Assessment of Genomic Diversity and Selective Pressures in Crossbred Dairy Cattle of Pakistan.

Improving the low productivity levels of native cattle breeds in smallholder farming systems is a pressing concern in Pakistan. Crossbreeding high milk-yielding holstein friesian (HF) breed with the adaptability and heat tolerance of Sahiwal cattle has resulted in offspring that are well-suited to local conditions and exhibit improved milk yield. The exploration of how desirable traits in crossbred dairy cattle are selected has not yet been investigated. This study aims to provide the first overview of the selective pressures on the genome of crossbred dairy cattle in Pakistan. A total of eighty-one crossbred, thirty-two HF and twenty-four Sahiwal cattle were genotyped, and additional SNP genotype data for HF and Sahiwal were collected from a public database to equate the sample size in each group. Within-breed selection signatures in crossbreds were investigated using the integrated haplotype score. Crossbreds were also compared to each of their parental breeds to discover between-population signatures of selection using two approaches: cross-population extended haplotype homozygosity and fixation index. We identified several overlapping genes associated with production, immunity, and adaptation traits, including U6, TMEM41B, B4GALT7, 5S_rRNA, RBM27, POU4F3, NSD1, PRELID1, RGS14, SLC34A1, TMED9, B4GALT7, OR2AK3, OR2T16, OR2T60, OR2L3, and CTNNA1. Our results suggest that regions responsible for milk traits have generally experienced stronger selective pressure than others.

A novel method to estimate the dead layer of HPGe detector for Monte Carlo FEPE computation.

In this study, a novel method to determine the surface and lateral dead layers of p-type HPGe detector is proposed to compute the full energy peak efficiency (FEPE). The method employed standard radioactive point sources 241Am, 133Ba and FEPE measurement at low energies to estimate the thickness of frontal and lateral dead layers. The method is simple to apply, requires only two standard radioactive sources to estimate the optimum thickness of frontal and lateral dead layers. The proposed method is validated by measuring the efficiency of various point sources and a volume source in the energy range from 59 to 1408 keV. The measured efficiencies agree to simulation with relative deviation less than 4.0% at each energy. The proposed detector model enables to calibrate the detector for environmental radioactivity measurement without standard volume sources.

Transcriptome diversity assessment of Gossypium arboreum (FDH228) leaves under control, drought and whitefly infestation using PacBio long reads.

Alternative splicing (AS) and alternative polyadenylation (APA) are common mechanisms in eukaryotes to increase the complexity of transcriptomes and subsequently proteomes. Analysis of long reads transcriptomics data can result in the discovery of novel transcripts, splice sites, AS or APA events. Gossypium arboreum is an important cultivated cotton species and a putative contributor of the A sub-genome to the modern tetraploid cotton; and inherently tolerant to several biotic and abiotic stresses. Specifically, its variety 'FDH228' is considered to be an important resistance source. In this study, we sequenced the G. arboreum (var. FDH228) transcriptome using PacBio IsoSeq and illumina short read sequencing under three different conditions i.e. untreated/healthy, treated with biotic stress through whitefly infestation, and treated with abiotic stress via water deprivation, for the discovery and surveying of canonical and non-canonical AS, APA and transcript fusion events. We were able to obtain 15,419 unique transcripts from all samples representing 11,343 genes, out of which 10,832 were annotated and 520 were novel with respect to the published reference genome. These transcripts were grouped into different structural categories including 60 Antisense, 11,959 having a full-splice match, 999 with incomplete-splice match, 30 fusion transcripts, 177 genic, 479 intergenic, 771 novels in the catalog, and 944 Novel but not found in the catalog. Subsequently, randomly selected candidate transcripts were experimentally validated using qRT-PCR. Our comprehensive identification of canonical and non-canonical splicing events, and novel and fusion transcripts aids in the understanding of the resistance mechanisms for this specific germplasm.

Toxicity assessment of emamectin benzoate and its commercially available formulations in Pakistan by in vivo and in vitro assays.

Emamectin benzoate (EMB) is generally considered a safe insecticide in agriculture and veterinary practices, yet, it can cause cytotoxic and genotoxic effects. Hence, the aim of this study was to evaluate toxic effects of 80% EMB and its commercially used formulations (Tycon 1.9% EC and Tycon plus 5% EW) in Pakistan and tested for acute toxicity in albino rats, rabbits and fish (Labeo rohita). Genotoxicity was investigated by in vivo comet assay and bone marrow micronucleues test in the rats. In vitro mutagenicity was tested in Salmonella typhimurium TA98 and TA100. The tested EMB formulations were found moderately toxic (oral LD50: 122-168 mg/kg), causing severe eye irritation in rabbits, highly toxic to fish (LC50: 9-43 µg/L) and found non mutagenic. Oral administrations of EMB (80% and 5%) at 100 mg/kg of body weight to male rats reduced red blood cells, hemoglobin, and slightly increased the blood glucose, urea and liver enzymes levels but had no significant damage to DNA. EMB induced bone marrow toxicity was observed as reduction of polychromatic erythrocytes. Overall, EMB exposure was highly toxic to fish, and caused hemo- and hepatotoxicity in rats. These findings warrant cautious use of EMB formulations in agrochemicals and veterinary medicine.

Development of a LAMP assay using a portable device for the real-time detection of cotton leaf curl disease in field conditions.

Cotton production is seriously affected by the prevalent cotton leaf curl disease (CLCuD) that originated from Nigeria (Africa) to various parts of Asia including Pakistan, India, China and Philippines. Due to CLCuD, Pakistan suffers heavy losses approximately 2 billion USD per annum. Numerous reports showed that CLCuD is associated with multiple species of begomoviruses, alphasatellites and a single species of betasatellite, that is 'Cotton leaf curl Multan betasatellite' (CLCuMuB). The most prevalent form of CLCuD is the combination of 'Cotton leaf curl Kokhran virus'-Burewala strain (CLCuKoV-Bur) and CLCuMuB. Thus, the availability of an in-field assay for the timely detection of CLCuD is important for the control and management of the disease. In this study, a robust method using the loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CLCuD. Multiple sets of six primers were designed based on the conserved regions of CLCuKoV-Bur and CLCuMuB-ßC1 genes. The results showed that the primer set targeting the CLCuMuB-ßC1 gene performed best when the LAMP assay was performed at 58°C using 100 ng of total plant tissue DNA as a template in a 25 µl reaction volume. The limit of detection for the assay was as low as 22 copies of total purified DNA template per reaction. This assay was further adapted to perform as a colorimetric and real-time LAMP assay which proved to be advantageously applied for the rapid and early point-of-care detection of CLCuD in the field. Application of the assay could help to prevent the huge economic losses caused by the disease and contribute to the socio-economic development of underdeveloped countries.