Target prediction of candidate miRNAs from Oryza sativa for silencing the RYMV genome.

In order to maintain a consistent supply of rice globally, control of pathogens affecting crop production is a matter of due concern. Rice yellow mottle virus(RYMV) is known to cause a variety of symptoms which can result in reduced yield. Four ORFs can be identified in the genome of RYMV encoding for P1 (ORF1), Polyprotein (processed to produce VPg, protease, helicase, RdRp4) (ORF2), putative RdRp (ORF3) and capsid/coat protein (ORF4). This research was aimed at identifying genome encoded miRNAs of O. sativa that are targeted to the genome of Rice Yellow Mottle Virus (RYMV). A consensus of four miRNA target prediction algorithms (RNA22, miRanda, TargetFinder and psRNATarget) was computed, followed by calculation of free energies of miRNA-mRNA duplex formation. A phylogenetic tree was constructed to portray the evolutionary relationships between RYMV strains isolated to date. From the consensus of algorithms used, a total of seven O. sativa miRNAs were predicted and conservation of target site was finally evaluated. Predicted miRNAs can be further evaluated by experiments involving the testing of the success of in vitro gene silencing of RYMV genome; this can pave the way for development of RYMV resistant rice varieties in the future.

In silico MCMV Silencing Concludes Potential Host-Derived miRNAs in Maize.

Maize Chlorotic Mottle Virus (MCMV) is a deleterious pathogen which causes Maize Lethal Necrosis Disease (MLND) that results in substantial yield loss of Maize crop worldwide. The positive-sense RNA genome of MCMV (4.4 kb) encodes six proteins: P32 (32 kDa protein), RNA dependent RNA polymerases (P50 and P111), P31 (31 kDa protein), P7 (7 kDa protein), coat protein (25 kDa). P31, P7 and coat protein are encoded from sgRNA1, located at the 3'end of the genome and sgRNA2 is located at the extremity of the 3'genome end. The objective of this study is to locate the possible attachment sites of Zea mays derived miRNAs in the genome of MCMV using four diverse miRNA target prediction algorithms. In total, 321 mature miRNAs were retrieved from miRBase (miRNA database) and were tested for hybridization of MCMV genome. These algorithms considered the parameters of seed pairing, minimum free energy, target site accessibility, multiple target sites, pattern recognition and folding energy for attachment. Out of 321 miRNAs only 10 maize miRNAs are predicted for silencing of MCMV genome. The results of this study can hence act as the first step towards the development of MCMV resistant transgenic Maize plants through expression of the selected miRNAs.

Correction: Deletion at the GCNT2 Locus Causes Autosomal Recessive Congenital Cataracts.

[This corrects the article DOI: 10.1371/journal.pone.0167562.].

Deletion at the GCNT2 Locus Causes Autosomal Recessive Congenital Cataracts.

PURPOSE: The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree. METHODS: All participating individuals underwent a detailed ophthalmic examination. Each patient's medical history, particularly of cataracts and other ocular abnormalities, was compiled from available medical records and interviews with family elders. Blood samples were donated by all participating family members and used to extract genomic DNA. Genetic analysis was performed to rule out linkage to known arCC loci and genes. Whole-exome sequencing libraries were prepared and paired-end sequenced. A large deletion was found that segregated with arCC in the family, and chromosome walking was conducted to estimate the proximal and distal boundaries of the deletion mutation. RESULTS: Exclusion and linkage analysis suggested linkage to a region of chromosome 6p24 harboring GCNT2 (glucosaminyl (N-acetyl) transferase 2) with a two-point logarithm of odds score of 5.78. PCR amplifications of the coding exons of GCNT2 failed in individuals with arCC, and whole-exome data analysis revealed a large deletion on chromosome 6p in the region harboring GCNT2. Chromosomal walking using multiple primer pairs delineated the extent of the deletion to approximately 190 kb. Interestingly, a failure to amplify a junctional fragment of the deletion break strongly suggests an insertion in addition to the large deletion. CONCLUSION: Here, we report a novel insertion/deletion mutation at the GCNT2 locus that is responsible for congenital cataracts in a large consanguineous family.

Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts.

PURPOSE: To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family. METHODS: All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model. RESULTS: Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points. CONCLUSION: A novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts.

Prediction of Host-Derived miRNAs with the Potential to Target PVY in Potato Plants.

Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe. PVY reduces the yield and quality of potato cultivars. During the last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in the PVY genome. The PVY genome is approximately 9 thousand nucleotides, which transcribes the following 6 genes:CI, NIa, NIb-Pro, HC-Pro, CP, and VPg. A total of 343 mature miRNAs were retrieved from the miRBase database and were examined for their target sequences in PVY genes using the minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. The identified potato miRNAs against viral mRNA targets have antiviral activities, leading to translational inhibition by mRNA cleavage and/or mRNA blockage. We found 86 miRNAs targeting the PVY genome at 151 different sites. Moreover, only 36 miRNAs potentially targeted the PVY genome at 101 loci. The CI gene of the PVY genome was targeted by 32 miRNAs followed by the complementarity of 26, 19, 18, 16, and 13 miRNAs. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h, and miR5303d) that could target the CI, NIa, NIb-Pro, HC-Pro, CP, and VPg genes of PVY. The predicted miRNAs can be used for the development of PVY-resistant potato crops in the future.

Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered "white gold" because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton.

Mutations in the ß-subunit of rod phosphodiesterase identified in consanguineous Pakistani families with autosomal recessive retinitis pigmentosa.

PURPOSE: This study was designed to identify pathogenic mutations causing autosomal recessive retinitis pigmentosa (RP) in consanguineous Pakistani families. METHODS: Two consanguineous families affected with autosomal recessive RP were identified from the Punjab Province of Pakistan. All affected individuals underwent a thorough ophthalmologic examination. Blood samples were collected, and genomic DNAs were extracted. Exclusion analysis was completed, and two-point LOD scores were calculated. Bidirectional sequencing of the ß subunit of phosphodiesterase 6 (PDE6ß) was completed. RESULTS: During exclusion analyses both families localized to chromosome 4p, harboring PDE6ß, a gene previously associated with autosomal recessive RP. Sequencing of PDE6ß identified missense mutations: c.1655G>A (p.R552Q) and c.1160C>T (p.P387L) in families PKRP161 and PKRP183, respectively. Bioinformatic analyses suggested that both mutations are deleterious for the native three-dimensional structure of the PDE6ß protein. CONCLUSIONS: These results strongly suggest that mutations in PDE6ß are responsible for the disease phenotype in the consanguineous Pakistani families.

Mapping of a new locus associated with autosomal recessive congenital cataract to chromosome 3q.

PURPOSE: To localize the disease interval for autosomal recessive congenital cataracts in a consanguineous Pakistani family. METHODS: All affected individuals underwent detailed ophthalmologic examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was performed with fluorescently-labeled microsatellite markers on genomic DNA from affected and unaffected family members and logarithm of odds (LOD) scores were calculated. RESULTS: Clinical records and ophthalmological examinations suggested that affected individuals have bilateral congenital cataracts. Genome-wide linkage analysis localized the critical interval to chromosome 3q with a maximum LOD score of 3.87 at θ=0; with marker D3S3609. Haplotype analyses refined the critical interval to a 23.39 cM (18.01 Mb) interval on chromosome 3q, flanked by D3S1614 proximally and D3S1262, distally. CONCLUSIONS: Here, we report a new locus for autosomal recessive congenital cataract localized to chromosome 3q in a consanguineous Pakistani family.

A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.

Ectopia lentis in a consanguineous pakistani family and a novel locus on chromosome 8q.

OBJECTIVE: To investigate the genetic basis and molecular characteristics of the isolated form of ectopia lentis. METHODS: We ascertained a consanguineous Pakistani family with multiple individuals with ectopia lentis. All affected as well as unaffected members with isolated ectopia lentis underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was completed with 382 polymorphic microsatellite markers, and logarithm of odds (LOD) scores were calculated. RESULTS: Maximum 2-point LOD scores of 5.68 and 2.88 at theta = 0 were obtained for markers D8S285 and D8S260, respectively, during the genome-wide scan. Additional microsatellite markers refined the disease locus to a 16.96-cM (14.07-Mb) interval flanked by D8S1737 proximally and D8S1117 distally. CONCLUSIONS: We report on a new locus for nonsyndromic autosomal recessive ectopia lentis on chromosome 8q11.23-q13.2 in a consanguineous Pakistani family. Clinical Relevance Identification of genetic loci and genes involved in ectopia lentis will enhance our understanding of the disease at a molecular level, leading to better genetic counseling and family screening and possible future development of better treatment.

Autosomal recessive congenital cataract in consanguineous Pakistani families is associated with mutations in GALK1.

PURPOSE: To identify the pathogenic mutations responsible for autosomal recessive congenital cataracts in consanguineous Pakistani families. METHODS: All affected individuals underwent detailed ophthalmologic and medical examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was performed with polymorphic microsatellite markers on genomic DNA from affected and unaffected family members and logarithm of odds (LOD) scores were calculated. All coding exons of galactokinase (GALK1) were sequenced to identify pathogenic lesions. RESULTS: Clinical records and ophthalmological examinations suggested that affected individuals have nuclear cataracts. Linkage analysis localized the critical interval to chromosome 17q with a maximum LOD score of 5.54 at theta=0, with D17S785 in family PKCC030. Sequencing of GALK1, a gene present in the critical interval, identified a single base pair deletion: c.410delG, which results in a frame shift leading to a premature termination of GALK1: p.G137fsX27. Additionally, we identified a missense mutation: c.416T>C, in family PKCC055 that results in substitution of a leucine residue at position 139 with a proline residue: p.L139P, and is predicted to be deleterious to the native GALK1 structure. CONCLUSIONS: Here, we report pathogenic mutations in GALK1 that are responsible for autosomal recessive congenital cataracts in consanguineous Pakistani families.

A new locus for autosomal recessive congenital cataract identified in a Pakistani family.

PURPOSE: To identify the disease locus for autosomal recessive congenital cataract in a consanguineous Pakistani family. METHODS: All affected individuals underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was performed with polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and logarithm of odds (LOD) scores were calculated. RESULTS: The clinical records and ophthalmological examinations suggested that all affected individuals have nuclear cataracts. Maximum LOD scores of 5.01, 4.38, and 4.17 at theta=0 were obtained with markers D7630, D7S657, and D7S515, respectively. Fine mapping refined the critical interval and suggested that markers in a 27.78 cM (27.96 Mb) interval are flanked by markers D7S660 and D7S799, which co-segregate with the disease phenotype in family PKCC108. CONCLUSIONS: We have identified a new locus for autosomal recessive congenital cataract, localized to chromosome 7q21.11-q31.1 in a consanguineous Pakistani family.

Mapping of a novel locus associated with autosomal recessive congenital cataract to chromosome 8p.

PURPOSE: To identify the disease locus for autosomal recessive congenital cataracts in a consanguineous Pakistani family. METHODS: All affected individuals underwent a detailed ophthalmologic examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was completed with fluorescently-labeled microsatellite markers on genomic DNA from affected and unaffected family members. Logarithms of odds (LOD) scores were calculated under a fully penetrant autosomal recessive model of inheritance. RESULTS: Ophthalmic examination suggested that affected individuals have bilateral cataracts. Linkage analysis localized the critical interval to chromosome 8p with LOD scores of 3.19, and 3.08 at θ=0, obtained with markers D8S549 and D8S550, respectively. Haplotype analyses refined the critical interval to 37.92 cM (16.28 Mb) region, flanked by markers, D8S277 proximally and D8S1734 distally. CONCLUSIONS: Here, we report a new locus for autosomal recessive congenital cataract mapped to chromosome 8p in a consanguineous Pakistani family.