Next-generation transcriptome sequencing, SNP discovery and validation in four market classes of peanut, Arachis hypogaea L.

Single-nucleotide polymorphisms, which can be identified in the thousands or millions from comparisons of transcriptome or genome sequences, are ideally suited for making high-resolution genetic maps, investigating population evolutionary history, and discovering marker-trait linkages. Despite significant results from their use in human genetics, progress in identification and use in plants, and particularly polyploid plants, has lagged. As part of a long-term project to identify and use SNPs suitable for these purposes in cultivated peanut, which is tetraploid, we generated transcriptome sequences of four peanut cultivars, namely OLin, New Mexico Valencia C, Tamrun OL07 and Jupiter, which represent the four major market classes of peanut grown in the world, and which are important economically to the US southwest peanut growing region. CopyDNA libraries of each genotype were used to generate 2 × 54 paired-end reads using an Illumina GAIIx sequencer. Raw reads were mapped to a custom reference consisting of Tifrunner 454 sequences plus peanut ESTs in GenBank, compromising 43,108 contigs; 263,840 SNP and indel variants were identified among four genotypes compared to the reference. A subset of 6 variants was assayed across 24 genotypes representing four market types using KASP chemistry to assess the criteria for SNP selection. Results demonstrated that transcriptome sequencing can identify SNPs usable as selectable DNA-based markers in complex polyploid species such as peanut. Criteria for effective use of SNPs as markers are discussed in this context.

ANCA-associated vasculitis presenting with isolated neurological manifestations in a patient with cocaine abuse: a case report and literature review.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of rare conditions predominantly affecting small vessels of skin, musculoskeletal, pulmonary, renal, and rarely central and peripheral nervous systems. Isolated neurological manifestations of AAV are uncommon and challenging to diagnose. Cocaine has been reported as a potential trigger for the development of AAV. There are only a few case reports of isolated neurological involvement in cocaine-induced AAV with poorly characterized histopathological features. We present a unique case of AAV with isolated neurological manifestations presenting with multiple cranial neuropathies, leptomeningeal enhancement on imaging and histopathologic evidence of small-vessel vasculitis in the leptomeninges and brain and extensive dural fibrosis in a patient with cocaine abuse. The patient's progressive neurological deficits were controlled after starting immunosuppression with rituximab and prednisone. We also reviewed the literature to provide the diagnostic overview of AAV and evaluate intervention options. To our knowledge, this is the first case of AAV with isolated neurological manifestations and histopathologic evidence of small-vessel vasculitis in a patient with cocaine abuse. Patients with multiple cranial neuropathies and meningeal involvement should be screened for AAV, especially if they have a history of cocaine abuse.

Less-Invasive Approach to Early-Onset Scoliosis-Surgical Technique for Magnetically Controlled Growing Rod (MCGR) Based on Treatment of 2-Year-Old Child with Severe Scoliosis.

BACKGROUND: Spinal deformities in children can be caused by various etiologies, such as congenital, syndromic, neuromuscular, or idiopathic. Early-onset scoliosis (EOS) is diagnosed before the age of ten years, and when the curvature continues to progress and exceeds a Cobb angle of 60-65 degrees, surgical treatment should be considered. Initial minimally invasive surgery and the implantation of magnetically controlled growing rods (MCGRs) allows for the noninvasive distraction of the spine, growing, and avoids multiple operations associated with the classic distractions of standard growing rods. CASE PRESENTATION: A 2-year-old girl was admitted to our clinic with rapidly progressive thoracic scoliosis. The major curve of the thoracic spine Cobb angle was 122° at 30 months. No congenital deformities were detected. The surgical technique was the less-invasive percutaneous and subfascial implantation of MCGRs, without long incisions on the back and the non-invasive ambulatory lengthening of her spine over the next 4 years. CONCLUSIONS: MCGR is a safe procedure for EOS patients. It is extremely effective at correcting spinal deformity; controlling the growth and curvature of the spine as the child develops during growth; reducing the number of hospitalizations and anesthesia; and minimizing the physical and mental burden of young patients, parents, and their families.

No Benefits in Using Magnetically Controlled Growing Rod as Temporary Internal Distraction Device in Staged Surgical Procedure for Management of Severe and Neglected Scoliosis in Adolescents.

BACKGROUND: Severe spinal curvatures (SSCs) in children and adolescents have long been treated with preoperative Halo traction, in its various variations. There are also several radical techniques available for the management of neglected SSCs, such as osteotomies; however, these can be risky. Comparing the treatment outcomes when using preoperative Halo Gravity Traction (HGT) against the use of a Magnetically Controlled Growing Rod (MCGR) as a temporary internal distraction (TID) device, we evaluated the differences in surgical and radiological outcomes. METHODS: We conducted a retrospective study of 30 patients with SSCs, treated with HGT followed by posterior spinal fusion (PSF; Group 1, n = 18) or treated using a temporary MCGR as a TID followed by PSF (Group 2, n = 12). All patients underwent surgical treatment between 2016 and 2022. The inclusion criteria were SSC > 90°, flexibility < 30%, and the use of preoperative HGT followed by PSF or the two-stage surgical procedure with initial TID rod placement (Stage 1) followed by PSF (Stage 2). The evaluated parameters were as follows: rib hump, trunk height, and radiographic outcomes. All parameters were collected preoperatively, after the initial surgery, after final correction and fusion, and during the final follow-up. RESULTS: In Group 1, we evaluated 18 patients with a mean age of 15.5 years; in Group 2, we evaluated 12 patients with a mean age of 14.2 years. The interval between the staged procedures averaged 32.7 days. The mean preoperative main curves (MC) were 118° and 112° in Group 1 and Group 2, respectively. After definitive surgery, the MC was corrected to 42° and 44° in G1 and G2, respectively. The mean percentage correction of the MC was similar in both groups (65% vs. 61% in G1 and G2, respectively). The mean preoperative thoracic kyphosis was 92.5° in G1 and 98° in G2, corrected to 43.8° in G1 and 38.8° in G2. Trunk height increased by 9 cm on average. CONCLUSIONS: There are no benefits in using a MCGR as a temporary internal distraction device in the management of neglected scoliosis in adolescents. Surgical treatment of severe scoliosis may be safe, with a reduced risk of potential complications, when using preoperative HGT. A specific intraoperative complication when using a MCGR as a temporary internal distraction device was a 50% risk of transient neuromonitoring changes, due to significant force applied to the spine and radical distraction of the spine. We achieved similar clinical, radiographic, and pulmonary function outcomes for both techniques. The use of HGT causes less blood loss with a shorter overall time under anesthesia. Partial correction significantly aids the subsequent operation by facilitating a gradual reduction in the curvature, thereby reducing the difficulty of surgical treatment and the risk of neurological deficits.

The Child's Age and the Size of the Curvature Do Not Affect the Accuracy of Screw Placement with the Free-Hand Technique in Spinal Deformities in Children and Adolescents.

BACKGROUND: The current method of treatment of spinal deformities would be almost impossible without pedicle screws (PS) placement. There are only a few studies evaluating the safety of PS placement and possible complications in children during growth. The present study was carried out to evaluate the safety and accuracy of PS placement in children with spinal deformities at any age using postoperative computed tomography (CT) scans. METHODS: 318 patients (34 males and 284 females) who underwent 6358 PS fixations for pediatric spinal deformities were enrolled in this multi-center study. The patients were divided into three age groups: less than 10 years old, 11-13 years old, and 14-18 years old. These patients underwent postoperative CT scans and were analyzed for pedicle screw malposition (anterior, superior, inferior, medial, and lateral breaches). RESULTS: The breach rate was 5.92% for all pedicles. There were 1.47% lateral and 3.12% medial breaches for all pedicles with tapping canals, and 2.66% lateral and 3.84% medial breaches for all pedicles without a tapping canal for the screw. Of the 6358 screws placed in the thoracic, lumbar, and sacral spine, 98% of the screws were accurately placed (grade 0, 1, and juxta pedicular). A total of 56 screws (0.88%) breached more than 4 mm (grade 3), and 17 (0.26%) screws were replaced. No new and permanent neurological, vascular, or visceral complications were encountered. CONCLUSIONS: The free-hand technique for pedicle screw placement in the acceptable and safety zone in pedicles and vertebral bodies was 98%. No complications associated with screw insertion in growth were noted. The free-hand technique for pedicle screw placement can be safely used in patients at any age. The screw accuracy does not depend on the child's age nor the size of the deformity curve. Segmental instrumentation with posterior fixation in children with spinal deformities can be performed with a very low complication rate. Navigation of the robot is only an auxiliary tool in the hands of the surgeons, and the result of the work ultimately depends on the surgeons.

Genome-wide approaches delineate the additive, epistatic, and pleiotropic nature of variants controlling fatty acid composition in peanut (Arachis hypogaea L.).

The fatty acid composition of seed oil is a major determinant of the flavor, shelf-life, and nutritional quality of peanuts. Major QTLs controlling high oil content, high oleic content, and low linoleic content have been characterized in several seed oil crop species. Here, we employ genome-wide association approaches on a recently genotyped collection of 787 plant introduction accessions in the USDA peanut core collection, plus selected improved cultivars, to discover markers associated with the natural variation in fatty acid composition, and to explain the genetic control of fatty acid composition in seed oils. Overall, 251 single nucleotide polymorphisms (SNPs) had significant trait associations with the measured fatty acid components. Twelve SNPs were associated with two or three different traits. Of these loci with apparent pleiotropic effects, 10 were associated with both oleic (C18:1) and linoleic acid (C18:2) content at different positions in the genome. In all 10 cases, the favorable allele had an opposite effect-increasing and lowering the concentration, respectively, of oleic and linoleic acid. The other traits with pleiotropic variant control were palmitic (C16:0), behenic (C22:0), lignoceric (C24:0), gadoleic (C20:1), total saturated, and total unsaturated fatty acid content. One hundred (100) of the significantly associated SNPs were located within 1000 kbp of 55 genes with fatty acid biosynthesis functional annotations. These genes encoded, among others: ACCase carboxyl transferase subunits, and several fatty acid synthase II enzymes. With the exception of gadoleic (C20:1) and lignoceric (C24:0) acid content, which occur at relatively low abundance in cultivated peanuts, all traits had significant SNP interactions exceeding a stringent Bonferroni threshold (α = 1%). We detected 7682 pairwise SNP interactions affecting the relative abundance of fatty acid components in the seed oil. Of these, 627 SNP pairs had at least one SNP within 1000 kbp of a gene with fatty acid biosynthesis functional annotation. We evaluated 168 candidate genes underlying these SNP interactions. Functional enrichment and protein-to-protein interactions supported significant interactions (P-value < 1.0E-16) among the genes evaluated. These results show the complex nature of the biology and genes underlying the variation in seed oil fatty acid composition and contribute to an improved genotype-to-phenotype map for fatty acid variation in peanut seed oil.

Next-generation sequencing reveals novel mutations in a collision tumor of glioblastoma and meningioma.

Primary intracranial collision tumors are rare in patients without predisposing factors. We report such a case in a 42-year-old female who presented with headaches and altered mental status. Imaging revealed a single heterogeneous, rim-enhancing lesion in the left parieto-occipital periventricular region, involving the corpus callosum. Stereotactic biopsy demonstrated glioblastoma. Subsequent tumor resection showed histologic evidence of glioblastoma and meningioma. Next-generation sequencing was performed on both tumor components. The glioblastoma exhibited a CDKN2A homozygous deletion and novel missense mutations in TAF1L and CSMD3, while no definitive genetic alterations were identified in the meningioma. Next-generation sequencing may yield insight into molecular drivers of intracranial collision tumors and aid in identifying future therapeutic targets.

Use of Targeted Amplicon Sequencing in Peanut to Generate Allele Information on Allotetraploid Sub-Genomes.

The use of molecular markers in plant breeding has become a routine practice, but the cost per accession can be a hindrance to the routine use of Quantitative Trait Loci (QTL) identification in breeding programs. In this study, we demonstrate the use of targeted re-sequencing as a proof of concept of a cost-effective approach to retrieve highly informative allele information, as well as develop a bioinformatics strategy to capture the genome-specific information of a polyploid species. SNPs were identified from alignment of raw transcriptome reads (2 × 50 bp) to a synthetic tetraploid genome using BWA followed by a GATK pipeline. Regions containing high polymorphic SNPs in both A genome and B genomes were selected as targets for the resequencing study. Targets were amplified using multiplex PCR followed by sequencing on an Illumina HiSeq. Eighty-one percent of the SNP calls in diploids and 68% of the SNP calls in tetraploids were confirmed. These results were also confirmed by KASP validation. Based on this study, we find that targeted resequencing technologies have potential for obtaining maximum allele information in allopolyploids at reduced cost.

Genotypic Characterization of the U.S. Peanut Core Collection.

Cultivated peanut (Arachis hypogaea) is an important oil, food, and feed crop worldwide. The USDA peanut germplasm collection currently contains 8,982 accessions. In the 1990s, 812 accessions were selected as a core collection on the basis of phenotype and country of origin. The present study reports genotyping results for the entire available core collection. Each accession was genotyped with the Arachis_Axiom2 SNP array, yielding 14,430 high-quality, informative SNPs across the collection. Additionally, a subset of 253 accessions was replicated, using between two and five seeds per accession, to assess heterogeneity within these accessions. The genotypic diversity of the core is mostly captured in five genotypic clusters, which have some correspondence with botanical variety and market type. There is little genetic clustering by country of origin, reflecting peanut's rapid global dispersion in the 18th and 19th centuries. A genetic cluster associated with the hypogaea/aequatoriana/peruviana varieties, with accessions coming primarily from Bolivia, Peru, and Ecuador, is consistent with these having been the earliest landraces. The genetics, phenotypic characteristics, and biogeography are all consistent with previous reports of tetraploid peanut originating in Southeast Bolivia. Analysis of the genotype data indicates an early genetic radiation, followed by regional distribution of major genetic classes through South America, and then a global dissemination that retains much of the early genetic diversity in peanut. Comparison of the genotypic data relative to alleles from the diploid progenitors also indicates that subgenome exchanges, both large and small, have been major contributors to the genetic diversity in peanut.

Development of a Peanut Canopy Measurement System Using a Ground-Based LiDAR Sensor.

Plant architecture characteristics contribute significantly to the microclimate within peanut canopies, affecting weed suppression as well as incidence and severity of foliar and soil-borne diseases. However, plant canopy architecture is difficult to measure and describe quantitatively. In this study, a ground-based LiDAR sensor was used to scan rows of peanut plants in the field, and a data processing and analysis algorithm was developed to extract feature indices to describe the peanut canopy architecture. A data acquisition platform was constructed to carry the ground-based LiDAR and an RGB camera during field tests. An experimental field was established with three peanut cultivars at Oklahoma State University's Caddo Research Station in Fort Cobb, OK in May and the data collections were conducted once each month from July to September 2015. The ground-based LiDAR used for this research was a line-scan laser scanner with a scan-angle of 100°, an angle resolution of 0.25°, and a scanning speed of 53 ms. The collected line-scanned data were processed using the developed image processing algorithm. The canopy height, width, and shape/density were evaluated. Euler number, entropy, cluster count, and mean number of connected objects were extracted from the image and used to describe the shape of the peanut canopies. The three peanut cultivars were then classified using the shape features and indices. A high correlation was also observed between the LiDAR and ground-truth measurements for plant height. This approach should be useful for phenotyping peanut germplasm for canopy architecture.