Strategies to identify long noncoding RNAs involved in gene regulation

Long noncoding RNAs (lncRNAs) have been detected in nearly every cell type and found to be fundamentally involved in many biological processes. The characterization of lncRNAs has immense potential to advance our comprehensive understanding of cellular processes and gene regulation, along with implications for the treatment of human disease. The recent ENCODE (Encyclopedia of DNA Elements) study reported 9,640 lncRNA loci in the human genome, which corresponds to around half the number of protein-coding genes. Because of this sheer number and their functional diversity, it is crucial to identify a pool of potentially relevant lncRNAs early on in a given study. In this review, we evaluate the methods for isolating lncRNAs by immunoprecipitation and review the advantages, disadvantages, and applications of three widely used approaches ?C microarray, tiling array, and RNA-seq ?C for identifying lncRNAs involved in gene regulation. We also look at ways in which data from publicly available databases such as ENCODE can support the study of lncRNAs.     

Genome-wide identification of the mitogen-activated protein kinase (MAPK) family in cotton (Gossypium hirsutum) reveals GhMPK6 involved in fiber elongation

Plant Molecular Biology - Mitogen-activated protein kinases (MAPKs) are important in regulating plant development as well as stress response. In this study, we genome-widely identified 56 MAPK...     

Simple sequence repeat genetic linkage maps of A-genome diploid cotton (Gossypium arboreum)

This study introduces the construction of the first intraspacific genetic linkage map of the A-genome diploid cotton with newly developed simple sequence repeat (SSR) markers using 189 F2 plants derived from the cross of two Asiatic parents were detected using 6 092 pairs of SSR primers. Two-hundred and sixty-eight pairs of SSR pdmers with better polymorphisms were picked out to analyze the F2 population. In total, 320 polymorphic bands were generated and used to construct a linkage map with JoinMap3.0. Two-hundred and sixty-seven loci, Including three phenotypic traits were mapped at a logarithms of odds ratio (LOD) ≥ 3.0 on 13 linkage groups. The total length of the map was 2 508.71 cM, and the average distance between adjacent markers was 9.40 cM. Chromosome assignments were according to the association of linkages with our backbone tetraploid specific map using the 89 similar SSR loci. Comparisons among the 13 suites of orthologous linkage groups revealed that the A-genome chromosomes are largely collinear with the At and Dt sub-genome chromosomes. Chromosomes associated with inversions suggested that allopolyploidization was accompanied by homologous chromosomal rearrangement. The inter-chromosomal duplicated loci supply molecular evidence that the A-genome diploid Asiatic cotton is paleopolyploid.     

Introgression of cotton leaf curl virus-resistant genes from Asiatic cotton (Gossypium arboreum) into upland cotton (G. hirsutum)

Cotton is under the constant threat of leaf curl virus, which is a major constraint for successful production of cotton in the Pakistan. A total of 3338 cotton genotypes belonging to different research stations were screened, but none were found to be resistant against the Burewala strain of cotton leaf curl virus (CLCuV). We explored the possibility of transferring virus-resistant genes from Gossypium arboreum (2n = 26) into G. hirsutum (2n = 52) through conventional breeding techniques. Hybridization was done manually between an artificial autotetraploid of G. arboreum and an allotetraploid G. hirsutum, under field conditions. Boll shedding was controlled by application of exogenous hormones, 50 mg/L gibberellic acid and 100 mg/L naphthalene acetic acid. Percentage pollen viability in F(1) hybrids was 1.90% in 2(G. arboreum) x G. hirsutum and 2.38% in G. hirsutum x G. arboreum. Cytological studies of young buds taken from the F(1) hybrids confirmed that they all were sterile. Resistance against CLCuV in the F(1) hybrids was assessed through grafting, using the hybrid plant as the scion; the stock was a virus susceptible cotton plant, tested under field and greenhouse conditions. All F(1) cotton hybrids showed resistance against CLCuV, indicating that it is possible to transfer resistant genes from the autotetraploid of the diploid donor specie G. arboreum into allotetraploid G. hirsutum through conventional breeding, and durable resistance against CLCuV can then be deployed in the field.     

Genetic modification of Gossypium arboreum universal stress protein (GUSP1) improves drought tolerance in transgenic cotton (Gossypium hirsutum)

Cotton crop suffers shortage of irrigation water at reproductive stage which reduces the yield and fibre quality. Universal stress proteins belong to Pfam00582 which enables several plants to cope with multiple stresses via ATP binding. GUSP1 (Gossypium arboreum USP) is one of such proteins; its amino acids were mutated after in silico simulations including homology modeling and molecular docking analysis. Transgenic cotton plants were developed through Agrobacterium mediated genetic transformation by using mutated pmGP1 and non mutated pGP1 constructs under CaMV35S promoter. PCR and semi-quantitative PCR analyses confirmed the amplification and expression of transgene in transgenic plants. It was revealed that leaf relative water content, total chlorophyll content, CO₂ assimilation as net photosynthesis, stomatal conductance, total soluble sugars and proline content was significantly increased at P ≤ 0.0001 and P ≤ 0.001 in both the pmGP1 and pGP1 transgenic plants as compared to non transgenic control plants. Moreover, relative membrane permeability and the transpiration rate were reduced significantly at P ≤ 0.0001 and P ≤ 0.001 respectively in transgenic plants under drought stress. Furthermore, the T1 transgenic seedlings containing pmGP1 mutated construct showed longer roots under desiccation stress imposed by 5% PEG. Transgene inheritance into the T1 progeny plants was confirmed by amplification through PCR and integration through Southern blot. Hence, our results pave the way to utilize the mutagenized known genes for increasing endurance of plants under drought stress. This will help to increase our understanding of drought tolerance/ sensitivity in cotton plants at the molecular level.Supplementary Information The online version contains supplementary material available at 10.1007/s12298-021-01048-5.     

Identification and characterization of microRNAs in Asiatic cotton (Gossypium arboreum L.)

To date, no miRNAs have been identified in the important diploid cotton species although there are several reports on miRNAs in upland cotton. In this study, we identified 73 miRNAs, belonging to 49 families, from Asiatic cotton using a well-developed comparative genome-based homologue search. Several of the predicted miRNAs were validated using quantitative real time PCR (qRT-PCR). The length of miRNAs varied from 18 to 22 nt with an average of 20 nt. The length of miRNA precursors also varied from 46 to 684 nt with an average of 138 ±120 nt. For a majority of Asiatic cotton miRNAs, there is only one member per family; however, multiple members were identified for miRNA 156, 414, 837, 838, 1044, 1533, 2902, 2868, 5021 and 5142 families. Nucleotides A and U were dominant, accounted for 62.95%, in the Asiatic cotton pre-miRNAs. The Asiatic cotton pre-miRNAs had high negative minimal folding free energy (MFE) and adjusted MFE (AMFE) and high MFE index (MFEI). Many miRNAs identified in Asiatic cotton suggest that miRNAs also play a similar regulatory mechanism in diploid cotton.     

Changes in the level of tubulin subunits during development of cotton (Gossypium hirsutum) fiber

The levels of tubulin protein in developing cotton ( Gossypium hirsutum L. cv. Stoneville 825) fibers were measured from 8 to 28 days post-anthesis using commercially available monoclonal antibodies against alpha- and beta-tubulin. As the monoclonal antibodies against alpha- and beta-tubulin were prepared from yeast tubulin and chick brain tubulin, respectively, indirect immunofluorescence microscopy was used to establish that the two monoclonal antibodies recognized microtubule structures in cotton fibers. Western blots of electrophoretically separated proteins in crude extracts of cotton roots and fibers showed that single polypeptides with the expected apparent molecular weight for tubulin subunits were recognized by the antisera. An enzyme-linked immunosorbent assay was used to quantify tubulin levels. From 10 to 20 days post-anthesis the level of tubulin protein increases approximately three-fold. After 20 days post-anthesis, the amount of tubulin relative to total fiber protein reaches a plateau or decreases slightly. The rapid rise in tubulin is correlated with the elongation of the fiber and an increase in cellulose synthesis.