H9 Gene Research Articles

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

Field efficacy of HVT-H9 vaccination after natural infection of H9N2 avian influenza virus in broilers

H9N2 subtype avian influenza virus (AIV) has been persistently circulating in China. It causes huge economic losses to the poultry industry and poses a great threat to public health. Previously, we constructed a turkey herpesvirus live vector vaccine candidate strain expressing an H9 gene, HVT-H9. Results showed that immunisation with HVT-H9 could provide good immunity in specific pathogen free (SPF) chickens. In this study, field-bred Arbour Acres plus (AA+) broilers were additionally immunised with HVT-H9 at one day of age. Then, broilers were naturally infected with H9N2 AIV. During the endemic period, death occurred in flocks without HVT-H9 immunisation and the mortality rate increased rapidly, forming a clear death wave. However, HVT-H9 vaccination prevented broiler mortality. Etiological tests and serological tests showed that broilers were positive for H9N2 AIV. Collectively, HVT-H9 immunisation provided good immunity for broilers in the field by inhibiting H9N2 virus infection and transmission.

Improved Whole Gamma Irradiated Avian Influenza Subtype H9N2 Virus Vaccine Using Trehalose and Optimization of Vaccination Regime on Broiler Chicken

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log2. The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p< 0.05). After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant (p > 0.05) among the vaccinated groups. In addition, the viral load of <100.5 TCID50/ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime.

Identification and isolation of low pathogenic avian influenza (LPAI) virus from chicken farms in Iraq

The objective of the study was Investigation and isolation of the Low Pathogenic Avian Influenza virus in poultry farms (Broilers and layers) in different provinces in Iraq over the period from August 2020 to June 2021. A total of 241 samples, including (Larynx, trachea &amp; lunge) were collected from 31 poultry farms suspected to be infected with avian influenza and distributed in different provinces including Baghdad, Babylon, Wasit, Karbala &amp; Salahuddin provinces. Collected samples were prepared firstly then viral RNA was extracted by a special viral RNA extraction kit and amplified by using primers for M gene-specific for type A influenza virus and primers specific for H9 gene of LPAI in Real-time RT-PCR which showed that from (31) farms suspected to have low pathogenic avian influenza, six farms (19.35%) were positive H9 LPAI.

Semi-Scavenging Poultry as Carriers of Avian Influenza Genes.

Ducks are the natural reservoir of influenza A virus and the central host for the avian influenza virus (AIV) subtype H5N1, which is highly pathogenic. Semi-scavenging domestic ducks allow for the reemergence of new influenza subtypes which could be transmitted to humans. We collected 844 cloacal swabs from semi-scavenging ducks inhabiting seven migratory bird sanctuaries of Bangladesh for the molecular detection of avian influenza genes. We detected the matrix gene (M gene) using real-time RT–PCR (RT–qPCR). Subtyping of the AIV-positive samples was performed by RT–qPCR specific for H5, H7, and H9 genes. Out of 844 samples, 21 (2.488%) were positive for AIV. Subtyping of AIV positive samples (n = 21) revealed that nine samples (42.85%) were positive for the H9 subtype, five (23.80%) were positive for H5, and seven (33.33%) were negative for the three genes (H5, H7, and H9). We detected the same genes after propagating the virus in embryonated chicken eggs from positive samples. Semi-scavenging ducks could act as carriers of pathogenic AIV, including the less pathogenic H9 subtype. This can enhance the pathogenicity of the virus in ducks by reassortment. The large dataset presented in our study from seven areas should trigger further studies on AIV prevalence and ecology.

A multiplex real-time RT-PCR method for detecting H5, H7 and H9 subtype avian influenza viruses in field and clinical samples

In recent years, H5 and H7 subtypes of highly pathogenic avian influenza viruses (HPAIVs) have been identified in poultry worldwide, resulting in large economic losses to poultry production. Furthermore, H9N2 low pathogenic AIVs are reported to provide internal genes for generating novel reassortant AIVs, leading to potential pandemic risks. To establish an accurate, sensitive and convenient diagnostic method for H5, H7 and H9 subtype AIVs in Eurasian lineage, four groups of specific primers and probes were designed based on the conserved fragments of M, H5, H7 and H9 genes, and a multiplex real-time RT-PCR (RRT-PCR) method was established. High sensitivity was achieved for the multiplex RRT-PCR approach, with a detection limit of 1-10 copies (plasmid DNA) per reaction. The specificity of the method was evaluated using diverse subtypes of AIVs and other avian respiratory viruses isolated in eastern China over the last 9 years. Compared with virus isolation, a higher consistency was achieved when assessing 135 field samples and 126 clinical samples. The results showed that the multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.

Development and application of a visual microarray for synchronously detecting H5N1, H7N9 and H9N2 avian influenza virus RNA

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH2O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 μL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 μg/mL for 30 min. After the supplementary of 200 μL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature.The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 μmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 μg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2.

Isolation and Identification of H9N2 influenza and Newcastle disease viruses co-infections in chicken

Co-infections with Newcastle disease (ND) and avian influenza (AI) highly pathogenic viruses causes high mortality and high economic losses. In this study, 322 samples were collected from 50 chicken flocks suffering from respiratory symptoms, severe drop in egg production and mortalities from December, 2013 to December, 2014. Real time polymerase chain reaction (RT-PCR) was performed to detect the M protein of H9 gene of AI and F gene of velogenic ND virus (vNDV) co-infection. H9 PCR was 20 and 34 in cloacal and pharyngeal swabs with total percentage of 6.2 % and 10.5%, respectively. The H9 sequences showed low pathogenic RSSR/GLF motif. The phylogenetic tree showed that H9 sequences belong to the G1 lineage which clustered with 2015 -2016 Egyptian H9N2. Partial sequences of the Fusion gene revealed that the isolated NDV isolates belong to class II genotype VIIj. The cleavage site of the F protein showed the presence of a polybasic amino acid motif (112RRQKRF117) suggesting that our two isolates were vNDV strain. The molecularly identified H9 (1 isolate) and NDV Genotype VII (2 isolates) were named AI/CHICKEN/EGYPT/48Ob/NRC-CU/2014(H9), NDV/CHICKEN/EGYPT/48Ob/NRC-CU/2014, and NDV/CHICKEN/EGYPT/ 66Oc/NRC-CU/2014, and nucleotide sequences were submitted to GenBank and given accession numbers MG966504, MG966505, and MG966506, respectively. Conclusion: Natural mixed infections with AIV H9N2 and virulent NDV genotype VIIj viruses were detected in chicken flocks suffering from respiratory symptoms and mortalities in Egypt.

Expression and functional analysis of FaPH01;H9 gene of strawberry (Fragariaxananassa)

Although the phosphate 1 (PHO1) gene family has been implicated in inorganic phosphate transport and homeostasis, the underlying mechanism of this gene in the strawberry has not yet been revealed. In the present study, we analyzed the expression of the PH01;H9 gene in the strawberry (Fragariaxananassa), revealing the involvement of this gene in the regulation of phosphorus (P) content. The coding sequence (CDS) of the PH01;H9 gene, was isolated from the cultivated strawberry ‘Sachinoka’ and named as FaPH01;H9. The full-length CDS of this gene was 2292 bp, encoding 763 amino acids, and the protein contained both SYG1/Pho81/XPR1 (SPX) and ERD1/XPR1/SYG1 (EXS) domains, which were involved in phosphate (Pi) signaling. Real-time reverse transcription-polymerase chain reaction (RT-PCR) data suggested that the level of FaPHO1;H9 expression was consistent with the P content in different organs, except for the petiole. Particularly, its expression level was also correlated with P content in fruits of different developmental stages. The expression of FaPHO1;H9 was also consistent with P content in leaves under different concentrations of P fertilizer application. Furthermore, transgenic Arabidopsis lines were generated, and the P content in Arabidopsis plants over-expressing FaPH01;H9 was significantly higher than that in wild-type plants. Therefore, we proposed that FaPH01;H9 functions in P transport.

Molecular and Serological Detection of Avian Influenza H9N2 Virus in Asymptomatic Commercial Layers in Faisalabad District, Punjab

The present study aimed at measuring the antibody titres against H9N2 influenza viruses and its molecular detections in asymptomatic commercial layers from Faisalabad district of Punjab, Pakistan. Overall 120 blood samples, 24 tissue samples from each organ (trachea, lung and intestine) were collected from the 12 commercial layer flocks selected randomly at the age of 35-50 weeks without any direct clinical manifestation of avian influenza. Serum collected from 120 birds was tested for antibodies against H9N2 by using Haemagglutination Inhibition assay. Calculated geometric mean titers of 5.37 revealed the infectivity of the flock with H9N2 Influenza virus. To investigate the presence of virus in the study population, trachea, lung and intestine tissue samples were processed for RNA isolation and subjected to molecular detection of H9 gene of H9N2 subtypes using one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Out of 24 samples taken from each organ, 22, 21, and 18 samples from trachea, lungs, and intestine showed viral RNA, respectively. Taken together, results showed that the H9N2 is endemic and widely distributed in asymptotic layers. Furthermore results indicated that H9N2 subtype may survive in layers without showing any symptoms.

Intranasal vaccination with H5, H7 and H9 hemagglutinins co-localized in a virus-like particle protects ferrets from multiple avian influenza viruses

Avian influenza H5, H7 and H9 viruses top the World Health Organization's (WHO) list of subtypes with the greatest pandemic potential. Here we describe a recombinant virus-like particle (VLP) that co-localizes hemagglutinin (HA) proteins derived from H5N1, H7N2, and H9N2 viruses as an experimental vaccine against these viruses. A baculovirus vector was configured to co-express the H5, H7, and H9 genes from A/Viet Nam/1203/2004 (H5N1), A/New York/107/2003 (H7N2) and A/Hong Kong/33982/2009 (H9N2) viruses, respectively, as well as neuraminidase (NA) and matrix (M1) genes from A/Puerto Rico/8/1934 (H1N1) virus. Co-expression of these genes in Sf9 cells resulted in production of triple-subtype VLPs containing HA molecules derived from the three influenza viruses. The triple-subtype VLPs exhibited hemagglutination and neuraminidase activities and morphologically resembled influenza virions. Intranasal vaccination of ferrets with the VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with H5N1, H7N2, and H9N2 viruses.

Characterization of new loci for Hessian fly resistance in common wheat

The discovery of several new loci for resistance to Hessian fly was reported here. QHf.uga-6AL, the late HR61 was recognized from wheat cultivar 26R61 on the distal end of 6AL with resistance to both biotypes E and vH13. It is the first gene or QTL found on this particular chromosome. QHf.uga-3DL and QHf.uga-1AL, physically assigned to the deletion bins 3DL2-0.27-0.81 and 1AL1-0.17-0.61, respectively, were detected for resistance to biotype vH13. Both QTL should represent new loci for Hessian fly resistance and the latter was detectable only in the late seedling stage when tolerance was evident. In addition, QHf.uga-6DS-C and QHf.uga-1AS had minor effect and were identified from the susceptible parent AGS 2000 for resistance to biotype E and vH13, respectively. QHf.uga-6DS-C is different from the known gene H13 on 6DS and QHf.uga-1AS is different from H9 gene cluster on 1AS. These loci also might be new components of Hessian fly resistance, although their LOD values were not highly significant. The QTL detections were all conducted on a RIL mapping population of 26R61/AGS 2000 with good genome coverage of molecular markers. The strategy used in the current study will serve as a good starting point for the discovery and mapping of resistance genes including tolerance to the pest and the closely linked markers will certainly be useful in selecting or pyramiding of these loci in breeding programs.

A multiplex reverse transcription-PCR assay for the detection of influenza A virus and differentiation of the H1, H3, H5 and H9 subtypes

A multiplex reverse transcription-PCR (mRT-PCR) assay was developed for the rapid detection of influenza A viruses. The assay simultaneously differentiated H1, H3, H5 and H9 hemagglutinin subtypes in a single reaction mixture. Five sets of specific primers targeted to the M, H1, H3, H5 and H9 genes were used in this assay. The amplified products were visualized by agarose gel electrophoresis. The sizes of the PCR amplified fragments were 612 bp for H1, 187 bp for H3, 338 bp for H5, 289 bp for H9 and 239 bp for M. The detection limit of the viral RNA template was 1 ng for the H1, H3 and H5 subtypes and 0.1 ng for the H9 subtype. Nonspecific product bands from RNAs of other viral pathogens were not amplified. The sensitivity analysis demonstrated that the mRT-PCR assay is as sensitive as conventional RT-PCR and 10 times less sensitive than SYBR Green real-time RT-PCR. In conclusion, the mRT-PCR assay developed in this study was able to type influenza A viruses and simultaneously differentiate H1, H3, H5 and H9 subtypes in both human and avian clinical specimens, and thus, the mRT-PCR assay could be a rapid, convenient and relatively inexpensive molecular diagnostic tool for large-scale screening of clinical samples.

Avian influenza virus H9 subtype in poultry flocks in Jordan

Avian influenza virus (AIV) has been recognized as one of the most important pathogens in poultry. This study was designed to investigate the prevalence of AIV H9 subtype in commercial chicken flocks in Jordan by serological and molecular methods. Serum samples from 180 chicken flocks (120 broilers and 60 layers) free from respiratory symptoms, were examined by hemagglutination inhibition (HI) test for specific antibodies against AIV H9 subtype, and 83 chicken flocks (60 broilers and 23 layers) with respiratory symptoms, were examined by reverse transcription-polymerase chain reaction (RT-PCR) using universal primers for influenza A viruses, then specific primers targeting AIV H9 gene were used for the flocks that were positive by universal primers. Overall, 65 out of 120 broiler flocks (54.2%), and 47 out of 60 layer flocks (78.3%) were positive for AIV H9 subtype antibodies. Nucleic acid of influenza A viruses was detected in 31 out of 60 broiler flocks (51.7%), and 15 out of 23 layer flocks (65.2%). AIV H9 subtype was detected in all flocks that were positive for influenza A viruses. The current study confirmed the endemic nature of AIV H9 subtype in broiler and layer flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of influenza and other viruses. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this virus.

Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes

Avian influenza viruses (AIVs) are endemic in wild birds and, if transmitted to poultry, can cause serious economic losses. The aim of this study was to develop a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of influenza virus type A, including H5 and H9 subtypes. The selected primers and various labeled TaqMan reporter probes corresponding to matrix, H5, and H9 genes were used in a multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. The results showed that the multiplex real-time RT-PCR assay can be applied to detect RNA of influenza virus type A including H5 and H9 subtypes with a high specificity and a sensitivity of 10 copies per reaction. As a result of its short turnaround times and a high specificity and sensitivity, the assay is very suitable for large-scale screening during AIV outbreaks.

H22, a major resistance gene to the Hessian fly (Mayetiola destructor), is mapped to the distal region of wheat chromosome 1DS

H22 is a major resistance gene conferring high-level of antibiosis to Hessian fly [Mayetiola destructor (Say)] larvae. It was previously assigned to wheat chromosome 1D through monosomic analysis (Raupp et al. in J Hered 84:142-145, 1993). The objective of this study was to identify molecular markers that can be used for marker-assisted selection for wheat breeding, and to further map this gene toward map-based cloning. Forty-five simple sequence repeat (SSR) and sequence-tagged site (STS) markers specific to chromosome 1D were evaluated for linkage to H22 using a segregating population consisting of 192 F(2:3) families, which were derived from the cross Tugela-Dn1 x KS85WGRC01(H22). The STS Xhor2kv and SSR Xgdm33 are two flanking markers that are tightly linked to H22 at genetic distances of 0.3 and 1.0 cM, respectively. Five other SSR markers including Xgpw7082, Xwmc147, Xcfd15, Xwmc432 and Xwmc336 were also linked to H22 at the distance from 0.8 to 20.8 cM. Analysis of Chinese Spring (CS) deletion lines revealed that all the H22-linked markers are located distal to the breakpoint of del 1DS-5, indicating that the H22 gene is located at the distal 30% region on the short arm of wheat chromosome 1D. Genomic comparison suggested that the H22 gene is located in the same or similar chromosomal region as the leaf rust resistance genes Lr21 and Lr40 on 1DS, and orthologous to the H9 gene cluster of 1AS.

Influenza A viruses in feral Canadian ducks: extensive reassortment in nature.

The current dogma of influenza accepts that feral aquatic birds are the reservoir for influenza A viruses. Although the genomic information of human influenza A viruses is increasing, little of this type of data is available for viruses circulating in feral waterfowl. This study presents the genetic characterization of 35 viruses isolated from wild Canadian ducks from 1983 to 2000, as the first attempt at a comprehensive genotypic analysis of influenza viruses isolated from feral ducks. This study demonstrates that influenza virus genes circulating in Canadian ducks have achieved evolutionary stasis. The majority of these duck virus genes are clustered in distinct North American clades; however, some H6 and H9 genes are clustered with those from Eurasian viruses. Genes appeared to reassort in a random fashion. None of the genotypes identified remained present throughout all of the years examined and most PA and PB2 genes that crossed over into swine were clustered in one phylogenetic grouping. Additionally, matrix genes were identified that branch very early in the evolutionary tree. These findings demonstrate the diversity of the influenza virus gene pool in Canadian ducks, and suggest that genes which cluster in specific phylogenetic groupings in the PB2 and PA genes can be used for markers of viruses with the potential for crossing the species barrier. A more comprehensive study of this important reservoir is needed to provide further insight into the genomic composition of viruses that crossover the species barrier, which would be a useful component to pandemic planning.

Genotypic interaction between resistance genes in wheat and virulence genes in the Hessian fly Mayetiola destructor (Diptera: Cecidomyiidae)

The genotypic interaction between wheat resistance genes H3, H6, H7H8, H9 and virulence genes vH3, vH6, vH7vH8, vH9 of Hessian fly, Mayetiola destructor (Say), was studied in a growth chamber. Results showed that plants homozygous and heterozygous for the H3 gene expressed a high level of resistance against homozygous avirulent and heterozygous larvae carrying the vH3 virulence allele. The H7H8 genes were highly effective in the homozygous condition, but displayed a reduced level of resistance in the heterozygous condition. The H6 and H9 genes showed different levels of resistance against the reciprocal heterozygous larvae (vH6(a)vH6(A) versus vH6(A)vH6(a) and vH9(a)vH9(A) versus vH9(A)vH9(a)). Adults reared from vH6(a)vH6(A) and vH9(a)vH9(A) larvae were all males, consistent with the vH6 and vH9 X-linkage. Plants homozygous for H3, H6, H7H8, and H9 allowed for greater larval survival of heterozygous larvae, which suggests that avirulence to these resistance genes is incompletely dominant. Greater survival of homozygous avirulent larvae on heterozygous plants (H3h3, H6h6, H7h7H8h8, H9h9) suggests incomplete dominance of these wheat genes. Survival of heterozygous along with homozygous virulent larvae would reduce selection pressure for virulence in Hessian fly populations infesting fields of resistant wheat cultivars. This would be expected to slow the increase in frequency of virulence alleles that often results from deployment of resistant cultivars.

Assignment of the Melatonin-Related Receptor to Human Chromosome X (GPR50) and Mouse Chromosome X (Gpr50)

Recent efforts to clone further members of the melatonin receptor family have led to the identification of a novel G-protein-coupled receptor in human pituitary. This receptor, referred to as H9, is clearly related to high-affinity melatonin receptors yet unable to bind this hormone. We now report the cloning and expression of the cDNA encoding the H9 receptor in mice. The mouse clone encodes a protein of 591 amino acids that shares 74% amino acid identity with the human receptor and is unable to bind 2-[125I]iodomelatonin when transiently expressed in COS-7 cells. We also determined the chromosome loci of the human and mouse H9 receptor genes. Both genes were found to be X-linked: radiation hybrid mapping revealed that the human H9 gene (GPR50) is localized to Xq28. The mouse gene (Gpr50) was determined to lie in the proximal portion of chromosome X by means of interspecific backcross analysis. These loci might be relevant to genetically based neuroendocrine disorders.

Association of a DNA marker with Hessian fly resistance gene H9 in wheat

The Hessian fly [Mayetiola destructor (Say)] is a major pest of wheat (Triticum aestivum L.) and genetic resistance has been used effectively over the past 30 years to protect wheat against serious damage by the fly. To-date, 25 Hessian fly resistance genes, designated H1 to H25, have been identified in wheat. With near-isogenic wheat lines differing for the presence of an individual Hessian fly resistance gene, in conjunction with random amplified polymorphic DNA (RAPD) analysis and denaturing gradient-gel electrophoresis (DGGE), we have identified a DNA marker associated with the H9 resistance gene. The H9 gene confers resistance against biotype L of the Hessian fly, the most virulent biotype. The RAPD marker cosegregates with resistance in a segregating F2 population, remains associated with H9 resistance in a number of different T. aestivum and T. durum L. genetic backgrounds, and is readily detected by either DGGE or DNA gel-blot hybridization.