The Role of Deleterious Substitutions in Crop Genomes.

Populations continually incur new mutations with fitness effects ranging from lethal to adaptive. While the distribution of fitness effects of new mutations is not directly observable, many mutations likely either have no effect on organismal fitness or are deleterious. Historically, it has been hypothesized that a population may carry many mildly deleterious variants as segregating variation, which reduces the mean absolute fitness of the population. Recent advances in sequencing technology and sequence conservation-based metrics for inferring the functional effect of a variant permit examination of the persistence of deleterious variants in populations. The issue of segregating deleterious variation is particularly important for crop improvement, because the demographic history of domestication and breeding allows deleterious variants to persist and reach moderate frequency, potentially reducing crop productivity. In this study, we use exome resequencing of 15 barley accessions and genome resequencing of 8 soybean accessions to investigate the prevalence of deleterious single nucleotide polymorphisms (SNPs) in the protein-coding regions of the genomes of two crops. We conclude that individual cultivars carry hundreds of deleterious SNPs on average, and that nonsense variants make up a minority of deleterious SNPs. Our approach identifies known phenotype-altering variants as deleterious more frequently than the genome-wide average, suggesting that putatively deleterious variants are likely to affect phenotypic variation. We also report the implementation of a SNP annotation tool BAD_Mutations that makes use of a likelihood ratio test based on alignment of all currently publicly available Angiosperm genomes.

Fast neutron-induced structural rearrangements at a soybean NAP1 locus result in gnarled trichomes.

KEY MESSAGE: Three adjacent and distinct sequence rearrangements were identified at a NAP1 locus in a soybean mutant. Genetic dissection and validation revealed the function of this gene in soybean trichome development. A soybean (Glycine max (L.) Merr.) gnarled trichome mutant, exhibiting stunted trichomes compared to wild-type, was identified in a fast neutron mutant population. Genetic mapping using whole genome sequencing-based bulked segregant analysis identified a 26.6 megabase interval on chromosome 20 that co-segregated with the phenotype. Comparative genomic hybridization analysis of the mutant indicated that the chromosome 20 interval included a small structural variant within the coding region of a soybean ortholog (Glyma.20G019300) of Arabidopsis Nck-Associated Protein 1 (NAP1), a regulator of actin nucleation during trichome morphogenesis. Sequence analysis of the candidate allele revealed multiple rearrangements within the coding region, including two deletions (approximately 1-2 kb each), a translocation, and an inversion. Further analyses revealed that the mutant allele perfectly co-segregated with the phenotype, and a wild-type soybean NAP1 transgene functionally complemented an Arabidopsis nap1 mutant. In addition, mapping and exon sequencing of NAP1 in a spontaneous soybean gnarled trichome mutant (T31) identified a frame shift mutation resulting in a truncation of the coding region. These data indicate that the soybean NAP1 gene is essential for proper trichome development and show the utility of the soybean fast neutron population for forward genetic approaches for identifying genes.

Crossed pulmonary arteries associated with single atrium in an adult: a case report.

BACKGROUND: Crossed pulmonary arteries or single atrium is a rare form of cardiovascular anomaly. In previous studies, the anomalies are detected in infant or early adolescence, and infrequently seen in adult population. CASE PRESENTATION: We presented a case of the coexistence of two congenital anomalies in a 44-year-old woman who remained well tolerated and undiscovered until adulthood. Physical examination showed a grade III systolic murmur at the cardiac apex, and a grade II/III systolic murmur at left 2-3 intercostal space. An echocardiography revealed absence of atrial septal tissue. Dual-source CT angiography was performed for further evaluation of the great vessel. Except an enlarged single atrium, the imaging showed that the origination of the left pulmonary artery from the pulmonary trunk was superior to that of the right pulmonary artery. The branch pulmonary arteries then crisscrossed as they coursed to their respective lungs. The findings were illustrated by the right heart catheterization and then confirmed at surgery. CONCLUSIONS: To our knowledge, this is the first case report of crossed pulmonary arteries with single atrium as the only additional cardiac anomaly in an adult. Knowledge of this rare combination will be helpful in the differential diagnosis of congenital heart disease and assist the surgeon in treatment planning.

Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies.

BACKGROUND: Alfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata. Due to its large and complex genome, there are few genomic resources available for alfalfa improvement. RESULTS: A de novo transcriptome assembly from two alfalfa subspecies, M. sativa ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes were assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626 unique transcript sequences. Nodule-specific and transcripts involved in cell wall biosynthesis were identified. Statistical analyses identified 20,447 transcripts differentially expressed between the two subspecies. Pair-wise comparisons of each tissue combination identified 58,932 sequences differentially expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing transcript abundance in floral tissues of B47 and F56 identified expression differences in sequences involved in anthocyanin and carotenoid synthesis, which determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to each M. sativa subspecies (110,241) were identified. CONCLUSIONS: The Medicago sativa Gene Index 1.2 increases the expressed sequence data available for alfalfa by ninefold and can be expanded as additional experiments are performed. The MSGI 1.2 transcriptome sequences, annotations, expression profiles, and SNPs were assembled into the Alfalfa Gene Index and Expression Database (AGED) at http://plantgrn.noble.org/AGED/ , a publicly available genomic resource for alfalfa improvement and legume research.

High-frequency modification of plant genes using engineered zinc-finger nucleases.

An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is at present lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better meet the world's burgeoning need for food, fibre and fuel. Zinc-finger nucleases (ZFNs)-enzymes engineered to create DNA double-strand breaks at specific loci-are potent stimulators of gene targeting; for example, they can be used to precisely modify engineered reporter genes in plants. Here we demonstrate high-frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetolactate synthase genes (ALS SuRA and SuRB), for which specific mutations are known to confer resistance to imidazolinone and sulphonylurea herbicides. Herbicide-resistance mutations were introduced into SuR loci by ZFN-mediated gene targeting at frequencies exceeding 2% of transformed cells for mutations as far as 1.3 kilobases from the ZFN cleavage site. More than 40% of recombinant plants had modifications in multiple SuR alleles. The observed high frequency of gene targeting indicates that it is now possible to efficiently make targeted sequence changes in endogenous plant genes.

Zinc Finger Database (ZiFDB) v2.0: a comprehensive database of C2H2 zinc fingers and engineered zinc finger arrays.

ZiFDB (Zinc Finger Database, http://zifdb.msi.umn.edu) is a web-accessible database that houses information on individual C(2)H(2) zinc fingers (ZFs) and engineered zinc finger arrays (ZFAs). ZiFDB serves as a resource for biologists interested in engineering ZFAs for use as sequence-specific DNA-binding reagents. Here, we describe four new features of ZiFDB: (i) the database allows users to input new ZFs and ZFAs; (ii) a shadow database temporarily stores user-submitted data, pending approval by the database curator and subsequent loading into the persistent database; (iii) ZiFDB contains 181 Context-Dependent Assembly (CoDA) ZFAs, which were generated by this newly described ZFA engineering platform; and (iv) the database also now contains 319 F1F2 CoDA units and 334 F2F3 CoDA units that can be used to construct CoDA arrays. In total, the new release of ZiFDB contains 1226 ZFs and 1123 ZFAs.

An RNA-Seq based gene expression atlas of the common bean.

BACKGROUND: Common bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species. RESULTS: Combining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form. CONCLUSIONS: These data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.

Evaluating the association between vascular remodeling and plaque calcification patterns of the carotid artery and its effects on ischemic symptoms using CT angiography.

Background: Arterial remodeling is a compensatory mechanism of the vessel wall in response to atherosclerotic plaque growth. However, the clinical significance of vascular remodeling of carotid lesions remains unclear. Through this study, we aimed to evaluate the association between vascular remodeling and ischemic symptoms in patients with an internal carotid artery (ICA) stenosis degree ≥50%, considering the differences in plaque calcification patterns. Methods: This retrospective cross-sectional study included adult patients with moderate-to-severe proximal ICA stenosis associated with atherosclerotic plaques admitted to the Zhejiang Hospital between September 2018 and March 2023. Parameters such as lumen diameter, plaque calcification types, calcium scores, and calcification thickness were assessed using non-contrast and contrast-enhanced computed tomography angiography (CTA). The remodeling ratio (RR) was calculated by dividing the maximum distance of the proximal ICA between the inner border of the arterial lumen at the plaque site and the outer borders of the plaque by the luminal diameter. Atherosclerosis risk factors and medications were recorded. The Mann-Whitney U test or chi-square test was used to compare the differences between groups. Correlations were measured using Pearson's correlation coefficient. Predictors of ischemic symptoms were assessed using multivariable logistic regression analysis, with results expressed as odds ratio (ORs) with 95% confidence intervals (CIs). A P value less than 0.05 (two-sided) was considered to indicate statistical significance The differences in RR among plaque calcification types and the association between vascular remodeling and clinical symptoms were analyzed. Results: A total of 242 ICAs in 196 patients were included in this study, and 84 were symptomatic and 158 were asymptomatic. The RR in symptomatic ICA [median, 1.31 (interquartile range, 1.17-1.68)] was significantly greater than that in asymptomatic group [median, 1.20 (interquartile range, 1.05-1.45)], P=0.006). Significant differences in RR existed among plaque calcification types, among which type 5 and 6 plaques had the highest RR. About 71.5% (173/242) of all ICAs showed positive remodeling. Significant correlations were observed between RR and ischemic symptoms and between positive remodeling and calcification thickness (P<0.05 for all variables). On multivariable logistic regression analysis, calcification thickness remained significantly associated with positive remodeling of carotid arteries (OR 2.30; 95% CI: 1.06-5.01; P=0.036). Conclusions: Arterial remodeling exists in the ICA. A significant association between arterial positive remodeling and plaque calcification thickness was established. RR helps predict ischemic symptoms. The results of our study suggest that arterial remodeling serves as a novel measure to help ascertain the risk stratification of ischemic events in carotid atherosclerotic disease.

Cholinergic hyperintensity pathways are associated with cognitive performance in patients with asymptomatic carotid artery stenosis.

OBJECTIVES: We aimed to determine whether asymptomatic carotid artery stenosis (ACS) induced cognitive impairments were related to the cholinergic hyperintensity pathway. METHODS: This cross-sectional study included patients with moderate-to-severe ACS, who were categorized into mild cognitive impairment (MCI) and normal cognition groups on the basis of Montreal Cognitive Assessment (MoCA) scores. The cholinergic pathway hyperintensity scale (CHIPS), Fazekas, and medial temporal atrophy (MTA) scores were assessed. SPSS software was used for statistical analyses. RESULTS: A total of 117 ACS patients (70.89 ± 8.81 years) and 105 controls (67.87 ± 9.49 years) were evaluated (t = 2.46, p = 0.015). The ACS group showed a worse median Mini-Mental Status Examination (MMSE) score (z = -2.41, p = 0.016) and MoCA score (z = -3.51, p < 0.001), and a significantly higher median total CHIPS score (z = 4.88, p < 0.001) and mean Fazekas score (t = 2.39, p = 0.018). In the correlation analysis, the MoCA score showed a significant negative correlation with the CHIPS score (ρ = -0.41, p < 0.001) and Fazekas score (ρ = -0.31, p < 0.001) in ACS group. Logistic regression analyses suggested that CHIPS scores were risk factors for MCI in patients with ACS (odds ratio [OR] = 1.07, 95% Confidence Interval [CI]1.01-1.13 and controls (OR = 1.09, 95%CI 1.01-1.17), while the MTA and Fazekas scores showed no predictive power. The receiver operating characteristic curve showed that the area under the curve of the CHIPS score for predicting MCI was 0.71 in ACS group, but was only 0.57 in controls. CONCLUSIONS: Patients with ACS showed poorer cognitive performance and higher CHIPS and Fazekas scores. CHIPS, but not Fazekas, scores were risk factors for cognitive impairment and were a valuable factor to predict MCI in patients with ACS.

Application of the Kaiser score by MRI in patients with breast lesions by ultrasound and mammography.

PURPOSE This study aimed to verify whether the use of the Kaiser score can improve the diagnostic performance in breast magnetic resonance imaging (MRI) for suspicious lesions and avoid further invasive diagnostic approaches. METHODS This retrospective study enrolled 97 patients who underwent breast MRI before undergoing breast biopsy or surgery. Evaluations were conducted on all MRI images individually by 2 radiologists using the Kaiser score. Neither radiologist had the knowledge of the final histopathological diagnosis. The ability of the Kaiser score in diagnosis was established via a receiver performing characteristic (ROC) analysis, which was measured by the area under the ROC curve (AUC). Youden index was used to define the optimal cutoff value. Kaiser score categories were dichotomized into positive (cutoff score > 4) and negative scores (cutoff score ≤ 4). Cohen's kappa coefficient was used to analyze the inter-rater agreement. RESULTS Histopathology revealed 56 malignant and 41 benign lesions. The AUC for all lesions evaluated by the Kaiser score was 0.992 (95% CI: 0.981-1.0) and 0.958 (95% CI: 0.920-0.996) for 2 radiologists, respectively. Inter-rater agreement of the dichotomized Kaiser score was excellent (κ=0.894, P < .001). A total of 20 lesions (33.8%) previously categorized as BI-RADS 4 were reduced to BI-RADS 2/3 (19 benign lesions and 1 malignant lesion). CONCLUSION The Kaiser score is a valuable auxiliary diagnostic tool for improving the diagnostic ability of radiologists, whose experiences in breast MRI are diverse. In some cases, the application of the Kaiser score could possibly avoid unnecessary breast biopsies.

New cerebral microbleeds in AF patients on non-vitamin K oral anticoagulants or warfarin: One-year follow-up.

ABSTRACT: Anticoagulant treatment increases the risk of intracerebral hemorrhage (ICH), but whether the treatment, more specifically non-vitamin K oral anticoagulants (NOACs), increases the risk of cerebral microbleeds (CMBs) remains uncertain. We performed this study to investigate the development of new CMBs due to NOACs or warfarin treatment in patients with atrial fibrillation (AF).We prospectively recruited AF patients before anticoagulation from June 2016 to June 2018. We performed susceptibility-weighted imaging (SWI) examinations on all enrolled AF patients and re-examined SWI 1 year later. We compared demographic features and new CMBs between the NOACs group and the warfarin group. Univariate analysis of clinical factors was performed according to the development of new CMBs; and age, a HAS-B(L)ED score, warfarin use, and the presence of baseline CMBs were then selected for inclusion in the multivariate logistic regression model.A total of 72 AF patients were recruited, 29 of whom were assigned to the NOACs group and 43 to the warfarin group. Finally, 1 patient in the NOACs group (3.4%) and 9 patients (20.9%) in the warfarin group developed new CMBs after 1 year follow-up (P = .08). Univariate analysis showed that age, a HAS-B(L)ED score ≥4, the presence of baseline CMBs were associated with the development of new CMBs (P < .05). And multivariate regression analysis showed baseline CMBs (P = .03, odds ratio = 6.37, 95% confidence interval 1.15-35.36) was independently related to the increase in new CMBs.AF patients treated with NOACs may have a decreased trend in the development of new CMBs compared with those treated with warfarin. Baseline CMBs increased the frequency of new CMBs during anticoagulant treatment. The development of new CMBs in AF patients with anticoagulation requires further longitudinal studies with longer follow-up in larger samples.

Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays.

Zinc fingers are the most abundant DNA-binding motifs encoded by eukaryotic genomes and one of the best understood DNA-recognition domains. Each zinc finger typically binds a 3-nt target sequence, and it is possible to engineer zinc-finger arrays (ZFAs) that recognize extended DNA sequences by linking together individual zinc fingers. Engineered zinc-finger proteins have proven to be valuable tools for gene regulation and genome modification because they target specific sites in a genome. Here we describe ZiFDB (Zinc Finger Database; http://bindr.gdcb.iastate.edu/ZiFDB), a web-accessible resource that compiles information on individual zinc fingers and engineered ZFAs. To enhance its utility, ZiFDB is linked to the output from ZiFiT--a software package that assists biologists in finding sites within target genes for engineering zinc-finger proteins. For many molecular biologists, ZiFDB will be particularly valuable for determining if a given ZFA (or portion thereof) has previously been constructed and whether or not it has the requisite DNA-binding activity for their experiments. ZiFDB will also be a valuable resource for those scientists interested in better understanding how zinc-finger proteins recognize target DNA.

Predicting success of oligomerized pool engineering (OPEN) for zinc finger target site sequences.

BACKGROUND: Precise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN) is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs) designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable. RESULTS: Analysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity+ of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88%) and specificity+ (92%), but with reduced ROC AUC (0.77). Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT. CONCLUSION: ZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function successfully in vivo, substantially reducing the experimental effort required. ZiFOpT is freely available and incorporated in the Zinc Finger Targeter web server (http://bindr.gdcb.iastate.edu/ZiFiT).

Evaluation of multislice computed tomographic perfusion imaging and computed tomographic angiography on traumatic cerebral infarction.

OBJECTIVE: To evaluate the application value of multislice computed tomographic perfusion imaging (MSCTPI) and multislice computed tomographic angiography (MSCTA) on traumatic cerebral infarction. METHODS: MSCTA was performed on 10 patients who were initially diagnosed as traumatic cerebral infarction by normal conventional computed tomography (NCCT), among whom, 3 patients were examined by MSCTPI simultaneously. Reconstructed images of the intracranial artery were made with techniques of maximum intensity projection (MIP) and volume rendering (VR) from MSCTA scanning data. Then the graph of function of four parameters, regional cerebral blood flow (rCBF), regional cerebral blood volume (rCBV), mean transit time (MTT), and time to peak (TTP), acquired by the perfusing analysis software was obtained. RESULTS: Among the 10 patients with traumatic cerebral infarction, 6 showed complex type on NCCT, which depicted abnormality on MSCTA, and 4 showed simple type on NCCT, which had negative results on MSCTA. Among the 4 patients with abnormal great vessels, 2 suffered from stenosis or occlusion of the middle cerebral artery, 1 from spasm of the anterior cerebral artery, and 1 from spasm of the vertebral-basal artery. The image of MSCTPI of 1 patient with massive cerebral infarction on the right cerebral hemisphere confirmed by CT was smaller than those of the other patients, which showed occlusion of the ipsilateral middle cerebral artery on MSCTA. Among the 6 patients whose MSCTA showed no abnormality, 4 showed simple infarction and 2 showed complex infarction. The infarction focus of 5 patients occurred in the basal ganglia and 1 in the splenium of corpus callosum. Among the 2 cases of small cerebral infarction volume on NCCT, one was normal, the other showed hypoperfusion on MSCTPI and was normal on MSCTA. CONCLUSION: The combination of MSCTPI and MSCTA is very useful for evaluating the change of intracranial artery in ischemic regions and assessing the cerebral hemodynamic information of traumatic cerebral infarction.

A rare case of single right coronary artery with congenital absence of left coronary artery in an adult: a case report.

Single right coronary artery with congenital absence of left coronary artery is one of the rarest coronary artery anomalies. Most coronary anomalies are asymptomatic and incidental findings. We report a case of single right coronary artery with congenital absence of left coronary artery detected by coronary CT angiography. Physical examination revealed a well-nourished female with a blood pressure of 130/75 mmHg and a pulse rate of 56 beats per minute. The myocardial enzymes and blood lipid levels showed normal findings. The dynamic electrocardiogram revealed frequent ventricular premature beats. Dual-source CT angiography was performed for evaluation of coronary artery. The imaging showed a very large single coronary artery arising from the right coronary sinus of Valsalva, and demonstrated absence of the left coronary artery. Meanwhile, the findings were confirmed by coronary angiography.

A roadmap for functional structural variants in the soybean genome.

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits.

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly.

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.