In silico molecular and morphological analysis of rice blast resistant gene Pi-ta in Sri Lankan rice germplasm

Background Pi-ta is a major blast resistant gene, introgressed from indica rice varieties. In this study, diversity of the Pi-ta gene of 47 Sri Lankan rice accessions was studied by bioinformatics, and the results were validated with molecular and disease reaction assays. Sequences of rice accessions at the locus Os12g0281300 were retrieved from Rice SNP-Seek Database, and the coding sequence of reference Pi-ta gene of cultivar Tetep (accession no. GQ918486.1) was obtained from GenBank. Comparisons were made at nucleotide, amino acid, and protein structure level, and the 3D models predicted using Phyre2 software were superimposed using TM-align software. Results In silico analysis revealed that 10 accessions possessed resistant allele of the Pi-ta gene. The remaining accessions recorded high polymorphism in the leucine-rich domain resulting in 9 allele types, leading to single–amino acid substitutions at 27 different positions including a functional mutation of alanine to serine at the 918th amino acid position. None of the genotypes led to truncations in the amino acid sequence. The in silico analysis results were validated on 23 accessions comprising resistant and susceptible genotypes and another 25 cultivars from Northern Sri Lanka, by molecular assay using YL183/YL87 and YL155/YL87 resistant and susceptible allele-specific markers. Resistance of Pi-ta gene for the causal fungus, Magnaporthe oryzae, was further validated through pathogenicity assay. Conclusion The Pi-ta gene, especially the LRD region, revealed significant variations within Sri Lankan rice cultivars leading to high levels of resistance against blast. This information would be highly useful in breeding programmes for resistance against rice blast.

Marker-Assisted Pyramiding of Downy Mildew-Resistant Gene Ppa3 and Black Rot-Resistant Gene Xca1bo in Popular Early Cauliflower Variety Pusa Meghna

Cauliflower is an important extensively grown cool season vegetable in India. Black rot and downy mildew are major devastating diseases reducing yield and quality of the crop. To tackle these through host plant resistance, a marker-assisted backcross breeding method was followed to pyramid a black rot-resistant gene (Xca1bo) and a downy mildew-resistant gene (Ppa3) from donors BR-161 and BR-2, respectively, into the background of Pusa Meghna cauliflower cultivar. Marker-assisted backcross breeding was followed up to BC2 generation using SCAR marker ScOPO-04833 and SSR marker BoGMS0624 for black rot and downy mildew resistance genes in foreground selection, respectively. In background selection, at each stage of backcrossing, 47 parental polymorphic SSR markers were used. The graphical genotyping of the five two-gene (Xca1boXca1boPpa3Ppa3) homozygous BC2F2 plants showed an average recovery of 85.44% of the Pusa Meghna genome with highest genome recovery of 91.7%. The genome contribution of donor parents (BR-161 and BR-2) was 8.26 with 6.34% of residual heterozygousity. The backcross derived pyramided lines BC2F2:3-7-16 and BC2F2:3-7-33 showed high resistance to both the diseases and exhibited higher yield and vitamin C content as compared with recipient parent Pusa Meghna. It is, therefore, evident from this study that resistant genes can be introgressed successfully into a Pusa Meghna cultivar without any yield penalty, benefitting farmers with reduced input cost and consumers with chemical residue free produce. Besides, the pyramided lines carrying dominant resistant genes can be exploited in a hybridization programme to develop hybrid(s) in cauliflower.

Antibiotic Resistant Pattern and Resistant Gene Identification of Staphylococcus aureus from Chicken Farm in Bogor

Chicken is one of the important protein source in Indonesia. Moreover, the largest population of chicken layer and poultry in Indonesia is known situated at West Java province with Bogor manicipality as the main producer. The aims of this study were to determine the antibiotic resistance pattern of Staphylococcus aureus isolated from poultry and layer farm in Bogor. The study also identified gene encoded the resistance. Cloacal swab samples were collected from chicken broiler and layer farm in Bogor manicipality. The samples were then cultured in Mannitol Salt Agar (MSA) medium to obtain S. aureus. Suspected colony was then confirmed by biochemical test. Positive strains were tested against several antibiotics and the diameter of clear zone arround of colony was interpreted based on Clinical and Laboratory Standard Institute. Furthermore, the DNA from resistant strains were then extracted, followed by detection of the resistance gene by using polymerase chain reaction (PCR) method. A total of 14 isolates of S. aureus were positive from poultry farm, and 15 isolates from layer farm. Most of all were resistant to tetracycline, ampicillin, oxytetracycline, erythromycin and nalidixic acid. On the other hands, several strains were sensitive to gentamycin and chloramphenicol. The study showed 28 isolates out of them were multi-drug resistant. Resistant gene such as blaTEM, gyrA and tetA were also identified in some isolates except for ErmB gene which was found in isolates originated from poultry farm. In conclussion, S. aureus in both farm showed mostly multi-drug resistant to several antibiotics which were supported by identification of resistant gene among isolates.

Molecular Characteristics of Staphylococcus aureus From Food Samples and Food Poisoning Outbreaks in Shijiazhuang, China

As an opportunistic pathogen worldwide, Staphylococcus aureus can cause food poisoning and human infections. This study investigated the sequence typing, the penicillin (blaZ) and methicillin (mec) resistance profiles of S. aureus from food samples and food poisoning outbreaks in Shijiazhuang City, and the staphylococcal enterotoxin (SE) types of the S. aureus isolates from food poisoning. A total of 138 foodborne S. aureus isolates were distributed into 8 clonal complexes (CCs) and 12 singletons. CC1, CC5, CC8, CC15, CC97, CC59, CC398, CC88, and CC7 were the predominant CCs of foodborne S. aureus isolates. Moreover, CC59, CC15, and CC5 were the most prevalent CCs in food poisoning outbreaks. SEE was the most commonly detected SE in food poisoning isolates. One hundred thirty-three S. aureus isolates harbored the penicillin-resistant gene blaZ, and nine isolates carried the mec gene. The present study further explained the relationship between S. aureus and foods and food poisoning and indicated the potential risk of S. aureus infection.

Cloning Method for Stress-Resistant Gene of Conringia planisiliqua under Drought Stress

The low temperature, drought, high salt, and other environments influence crop production and development directly, so the gene cloning method has become an effective biological means. In order to effectively improve the cloning effect, a gene cloning method for Conringia planisiliqua based on mRNA differential display technology was proposed. Based on mRNA differential display technology, the gene of Conringia planisiliqua was transcribed. The present study expects gene cloning to be better than the traditional method. This will lay the basis for gene cloning and functional verification of the transcription and disease-resistant proteins in Conringia planisiliqua. According to homologous identification results, the homologous drought-resistant genes were determined and screened. The data of Conringia planisiliqua in the existing biological database were used to extract ESTs data of Conringia planisiliqua. Then, the heating environment was established and the concept of integral function was introduced to express the influence of growth environment of different genomes. The mass, momentum, energy, and turbulent flow situation of stress-resistant gene of Conringia planisiliqua during the growth were satisfied. Finally, the data search was carried out in the NCBI database and gene cloning was achieved by ESTs data sequence. Experimental results show that the proposed method can effectively reduce the gene data fitting and improve the quantity of gene fragments cloned in a cycle, so the overall cloning effect is better.