Prophylactic and therapeutic antibiotic treatments have the potential to increase excretion of antibiotic resistance genes (ARGs) by dairy cattle through selection pressure on the gut microbiome. The objective of these studies was to evaluate the effect of cephapirin benzathine administered prophylactically at the end of lactation and pirlimycin hydrochloride administered therapeutically during a clinical mastitis infection on the abundance and relative abundance of ARGs in dairy cow feces. For prophylactic treatment using cephapirin benzathine, nineteen end-of-lactation cows were used. Treatment cows (n = 9) received cephapirin benzathine as an intramammary infusion prior to dry-off, and control cows (n =10) received no antibiotics. All cows received an internal non-antibiotic teat sealant. Fecal grab samples were collected for each cow on d -2 (baseline, used as covariate), d 1, 3, 5, 7, and once per week until d 49. Fecal samples were collected in sterile containers, then freeze-dried and subject to DNA extraction. The abundance of ampC, blaCMY-2, ermB, sul1, tetO, tetW, integrase-specific gene int1, and 16S rRNA were quantified using quantitative polymerase chain reaction (qPCR). The genes ampC and blaCMY-2 encode resistance to ß-lactam antibiotics, ermB to macrolides, sul1 to sulfonamides, tetO and tetW to tetracyclines, and int1 a class-1 integrase gene that facilitates horizontal transfer of ARGs across bacteria. The 16S rRNA gene was used as a representation of bacterial population. Absolute abundance was defined as number of ARG copies per gram of freeze-dried feces, while relative abundance was defined as ARG copy numbers per copy of 16S rRNA gene, which is indicative of the proportion of bacteria carrying ARGs. Non-normal data were logarithmically transformed and were statistically analyzed using PROC GLIMMIX in SAS 9.2. Abundance and relative abundance of sul1 and blaCMY-2 were below the limit of quantification in most samples and therefore not suitable for statistical comparisons. The int1 gene was not detectable in any sample. There were significant interactions between treatment and day for the abundance and relative abundance of ampC, tetO, and tetW. The abundance and relative abundance of ampC increased with time in control cows while remaining constant in antibiotic treated cows through the dry period. Antibiotics may act to stabilize the gut microbiome in response to diet and housing changes. There was a significant main effect of treatment for ermB with a significantly greater proportion of bacteria carrying ermB in control cows when compared to antibiotic treated cows. The tetracycline resistance genes tetO and tetW behaved similarly with a significant treatment by day interaction for the abundance and relative abundance of both genes. The relative abundance of both tetO and tetW were greater in control cows when compared to antibiotic treated cows on days 3, 5, 7, and 14. The abundance of both tetO and tetW resistance genes increased in antibiotic treated cows from day 1 to 49. There was also a significant increase in tetW relative abundance when comparing day 1 to 49. Administering long-acting antibiotics as intramammary dry treatment changed fecal bacteria composition during the dry period perhaps by stabilizing GI bacteria through dietary and housing changes. However, the use of prophylactic dry cow treatment does not uniformly or predictably lead to changes in fecal ARGs. In a second study, after clinical mastitis detection and identification, 6 lactating dairy cows received therapeutic mastitis treatment (pirlimycin hydrochloride as an intramammary infusion). Fecal grab samples were collected from each cow on d 0, 3, 9, and 12. Collection and analytical methods were as previously described. Abundance and relative abundance of sul1 and blaCMY-2 were again below the limit of quantification and therefore not suitable for statistical comparison. The int1 gene was not detected in any sample. The abundance of 16S rRNA genes decreased with day and relative abundance ermB, tetO, and tetW increased with day. There was no significant effect of day on the relative abundance of ampC or the abundance of ampC, ermB, tetO, and tetW in feces of cows with clinical mastitis. Administering fast-acting antibiotics as therapeutic intramammary mastitis treatment to dairy cows increased the relative abundance (gene copies per 16S rRNA) of selected ARGs but not the total abundance of ARGs in feces. The use of antibiotics for prevention and treatment of bacterial infections does not uniformly or predictably increase ARGs. / Master of Science

No description available.

No description available.

Twenty-eight Holstein and crossbred calves of both genders were used to evaluate the effect of milk replacer antibiotics on abundance of selected antibiotic resistance genes (ARG) in the feces. Calves were blocked by breed, gender, and birth order, and assigned to one of three treatments at birth. Treatments were control (containing no antibiotics in the milk replacer), subtherapeutic (neomycin sulfate and oxytetracycline hydrochloride each fed at 10 mg/calf/d), and therapeutic (no antibiotics in the milk replacer until d 36, then neomycin sulfate and oxytetracycline hydrochloride each fed at 1000 mg/calf/d for 14 d). Calves were fed milk replacer twice daily at 0600 h and 1800 h. Fecal and respiratory scores and rectal temperatures were recorded daily. Calves were weighed at birth and weaning to calculate average daily gain. Beginning at six weeks of age fecal grab samples were collected from heifers at 0600 h, 1400 h, 2000 h, and 2400 h for 7 d, while bull calves were placed in metabolism crates for collection of all feces and urine. DNA was extracted from feces, and ARG corresponding to the tetracyclines (tetC, tetG, tetO, tetW, and tetX), macrolides (ermB, ermF), and sulfonamides (sul1, sul2) classes of antibiotics along with the class I integron gene, intI1, were measured by quantitative polymerase chain reaction (qPCR). No tetC or intI was detected. There was no significant effect of antibiotic treatment on the absolute abundance (gene copies/ g wet manure) of any of the ARG except ermF, which was lower in the antibiotic-treated calf manure probably because host bacterial cells carrying ermF were not resistant to tetracycline or neomycin. All ARG except tetC and intI were detectable in feces from 6 weeks onwards, and tetW and tetG significantly increased with time (P < 0.10), even in control calves. Overall, the majority of ARG analyzed for were present in the feces of the calves regardless of exposure to dietary antibiotic. Feed antibiotics had little effect on the ARG monitored; other methods for reducing the ARG pool should also be investigated. / Master of Science

Soybean mosaic virus (SMV) and peanut mottle virus (PMV) are potyviruses that can cause serious yield reductions in soybean [Glycine max (L.) Merr.]. Virus resistant soybean cultivars have been released with alleles at the Rsv1 and Rpv1 locus that confer resistance to SMV and PMV, respectively. A high-resolution map-based cloning approach was undertaken to isolate Rsv1 and Rpv1 from soybean, with hopes of providing insight into this host-pathogen relationship. A mapping population of 1,056 F2 individuals was constructed from the cross of the resistant cultivar PI 96983 (Rsv1 and Rpv1) by the susceptible cultivar Lee 68 (rsv1 and rpv1). Ninety-one of the 1,056 F2 individuals had a cross-over (recombination) in the chromosomal region between microsatellite, or simple sequence repeat (SSR) marker loci Hsp176 and Sat120, and these 91 recombinant lines (RLs) were selected for further genetic analysis. Genotypes of Rsv1 and Rpv1 for the 91 RLs were obtained by inoculating their F2:3 progeny with SMV-G1 and PMV-P1, respectively. The 91 RLs also were used for mapping one random amplified polymorphic DNA (RAPD), five SSR, and 21 restriction fragment length polymorphism (RFLP) markers. Included in these RFLP markers were seven resistance gene candidate (RGC) and five resistance gene candidate flanking (RGCF) markers. RGC probes encode a protein with homology to previously cloned plant disease resistance genes, and RGCF probes are sequences obtained from the flanking regions of candidate disease resistance genes. The resultant high-resolution map consisted of 41 marker loci detected by 27 molecular markers. Rsv1 and Rpv1 cosegregated with one or more RFLP bands detected by RGCF probes: GG27-1a, 3gG2SP, and/or T3G. Analyses of the disease reaction and molecular marker data from seven RLs suggested that the map position of Rsv1 should be at a locus different from that designated by the linkage analysis software, Mapmaker 3.0. Compared to the other 89 RLs, a high percentage (>34%) of F3 plants grown from four of these seven RLs gave a necrotic reaction when inoculated with SMV-G1. From this evidence, we believed that another locus independent of Rsv1 was involved in PI 96983's response to SMV-G1. The two loci conferring resistance to SMV-G1 were designated Rsv1a and Rsv1b. / Master of Science

The largest group of plant disease resistance (R) genes that share similar structures contains a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers and homology searches from public databases. Multiple combinations of degenerate primers were designed from three conserved motifs (one motif was used for a subgroup-specific primer design) in the NBS regions of R genes of various plants. All combinations of primer pairs were used to amplify genomic DNA from sorghum. TIR-specific primer combinations showed no PCR amplification in sorghum. Homology searches identified many NBS-encoding sequences among the expressed or genomic molecular database entries for sorghum. Motif analysis of the sorghum NBS sequences that were identified in this study revealed eight major conserved motifs plus two additional highly conserved motifs, but no TIR-specific motifs. Phylogenetic analysis of sorghum NBS sequences showed tree topology typical of NBS-LRR genes, including clustered nodes and longbranch lengths. Eleven distinct families of NBS sequences, representing a highly diverse sample, were isolated from Sorghum bicolor. With two exceptions, sorghum RGA families appeared to be closely related in sequence to at least one R-gene cloned from other species. In addition, deduced amino acid sequences of sorghum RGAs showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)- type R-genes. Mapping with sorghum RGA markers revealed one linkage group containing four out of ten randomly selected markers, suggesting non-random distribution of NBS sequences in the sorghum genome. Rice sequences homologous to sorghum NBS sequences were found from two-way BLAST searches. Some of them were shown to be orthologs, when determined by using phylogenetic approaches which combined five different evolution models and tree-building methods.

Weed control in chickpea (Cicer arietinum L.) is challenging because of poor crop competition ability and limited herbicide options. Development of chickpea varieties with resistance to different herbicide modes of action would be desirable. Resistance to imidazolinone (IMI) herbicides in chickpea has been previously identified, but the genetic inheritance and the mechanism were unknown. In many plant species, IMI resistance is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution. This changes the enzyme configuration at the herbicide binding site, preventing the herbicide attachment to the molecule. The main research objective was to genetically characterize chickpea resistance to imidazolinone herbicides. Two homologous AHAS genes, namely AHAS1 and AHAS2 sharing 80% similarity were identified in the chickpea genome. A point mutation in AHAS1 at cytosine 675 thymine 675 resulting in an amino acid substitution from alanine 205 to valine 205 confers the resistance to imidazolinone in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the herbicide response in the RIL population. This same population was used in molecular mapping where the major locus for herbicide resistance was mapped to chromosome 5. Segregation analysis demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. To study the synteny of AHAS across plant species, lentil (Lens culinaris) AHAS1 was sequenced. The same mutation that confers the resistance to imidazolinone in chickpea was also found in lentil. Phylogenetic analysis indicated independent clustering of AHAS1 and AHAS2 across pulse species. In vivo and in vitro AHAS enzyme activity analysis showed inhibition of AHAS activity in the susceptible genotype CDC Frontier over time and with the increasing imidazolinone concentrations. In contrast, the resistant genotype CDC Cory did not show AHAS inhibition under the same treatments. In summary, the simple genetic inheritance and the availability of KASP marker could aid in the development of chickpea varieties with resistance to imidazolinone herbicide.

Thesis (M.Sc.)--York University, 2007. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 129-130). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR32034

Bean rust, caused by the fungus Uromyces appendiculatus (Pers.:Pers) Unger, is a disease of common bean (Phaseolus vulgaris) prevalent in the Americas and Africa. The most cost-effective countermeasure to bean rust is genetic resistance. While 17 dominant rust resistance genes (named with Ur- symbol) have been identified in common bean, not all of these genes have been genetically fine-mapped. To expand our knowledge of rust resistance genes in common bean, Ur-6 was mapped in the common bean genome. A GWAS analyses suggested that Ur-6 is present on chromosome Pv07 of P. vulgaris. Two InDel markers tightly linked to Ur-6 were developed by F2 bi-parental mapping and may prove effective for marker-assisted selection in bean breeding programs in the future. Further, 25 candidate genes were identified and are the potential focus of future gene validation research.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-12T13:20:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_FlaviaAraujo_2015_VersãoFinal.pdf: 4388867 bytes, checksum: 1ebc175e90318442d9eb1b8a4a688b3c (MD5) / Made available in DSpace on 2016-07-12T13:20:39Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_FlaviaAraujo_2015_VersãoFinal.pdf: 4388867 bytes, checksum: 1ebc175e90318442d9eb1b8a4a688b3c (MD5) Previous issue date: 2015-03-02 / FACEPE / A cultura do feijão-caupi [Vigna unguiculata (L.) Walp.] apresenta importância econômica em nível internacional, entretanto ela é frequentemente acometida por uma diversidade de patógenos. Nesse contexto a família gênica NBS-LRR de genes de Resistência (R) se destaca devido ao seu papel fundamental na defesa das plantas contra o ataque de patógenos, sendo a maior e mais diversificada família desse grupo. O objetivo do presente estudo foi caracterizar genes NBS-LRR em feijão-caupi, desenvolver marcadores moleculares RGA (Resistance Gene Analogs) e validar genes diferencialmente expressos em uma interação planta-vírus. Inicialmente, sequências candidatas para genes NBS-LRR foram obtidas no banco de dados NordEST, procedendo-se com a anotação dos dados, tradução e identificação dos domínios conservados por meio de ferramentas in silico. Um total de 57 sequências NBS-LRR completas foi identificado em feijão-caupi. Como as proteínas codificadas pelos genes R apresentam domínios e motivos conservados, foi possível desenvolver marcadores RGAs usando as sequências de feijão-caupi como sonda contra o banco de Phaseolus vulgaris L. Foram desenhados 16 pares de iniciadores para P. vulgaris, identificando-se um percentual de 87,5% de transferibilidade para o feijão-caupi. Destes, dois foram polimórficos e apresentaram segregação mendeliana em uma população de mapeamento para o vírus do mosaico severo do feijão-caupi (CPSMV). Os 57 candidatos foram ancorados nos 20 pseudocromossomos de Glycine max (L.) Merr., verificando-se repetições in tandem deste grupo gênico. A análise de expressão gênica diferencial in silico foi realizada utilizando dados de RNAseq e SuperSAGE. A validação da expressão gênica via RT-qPCR foi através do desenho de primers, com os dados de SuperSAGE, onde três genes alvo apresentaram indução nos níveis de expressão após 16 horas da inoculação com o patógeno. Esses resultados mostram-se valiosos para o melhoramento genético do feijão-caupi. / The cowpea [Vigna unguiculata (L.) Walp.] culture has an international economic importance, however it is often affected by a diversity of pathogens. In this context the NBS-LRR family of resistance (R) genes stands out because of its key role in plant defense against pathogen attack, being the largest and most diverse family of this group. The present work aimed the characterization of NBS-LRR genes from cowpea, the development of RGA (resistance gene analogs) markers and the validation of differentially expressed genes in plant-virus interaction. Initially, NBS-LRR gene candidate sequences were obtained from NordEST database, proceeding with data annotation, translation and identification of conserved domains through in silico methods. A total of 57 NBS-LRR complete sequences were identified for cowpea. Since R-gene encoded proteins exhibit conserved domains and motifs, it was possible to develop RGA markers using cowpea sequences as probes against Phaseolus vulgaris L. Sixteen primer pairs of P. vulgaris were designed, from which 87.5% were transferable to cowpea. From those, two were polymorphic and showed Mendelian segregation in a mapping population for the cowpea severe mosaic virus (CPSMV). The 57 candidates were anchored in the 20 Glycine max (L.) Merr. pseudo-chromosomes, revealing in tandem repetitions for this group of gene. Differential gene expression analysis in silico was performed using data from RNAseq and SuperSAGE. The validation of gene expression by RT-qPCR was carried out after design of primers using SuperSAGE data, from which three target genes presented induction in expression levels at 16 hours after the pathogen inoculation. These results represent valuable data for the genetic improvement of cowpea.