkage group map distance did not differ among 18 of the 20 linkage groups; however, the SSRs were not uniformly spaced over a linkage group, clusters of SSRs with very limited recombination were frequently present. These clusters of SSRs may be indicative of gene-rich regions of soybean, as has been suggested by a number of recent studies, indicating the significant association of genes and SSRs. Development of SSR markers from map-referenced BAC clones was a very effective means of targeting markers to marker-scarce positions in the genome.