DNA Pooling Strategy Research Articles

Genotyping of DNA pools identifies untapped landraces and genomic regions to develop next-generation varieties.

Landraces, that is, traditional varieties, have a large diversity that is underexploited in modern breeding. A novel DNA pooling strategy was implemented to identify promising landraces and genomic regions to enlarge the genetic diversity of modern varieties. As proof of concept, DNA pools from 156 American and European maize landraces representing 2340 individuals were genotyped with an SNP array to assess their genome-wide diversity. They were compared to elite cultivars produced across the 20th century, represented by 327 inbred lines. Detection of selective footprints between landraces of different geographic origin identified genes involved in environmental adaptation (flowering times, growth) and tolerance to abiotic and biotic stress (drought, cold, salinity). Promising landraces were identified by developing two novel indicators that estimate their contribution to the genome of inbred lines: (i) a modified Roger's distance standardized by gene diversity and (ii) the assignation of lines to landraces using supervised analysis. It showed that most landraces do not have closely related lines and that only 10 landraces, including famous landraces as Reid's Yellow Dent, Lancaster Surecrop and Lacaune, cumulated half of the total contribution to inbred lines. Comparison of ancestral lines directly derived from landraces with lines from more advanced breeding cycles showed a decrease in the number of landraces with a large contribution. New inbred lines derived from landraces with limited contributions enriched more the haplotype diversity of reference inbred lines than those with a high contribution. Our approach opens an avenue for the identification of promising landraces for pre-breeding.

GENETIC DIVERSITY AMONG THE PHILIPPINE TRADITIONAL MAIZE (ZEA MAYS L.) POPULATIONS BASED ON SSR MARKERS

This paper reports the first genetic diversity analysis of Philippine traditional maize populations performed through a cost-effective DNA pooling strategy. The diversity among selected 100 traditional maize populations collected from Luzon, Visayas, and Mindanao was evaluated using twenty simple sequence repeats (SSR) markers at the Institute of Plant Breeding, University of the Philippines Los Baños, Laguna, Philippines. A total of 138 bands ranging from two to 12 bands per primer were detected. The average number of polymorphic alleles, polymorphism rate, effective multiplex ratio, marker index, resolving power, and expected heterozygosity are 6.283, 87.17%, 5.798, 4.104, 15.897, and 0.658, respectively. The polymorphism information content (PIC) varied between 0.141 to 0.848, with an average value of 0.620. A dendrogram was constructed with a dissimilarity coefficient ranging from 0.14 to 0.55 and a mean dissimilarity index of 0.425. Cluster analysis revealed 13 groups based on the result of Approximately Unbiased (AU) p-values from 10,000 bootstrap iterations. The cluster analysis enabled the classification of populations with ambiguous places of origin. Analysis of molecular variance (AMOVA) showed higher within-population diversity (70%) than among-population diversity (30%) with PhiPT (pairwise genetic differentiation metric) of 0.298 (P = 0.001). These results revealed the significant diversity of traditional maize populations in the Philippines and the power of SSR markers in diversity and cluster analyses despite the age of this marker technology. These findings will aid plant breeders in developing approaches towards knowledgeable and efficient execution of breeding programs using traditional maize populations.

A reference-guided TILLING by amplicon-sequencing platform supports forward and reverse genetics in barley

Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe, making it an attractive model for genetic studies in Triticeae crops. The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations. In this study, we report an ethyl methanesulfonate (EMS)-mutagenized population consisting of 8525 M3 lines in the barley landrace “Hatiexi” (HTX), which we complement with a high-quality de novo assembly of a reference genome for this genotype. The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase, depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis. We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING (targeting induced locus lesion in genomes) platform in barley. Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals. We discovered abundant allelic mutants for dozens of genes, including the barley Green Revolution contributor gene Brassinosteroid insensitive 1 (BRI1). As a proof of concept, we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy, demonstrating the value of this resource to support forward and reverse genetic studies in barley.

MLIP genotype as a predictor of pharmacological response in primary open-angle glaucoma and ocular hypertension

Predicting the therapeutic response to ocular hypotensive drugs is crucial for the clinical treatment and management of glaucoma. Our aim was to identify a possible genetic contribution to the response to current pharmacological treatments of choice in a white Mediterranean population with primary open-angle glaucoma (POAG) or ocular hypertension (OH). We conducted a prospective, controlled, randomized, partial crossover study that included 151 patients of both genders, aged 18 years and older, diagnosed with and requiring pharmacological treatment for POAG or OH in one or both eyes. We sought to identify copy number variants (CNVs) associated with differences in pharmacological response, using a DNA pooling strategy of carefully phenotyped treatment responders and non-responders, treated for a minimum of 6 weeks with a beta-blocker (timolol maleate) and/or prostaglandin analog (latanoprost). Diurnal intraocular pressure reduction and comparative genome wide CNVs were analyzed. Our finding that copy number alleles of an intronic portion of the MLIP gene is a predictor of pharmacological response to beta blockers and prostaglandin analogs could be used as a biomarker to guide first-tier POAG and OH treatment. Our finding improves understanding of the genetic factors modulating pharmacological response in POAG and OH, and represents an important contribution to the establishment of a personalized approach to the treatment of glaucoma.

Exploiting sorghum genetic diversity for enhanced aluminum tolerance: Allele mining based on the AltSB locus

Root damage due to aluminum (Al) toxicity restricts crop production on acidic soils, which are extensive in the tropics. The sorghum root Al-activated citrate transporter, SbMATE, underlies the Al tolerance locus, AltSB, and increases grain yield under Al toxicity. Here, AltSB loci associated with Al tolerance were converted into Amplification Refractory Mutation System (ARMS) markers, which are cost effective and easy to use. A DNA pooling strategy allowed us to identify accessions harboring rare favorable AltSB alleles in three germplasm sets while greatly reducing genotyping needs. Population structure analysis revealed that favorable AltSB alleles are predominantly found in subpopulations enriched with guinea sorghums, supporting a possible Western African origin of AltSB. The efficiency of allele mining in recovering Al tolerance accessions was the highest in the largest and highly diverse germplasm set, with a 10-fold reduction in the number of accessions that would need to be phenotyped in the absence of marker information. Finally, Al tolerant accessions were found to rely on SbMATE to exclude Al3+ from sensitive sites in the root apex. This study emphasizes gene-specific markers as important tools for efficiently mining useful rare alleles in diverse germplasm, bridging genetic resource conservation efforts and pre-breeding for Al tolerance.

Pool deconvolution approach for high-throughput gene mining from Bacillus thuringiensis.

Novel genes from Bacillus thuringiensis (Bt) are required for effective deployment in agriculture, human health, and forestry. In an improvement over conventional PCR-based screening, next generation sequencing (NGS) has been used for identification of new genes of potential interest from Bt strains, but cost becomes a constraint when several isolates are to be sequenced. We demonstrate the potential of a DNA pooling strategy known as pool deconvolution to identify commercially important toxin genes from 36 native Bt isolates. This strategy is divided into three steps: (a) DNA pooling, (b) short read sequence assembly followed by gene mining, and (c) host isolate identification. With this approach, we have identified insecticidal protein (ip) genes including nine three-domain (3D) cry genes, three cyt-type genes, three mtx genes (mosquitocidal toxin), and one bin and vip-type gene each. Three cry-type and three cyt-type genes were cloned, out of which, two cry-type genes, ip11 and ip13, were named as cry4Ca2 and cry52Ca1, respectively by the Bacillus thuringiensis nomenclature committee ( http://www.biols.susx.ac.uk/Home/Neil_Crickmore/BT/ ). Our results show that the pool deconvolution approach is well suited for high-throughput gene mining in bacteria.

Enabling Reverse Genetics in Medicago truncatula Using High-Throughput Sequencing for Tnt1 Flanking Sequence Recovery.

The genome sequence of Medicago truncatula was published and released in 2011. A Tnt1 insertional mutant population with 21,700 independently regenerated lines was completed in 2012 at The Samuel Roberts Noble Foundation. With an estimated 25 insertions per line, the Tnt1 mutant population harbors more than 500,000 insertions in the M. truncatula genome. Based on the genome size, average gene length, and random insertion of Tnt1into the genome, the mutant population affects about 90% of genes in the M. truncatula genome. Therefore, the mutant population enables functional characterization of most genes in the M. truncatula genome. From 2006 to 2011, we sequenced about 33,000 flanking sequence tags (FSTs) from 2600 Tnt1 lines using TAIL-PCR followed by TA cloning, plasmid isolation, and traditional Sanger sequencing. To accelerate FST sequencing, we developed a two-dimensional DNA pooling strategy coupled with next-generation sequencing and produced about 380,000 FSTs from all 21,700 lines in a relatively short time. All FSTs are BLAST searchable in a web-based database. One can quickly search the database to find M. truncatula mutant lines with Tnt1 insertions in most genes of interest.

GWAS study using DNA pooling strategy identifies association of variant rs4910623 in OR52B4 gene with anti-VEGF treatment response in age-related macular degeneration.

Pooled DNA based GWAS to determine genetic association of SNPs with visual acuity (VA) outcome in anti-vascular endothelial growth factor (anti-VEGF) treated neovascular age-related macular degeneration (nAMD) patients. We performed pooled DNA based GWAS on 285 anti-VEGF treated nAMD patients using high density Illumina 4.3 M array. Primary outcome was change in VA in Early Treatment Diabetic Retinopathy Study (ETDRS) letters after 6 months of anti-VEGF treatment (patients who lost ≥5 ETDRS letters classified as non-responders and all remaining classified as responders). GWAS analysis identified 44 SNPs of interest: 37 with strong evidence of association (p < 9 × 10−8), 2 in drug resistance genes (p < 5 × 10−6) and 5 nonsynonymous changes (p < 1 × 10−4). In the validation phase, individual genotyping of 44 variants showed three SNPs (rs4910623 p = 5.6 × 10−5, rs323085 p = 6.5 × 10−4 and rs10198937 p = 1.30 × 10−3) remained associated with VA response at 6 months. SNP rs4910623 also associated with treatment response at 3 months (p = 1.5 × 10−3). Replication of these three SNPs in 376 patients revealed association of rs4910623 with poor VA response after 3 and 6 months of treatment (p = 2.4 × 10−3 and p = 3.5 × 10−2, respectively). Meta-analysis of both cohorts (673 samples) confirmed association of rs4910623 with poor VA response after 3 months (p = 1.2 × 10−5) and 6 months (p = 9.3 × 10−6) of treatment in nAMD patients.

Genomic analyses of tropical beef cattle fertility based on genotyping pools of Brahman cows with unknown pedigree.

We introduce an innovative approach to lowering the overall cost of obtaining genomic EBV (GEBV) and encourage their use in commercial extensive herds of Brahman beef cattle. In our approach, the DNA genotyping of cow herds from 2 independent properties was performed using a high-density bovine SNP chip on DNA from pooled blood samples, grouped according to the result of a pregnancy test following their first and second joining opportunities. For the DNA pooling strategy, 15 to 28 blood samples from the same phenotype and contemporary group were allocated to pools. Across the 2 properties, a total of 183 pools were created representing 4,164 cows. In addition, blood samples from 309 bulls from the same properties were also taken. After genotyping and quality control, 74,584 remaining SNP were used for analyses. Pools and individual DNA samples were related by means of a "hybrid" genomic relationship matrix. The pooled genotyping analysis of 2 large and independent commercial populations of tropical beef cattle was able to recover significant and plausible associations between SNP and pregnancy test outcome. We discuss 24 SNP with significant association ( < 1.0 × 10) and mapped within 40 kb of an annotated gene. We have established a method to estimate the GEBV in young herd bulls for a trait that is currently unable to be predicted at all. In summary, our novel approach allowed us to conduct genomic analyses of fertility in 2 large commercial Brahman herds managed under extensive pastoral conditions.

Analysis of Common Single Nucleotide Polymorphisms in Complex Regional Pain Syndrome: Genome Wide Association Study Approach and Pooled DNA Strategy.

The objective of this study was to use a genome-wide association (GWAS) approach and pooled DNA strategy to search for new genomic loci associated with complex regional pain syndrome (CRPS). The study cohort consisted of 230 patients with established diagnosis of CRPS. The control group consisted of 230 age- and gender-matched subjects without chronic pain. We tested the association of common single nucleotide polymorphisms (SNPs), genotyped using a high-density microarray platform, with CRPS phenotype. This was followed by individual genotyping of the most significant SNPs identified in the microarray genomic scan, in both original discovery (N = 115) and independent verification (N = 115) groups of patients with CRPS, as well as in the appropriate matched control subjects. The results of our study provide no support for the initial hypothesis of the existence of an association between any investigated genomic targets (including GWAS for all genomic loci available on the microarray, and focused scan of the HLA locus on chromosome 6) and CRPS phenotype. Despite the fact that we interrogated about 83% of all of common SNPs in the human genome, we did not find evidence that any of the investigated common SNPs may be associated with CRPS phenotype.

Identification of genetic variation in the swine toll-like receptors and development of a porcine TLR genotyping array.

BackgroundToll-like receptors (TLR) are crucial in innate immunity for the recognition of a broad range of microbial pathogens and are expressed in multiple cell types. There are 10 TLR genes described in the pig genome.ResultsWith a twofold objective i.e. to catalogue genetic variants in porcine TLR genes and develop a genotyping array for genetic association studies on immune-related traits, we combined targeted sub-genome enrichment and high-throughput sequencing to sequence the 10 porcine TLR genes in 266 pigs from 10 breeds and wild boars using a DNA-pooling strategy. We identified 306 single nucleotide variants across the 10 TLR and 11 populations, 87 of which were novel. One hundred and forty-seven positions i.e. six stop-gains and 141 non-synonymous substitutions were predicted to alter the protein sequence. Three positions were unique to a single breed with alternative allele frequencies equal to or higher than 0.5. We designed a genotyping array for future applications in genetic association studies, with a selection of 126 variants based on their predicted impact on protein sequence. Since TLR4, TLR7 and TLR9 were underrepresented in this selection, we also included three variants that were located in the 3′UTR of these genes. We tested the array by genotyping 214 of the 266 sequenced pigs. We found that 93 variants that involved the 10 TLR genes were polymorphic in these animals. Twelve of these variants were novel. Furthermore, seven known variants that are associated with immune-related phenotypes are present on the array and can thus be used to test such associations in additional populations.ConclusionsWe identified genetic variations that potentially have an impact on the protein sequence of porcine TLR. A genotyping array with 80 non-synonymous, 10 synonymous and three 3′UTR polymorphisms in the 10 TLR genes is now available for association studies in swine populations with measures on immune-related traits.Electronic supplementary materialThe online version of this article (doi:10.1186/s12711-016-0206-0) contains supplementary material, which is available to authorized users.

A permutation test for oligoset DNA pooling studies.

Case-control association studies often suffer from population stratification bias. A previous triple combination strategy of stratum matching, genomic controlling, and multiple DNA pooling can correct the bias and save genotyping cost. However the method requires researchers to prepare a multitude of DNA pools—more than 30 case-control pooling sets in total (polyset). In this paper, the authors propose a permutation test for oligoset DNA pooling studies. Monte-Carlo simulations show that the proposed test has a type I error rate under control and a power comparable to that of individual genotyping. For a researcher on a tight budget, oligoset DNA pooling is a viable option.

Case-control genome-wide association study of persistent attention-deficit hyperactivity disorder identifies FBXO33 as a novel susceptibility gene for the disorder.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder with high heritability. At least 30% of patients diagnosed in childhood continue to suffer from ADHD during adulthood and genetic risk factors may play an essential role in the persistence of the disorder throughout lifespan. To date, genome-wide association studies (GWAS) of ADHD have been completed in seven independent datasets, six of which were pediatric samples and one on persistent ADHD using a DNA-pooling strategy, but none of them reported genome-wide significant associations. In an attempt to unravel novel genes for the persistence of ADHD into adulthood, we conducted the first two-stage GWAS in adults with ADHD. The discovery sample included 607 ADHD cases and 584 controls. Top signals were subsequently tested for replication in three independent follow-up samples of 2104 ADHD patients and 1901 controls. None of the findings exceeded the genome-wide threshold for significance (PGC<5e-08), but we found evidence for the involvement of the FBXO33 (F-box only protein 33) gene in combined ADHD in the discovery sample (P=9.02e-07) and in the joint analysis of both stages (P=9.7e-03). Additional evidence for a FBXO33 role in ADHD was found through gene-wise and pathway enrichment analyses in our genomic study. Risk alleles were associated with lower FBXO33 expression in lymphoblastoid cell lines and with reduced frontal gray matter volume in a sample of 1300 adult subjects. Our findings point for the first time at the ubiquitination machinery as a new disease mechanism for adult ADHD and establish a rationale for searching for additional risk variants in ubiquitination-related genes.

Haplotype-specific single-locus multiplex PCR assay for molecular identification of sea-bob shrimp, Xiphopenaeus kroyeri (Heller, 1862), cryptic species from the Southwest Atlantic using a DNA pooling strategy for simultaneous identification of multiple samples

This work describes a single-locus multiplex PCR assay based on partial COI mitochondrial gene polymorphisms for identification of two Atlantic cryptic species of the sea-bob shrimp, Xiphopenaeus kroyeri (Heller, 1862) that were recently identified using molecular approaches. Reliable identification of cryptic species of Xiphopenaeus spp. has fundamental implications for management and conservation of the sea-bob shrimp fishery stocks. The assay was developed based on sequence polymorphisms of 130 specimens of both species, comprising samples from Venezuela to the southern coast of Brazil, validated by the amplification of 368 adult shrimp samples from nine different locations and confirmed by direct sequencing. The methodology has been optimized to enable the identification of equi-molar mixtures of DNA from up to 10 individuals by PCR reaction, allowing the fast and cheap identification of many specimens for large scale studies on fisheries biology and population genetics. The DNA pooling strategy enabled the identification of a new locality of occurrence of Xiphopenaeus sp. II in the Brazilian coast, Caravelas, indicating that the species distribution may be continuous on the coast, and not disjoint as observed so far.

Identifying Potential Regions of Copy Number Variation for Bipolar Disorder.

Bipolar disorder is a complex psychiatric disorder with high heritability, but its genetic determinants are still largely unknown. Copy number variation (CNV) is one of the sources to explain part of the heritability. However, it is a challenge to estimate discrete values of the copy numbers using continuous signals calling from a set of markers, and to simultaneously perform association testing between CNVs and phenotypic outcomes. The goal of the present study is to perform a series of data filtering and analysis procedures using a DNA pooling strategy to identify potential CNV regions that are related to bipolar disorder. A total of 200 normal controls and 200 clinically diagnosed bipolar patients were recruited in this study, and were randomly divided into eight control and eight case pools. Genome-wide genotyping was employed using Illumina Human Omni1-Quad array with approximately one million markers for CNV calling. We aimed at setting a series of criteria to filter out the signal noise of marker data and to reduce the chance of false-positive findings for CNV regions. We first defined CNV regions for each pool. Potential CNV regions were reported based on the different patterns of CNV status between cases and controls. Genes that were mapped into the potential CNV regions were examined with association testing, Gene Ontology enrichment analysis, and checked with existing literature for their associations with bipolar disorder. We reported several CNV regions that are related to bipolar disorder. Two CNV regions on chromosome 11 and 22 showed significant signal differences between cases and controls (p < 0.05). Another five CNV regions on chromosome 6, 9, and 19 were overlapped with results in previous CNV studies. Experimental validation of two CNV regions lent some support to our reported findings. Further experimental and replication studies could be designed for these selected regions.

Frequent methylation of HOXA9 gene in tumor tissues and plasma samples from human hepatocellular carcinomas

Aberrant DNA methylation is associated with the development of hepatocellular carcinoma (HCC), suggesting that gene methylation could be a potential biomarker for detection of HCC. The aim of this study is to identify potential biomarkers in HCC. We used the Infinium methylation array and a DNA-pooling strategy to analyze the genome-wide methylation profile in HCC. Quantitative methylation-specific PCR (Q-MSP) was used to validate homeobox A9 (HOXA9) methylation in 29 normal controls, 100 HCC samples and adjacent non-tumor tissues and in 74 plasma samples, including 40 patients with HCC. Ten genes (HOXA9, NEUROG1, TNFRSF10C, IRAK3, GFPT2, ZNF177, DPYSL4, ELOVL4, FSD1, and CACNA1G) showed differences in methylation between controls and HCCs. Of these, HOXA9 was significantly hypermethylated in HCCs (76.7%; 23/30) compared with controls (3.4%; 1/29). In addition, combination analysis of two- and three-gene sets for HCC detection showed greater sensitivity (90%-96.7%) and comparable specificity (93.1%-96.6%) to each individual gene (33.3%-76.7% and 55.2%-100.0%). HOXA9 methylation was further validated by Q-MSP in two independent set of clinical samples including 100 HCC and paired non-tumor tissues. Further, HOXA9 methylation could be detected in plasma from HCC patients (n=40) but not in normal plasma (n=34) (p<0.0005). Combined testing (either parameter positive) for α-fetoprotein (AFP, a plasma protein biomarker) and HOXA9 methylation showed greater sensitivity (94.6%) for detection of HCC than AFP alone (75.7%). These data suggest that methylation of HOXA9 could be a helpful biomarker to assist in HCC detection.

New single nucleotide polymorphisms associated with supraventricular arrythmias in patients with pacemaker implantation: genome-wide association approach and pooled DNA strategy

Aims: The objective of this study was to use genome-wide association approach and pooled DNA strategy to search for new genomic loci associated with inter-individual differences in patients with pacemakers placed for sinus node dysfunction in form of sick sinus syndrome (SSS) or tachycardia-bradycardia syndrome (TB) and/or atrio-ventricular (AV) conduction disturbances (AV blocks). Methods and results: Study cohort consisted of 619 patients (recruited from the areas of central Poland) with pacemaker placement and 66 control patients (no pacemaker implantation or history of atrial arrhythmias). We tested association of single nucleotide polymorphisms (SNPs) genotyped using high density microarray platform with 3 groups separated on the basis of the diagnosis associated with pacemaker implantation (AV n=233, SSS n=171, and TB n=215), followed by individual genotyping of most significant SNPs identified in the microarray genomic scan. From the number of 23 initially genotyped SNPs we identified SNP rs2200733 located in the proximity of the PITX2 gene, which showed statistically significant differences between controls (MAF=11%) and 3 pacemaker groups (TB 24.2%; SSS 13.3% and AV 17.4%). The highest statistical significance (p value of 0.005 for minor allele frequency difference in individual genotyping) was observed for SNP in the TB group and AV group (p=0.016) when compared with control. Conclusions: Previous genome-wide association studies implicated a region of human chromosome 4q25 in familial atrial fibrillation (AF) and atrial flutter (AFl), approximately 150 kb distal to the PITX2 homeobox gene, a developmental left-right asymmetry (LRA) gene. In this respect the observed difference in allelic frequency for the investigated PITX2 polymorphism may be associated with the etiology underlying pacemaker placement in the TB group which was, in contrast to SSS group, associated with the frequent episodes of underlying AF. The genome-wide approach might provide an opportunity to identify novel candidate genes and pathways related to the diagnosis of cardiac arrhythmias and indication for the pacemaker implantation. In addition, our findings implicate PITX2 and PITX2-mediated LRA-signaling pathways in prevention of atrial arrhythmias.

Genomic architecture of bovine κ-casein and β-lactoglobulin

The objective of this study was to characterize the genetic architecture underlying the absolute concentrations of 2 important milk proteins, κ-casein (κ-CN) and β-lactoglobulin (β-LG), in a backcross population of (Holstein × Jersey) × Holstein cattle. A genome-wide association analysis was performed using a selective DNA pooling strategy and the Illumina BovineHD BeadChip assay [777,000 (777K) SNP markers; Illumina Inc., San Diego, CA]. After correction for multiple testing, 25 single nucleotide polymorphisms were found to be associated with κ-CN and 36 single nucleotide polymorphisms were associated with β-LG. A pathway association analysis revealed 15 Gene Ontology (GO) terms associated with the κ-CN trait and 28 GO terms associated with β-LG. In addition, several GO terms were associated with both milk proteins. Further analysis revealed that κ-CN and β-LG production is regulated by both kinase and phosphatase activity, including mechanisms regulating the extracellular matrix. These results are in concordance with the complex multihormonal process controlling the expression of milk proteins and interactions between mammary epithelial cells and extracellular matrix components. Although κ-CN and β-LG milk proteins are expressed by single genes, the results from this study showed that many loci are involved in the regulation of the concentration of these 2 proteins.

SMART – Sunflower Mutant population And Reverse genetic Tool for crop improvement

BackgroundSunflower (Helianthus annuus L.) is an important oilseed crop grown widely in various areas of the world. Classical genetic studies have been extensively undertaken for the improvement of this particular oilseed crop. Pertaining to this endeavor, we developed a “chemically induced mutated genetic resource for detecting SNP by TILLING” in sunflower to create new traits.ResultsTo optimize the EMS mutagenesis, we first conducted a “kill curve” analysis with a range of EMS dose from 0.5% to 3%. Based on the observed germination rate, a 50% survival rate i.e. LD50, treatment with 0.6% EMS for 8 hours was chosen to generate 5,000 M2 populations, out of which, 4,763 M3 plants with fertile seed set. Phenotypic characterization of the 5,000 M2 mutagenised lines were undertaken to assess the mutagenesis quality and to identify traits of interest. In the M2 population, about 1.1% of the plants showed phenotypic variations. The sunflower TILLING platform was setup using Endo-1-nuclease as mismatch detection system coupled with an eight fold DNA pooling strategy. As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations.ConclusionBased on the TILLING of FatA and SAD genes, we calculated the overall mutation rate to one mutation every 480 kb, similar to other report for this crop so far. As sunflower is a plant model for seed oil biosynthesis, we anticipate that the developed genetic resource will be a useful tool to identify novel traits for sunflower crop improvement.

New single nucleotide polymorphisms associated with differences in platelets reactivity in patients with type 2 diabetes treated with acetylsalicylic acid: genome-wide association approach and pooled DNA strategy

The objective of this study was to use genome-wide association approach and pooled DNA strategy to search for new genomic loci associated with inter-individual differences in platelet reactivity in the diabetic patients during acetylsalicylic acid (ASA) treatment. Study cohort consisted of 297 diabetic patients who had been taking ASA (75mg daily) for at least 3months. We tested association of single nucleotide polymorphisms (SNPs) genotyped using high density microarray platform with several platelet reactivity assays, followed by individual genotyping of most significant SNPs identified in the microarray genomic scan. The highest statistical significance (p value of 0.0001-0.008 in individual genotyping) was observed for SNP located within the regulatory G-protein signaling (RGS) 7 gene (rs2502448) using recessive genetic model. The diabetic patients on ASA treatment and homozygotes for its minor allele were characterized by increased odds ratio of at 3.45 (confidence interval: 1.82-6.53) for high on ASA platelet reactivity (i.e. impaired ASA response) when compared with homozygotes for wild-type allele. The genome-wide approach might provide an opportunity to identify novel candidate genes and pathways related to platelet activation in diabetic patients.