SNP discovery by high-throughput sequencing in soybean

<p>Abstract</p> <p>Background</p> <p>With the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology.</p> <p>Results</p> <p>A total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp.</p> <p>Conclusions</p> <p>We have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops.</p

Genetic Mapping of Soybean Cyst Nematode (Heterodera glycines) Resistance to Enhance Soybean Production in the United States [abstract]

Only abstract of poster available.Track V: BiomassSoybean cyst nematode (SCN, Heterodera glycines) is the most destructive pest of soybean in the United States, resulting in an annual extensive yield loss of approximately $1.5 billion in the United States alone. Breeding for resistance to SCN is the most effective approach to control this pest. However, most of commercial soybean varieties resistant to SCN were mainly derived from a few common resistant sources. The continuation of growing the same resistant cultivar(s) have resulted in SCN population shifts and loss of SCN resistance; thus it highlights a need of further investigation to mine new resistant genes from new resistant sources for soybean improvement. As a leading group on SCN research in the United States, the University of Missouri SCN researchers have been continuing the evaluation of exotic soybean germplasm for broad-based resistance to multi-HG types of SCN, the identification and mapping of novel quantitative trait loci (QTL)/gene(s), and the discovery of genetic markers for marker-assisted selection (MAS) programs. Using many plant introductions (PIs) with high resistance to multi-SCN HG types, we have developed genetic populations for molecular characterization and QTL mapping. These efforts led to the discovery of many novel QTL underlying the resistance to multi-SCN HG types. With sequence information using the genome-wide Illumina/Solexa sequencing technology, we have developed hundreds of genetic markers associated with the target QTL. Along with the soybean physical and genetic maps, these markers will provide a powerful genomics tool facilitating our efforts toward fine-mapping and positional cloning of candidate genes for SCN resistance. Moreover, the QTL associated genetic markers are greatly useful to incorporate novel resistant genes into new soybean varieties through the MAS approach. With SCN resistant soybean varieties, soybean yield and productivity will be increased and, in turn, enhance the seed oil production; which will significantly be an important source for the development of biofuel

Incidence, Risk Factors, and Outcomes of Rhegmatogenous Retinal Detachment after Intravitreal Injections of Anti-VEGF for Retinal Diseases: Data from the Fight Retinal Blindness! Registry

PURPOSE To report the estimated incidence, probability, risk factors, and 1-year outcomes of rhegmatogenous retinal detachment (RRD) in eyes receiving intravitreal injections (IVTs) of VEGF inhibitors for various retinal conditions in routine clinical practice. DESIGN Retrospective analysis of data from a prospectively designed observational outcomes registry: the Fight Retinal Blindness! PROJECT PARTICIPANTS Eyes of patients starting IVTs of VEGF inhibitors (ranibizumab, aflibercept, or bevacizumab) for neovascular age-related macular degeneration, diabetic macular edema, or retinal vein occlusion from January 1, 2006, to December 31, 2020. All eyes that developed RRD within 90 days of IVTs were defined as cases with RRD and were matched with control eyes. METHODS Estimated incidence, probability, and hazard ratios (HRs) of RRD were measured using Poisson regression, Kaplan-Meier survival curve, and Cox proportional hazards models. Locally weighted scatterplot smoothing curves were used to compare visual acuity (VA) between cases and matched controls. MAIN OUTCOME MEASURES Estimated incidence of RRD. RESULTS We identified 16 915 eyes of 13 792 patients who collectively received 265 781 IVTs over 14 years. Thirty-six eyes were reported to develop RRD over the study period. The estimated incidence (95% confidence interval [CI]) per year per 1000 patients and per 10 000 injections was 0.77 (0.54-1.07) and 1.36 (0.95-1.89), respectively. The probability of RRD did not significantly increase at each successive injection (P = 0.95) with the time of follow-up. Older patients (HR [95% CI] = 1.81 [1.21-3.62] for every decade increase in age, P < 0.01) were at a higher risk of RRD, whereas patients with good presenting VA (HR [95% CI] = 0.85 [0.70-0.98] for every 10-letter increase in VA, P = 0.02) were at a lower risk. Neither the type of retinal disease (P = 0.52) nor the VEGF inhibitor (P = 0.09) was significantly associated with RRD risk. Cases with RRD lost 3 lines of vision on average compared with the prior RRD VA and had significantly fewer injections than matched controls over the year after the RRD. CONCLUSIONS Rhegmatogenous retinal detachment is a rare complication of VEGF inhibitor IVT in routine clinical practice with poor visual outcomes at 1 year

Changes in 12-month outcomes over time for age-related macular degeneration, diabetic macular oedema and retinal vein occlusion

OBJECTIVES To identify whether the outcomes of neovascular age-related macular degeneration (nAMD), diabetic macular oedema (DMO) and retinal vein occlusion (RVO) in routine clinical practice have changed over time. METHODS We analysed 12-month outcomes in treatment-naïve eyes that started aflibercept or ranibizumab for nAMD (3802 eyes), DMO (975 eyes), Branch RVO (BRVO, 357 eyes), Central RVO (CRVO, 371 eyes) and Hemi-RVO (HRVO, 54 eyes) from 2015 and 2019 tracked in the prospectively designed observational Fight Retinal Blindness! Registry. RESULTS The mean VA change at 12-month for each year between 2015 and 2019 remained stable or otherwise showed no discernible trends over time in eyes with nAMD (+3.3 to +6 letters), DMO (+3.6 to +6.7 letters) and RVO (+10.3 to +11.7 letters for BRVO, +5.9 to +17.7 letters for CRVO and 10.2 to 20.7 letters for HRVO). The median number of VEGF-inhibitor injections in eyes that completed 12-month follow-up also remained stable at 8-9 for nAMD, 6-7 for DMO, 7-9 for RVO. Fewer eyes (<one-fourth) that started treatment between 2015 and 2018 and more eyes starting in 2019 did not complete 12-month's follow-up visit. The mean VA in non-completers at their last visit was higher than that of their baseline visit. CONCLUSIONS Treatment patterns and outcomes for nAMD, DMO and RVO in routine clinical practice have stabilised over the past 5 years at levels inferior to those reported by the pivotal phase 3 studies. A conscious effort to treat these conditions more intensively, or with longer lasting agents, would likely improve outcomes further in our patients

Incidence, risk factors and outcomes of submacular haemorrhage with loss of vision in neovascular age-related macular degeneration in daily clinical practice: data from the FRB! registry

PURPOSE The main purpose of the study was to report the estimated incidence, cumulative rate, risk factors and outcomes of submacular haemorrhage (SMH) with loss of vision in neovascular age-related macular degeneration (nAMD) receiving intravitreal injections (IVT) of vascular endothelial growth factor (VEGF) inhibitor in routine clinical practice. METHODS Retrospective analysis of treatment-naïve eyes receiving IVTs of VEGF inhibitors (ranibizumab, aflibercept or bevacizumab) for nAMD from 1 January 2010 to 31 December 2020 that were tracked the Fight Retinal Blindness! registry. Estimated incidence, cumulative rate and hazard ratios (HR) of SMH with loss of vision during treatment were measured using the Poisson regression, Kaplan-Meier survival curves and Cox proportional hazard models. RESULTS We identified 7642 eyes (6425 patients) with a total of 135 095 IVT over a 10-year period. One hundred five eyes developed SMH with loss of vision with a rate of 1 per 1283 injections (0.08% 95% confidence interval [95% CI] [0.06; 0.09]). The estimated incidence [95% CI] was 4.6 [3.8; 5.7] SMH with loss of vision per year per 1000 treated patients during the study. The cumulative [95% CI] rate of SMH per patient did not increase significantly with each successive injection (p = 0.947). SMH cases had a mean VA drop of around 6 lines at diagnosis, which then improved moderately to a 4-line loss at 1 year. CONCLUSIONS Submacular haemorrhage (SMH) with loss of vision is an uncommon complication that can occur at any time in eyes treated for nAMD in routine clinical practice, with only limited recovery of vision 1 year later

Three-year treatment outcomes of Aflibercept versus Ranibizumab for diabetic macular edema:: Data from the Fight Retinal Blindness! Registry

PURPOSE Compare the 3-year outcomes of ranibizumab versus aflibercept in eyes with diabetic macular edema in daily practice. METHODS This was a retrospective analysis of naive diabetic macular edema eyes starting intravitreal injections of ranibizumab (0.5 mg) or aflibercept (2 mg) from January 1, 2013 to December 31, 2017 that were collected in the Fight Retinal Blindness! Registry. RESULTS We identified 534 eyes (ranibizumab-267 and aflibercept-267) of 402 patients. The adjusted mean (95% confidence interval) visual acuity change of +1.3 (-0.1 to 4.2) letters in the ranibizumab group and +2.4 (-0.2 to 5.1) letters (P = 0.001) in the aflibercept group at 3 years was not clinically different. However, the adjusted mean CST change seemed to remain significantly different throughout the 3-year period with higher reductions in favor of aflibercept (-87.8 [-108.3 to -67.4] µm for ranibizumab vs. -114.4 [-134.4 to -94.3] for aflibercept; P < 0.01). When baseline visual impairment was moderate (visual acuity ≤68 Early Treatment Diabetic Retinopathy Study letters), we found a faster improvement in visual acuity in eyes treated with aflibercept up until 18 months of treatment than eyes treated with ranibizumab, which then stayed similar until 36 months of treatment, whereas there was no apparent difference when baseline visual impairment was mild (visual acuity ≥69 Early Treatment Diabetic Retinopathy Study letters). The rate of serious adverse events was low. CONCLUSION Aflibercept and ranibizumab were both effective and safe for diabetic macular edema over 3 years

Vascular endothelial growth factor inhibitors for predominantly Caucasian myopic choroidal neovascularization: 2-year treatment outcomes in clinical practice: data from the Fight Retinal Blindness! Registry

Purpose: To report the 24-month outcomes of vascular endothelial growth factor (VEGF) inhibitors for myopic choroidal neovascularization (mCNV) in predominantly Caucasian eyes in routine clinical practice. Methods: Retrospective analysis of treatment-na¿ıve eyes starting intravitreal injection of VEGF inhibitors of either bevacizumab (1.25 mg) or ranibizumab (0.5 mg) for mCNV from 1 January 2006 to 31 May 2018 that were tracked in the Fight Retinal Blindness! registry. Results: We identified 203 eyes (bevacizumab-85 and ranibizumab-118) of 189 patients. The estimated mean (95% CI) change in VA over 24 months for all eyes using longitudinal models was +8 (5, 11) letters with a median (Q1, Q3) of 3 (2, 5) injections given mostly during the first year. The estimated mean change in VA at 24 months was similar between bevacizumab and ranibizumab [+9 (5, 13) letters for bevacizumab versus +9 (6, 13) letters for ranibizumab; p = 0.37]. Both agents were also similar in the mCNV activity outcomes, treatment frequency and visit frequency. Conclusions: The 24-month treatment outcomes of VEGF inhibitors for mCNV were favourable in this largest series yet reported of predominantly Caucasian eyes in routine clinical practice,with approximately two lines of visual gain and amedian of three injections given mostly during the first year. These outcomes are similar to those reported for predominantly Asian eyes.Bevacizumab appeared to be as safeandeffective as ranibizumab. Key words: anti-VEGF therapy - Caucasian - high myopia - myopia - myopic choroidal neovascularization - optical coherence tomography - pathologic myopia - VEGF inhibitor

Hemiretinal vein occlusion 12-month outcomes are unique with vascular endothelial growth factor inhibitors: data from the Fight Retinal Blindness! Registry

BACKGROUND/AIMS To describe baseline characteristics and 12-month outcomes with vascular endothelial growth factor (VEGF) inhibitors of treatment-naïve hemiretinal vein occlusion (HRVO) compared with branch (BRVO) and central (CRVO) variants in routine clinical care. METHODS A database observational study recruited 79 HRVO eyes, 590 BRVO eyes and 344 CRVO eyes that initiated therapy over 10 years. The primary outcome was mean change in visual acuity (VA-letters read on a logarithm of minimal angle of resolution chart) at 12 months. Secondary outcomes included mean change in central subfield thickness (CST), injections and visits. RESULTS At baseline, mean VA in HRVO (53.8) was similar to CRVO (51.9; p=0.40) but lower than BRVO (59.4; p=0.009). HRVO eyes improved to match BRVO eyes from soon after treatment started through 12 months. Mean change in VA was greater in HRVO (+16.4) than both BRVO (+11.4; p=0.006) and CRVO (+8.5; p<0.001). Mean change in CST in HRVO (-231 µm) was similar to CRVO (-259 µm; p=0.33) but greater than BRVO eyes (-151 µm; p=0.003). The groups had similar median burdens of eight injections and nine visits. CONCLUSIONS HRVO generally experienced the greatest mean change in VA of the three types of RVO when treated with VEGF inhibitors, ending with similar 12-month VA and CST to BRVO despite starting closer to CRVO. Inclusion of HRVO in BRVO or CRVO cohorts of clinical trials would be expected to proportionally inflate and skew the visual and anatomic outcomes

Central retinal vein occlusion 36-month outcomes with anti-vascular endothelial growth factors: the Fight Retinal Blindness! registry

PURPOSE To analyze the 3-year outcomes in a broad population of patients starting vascular endothelial growth factor (VEGF) inhibitors for central retinal vein occlusion (CRVO) in routine clinical practice. DESIGN Observational database study PARTICIPANTS: 527 treatment-naïve CRVO eyes that commenced VEGF inhibitors between December 1, 2010-2018 tracked in the Fight Retinal Blindness! registry. METHODS Longitudinal models were used to plot changes in visual acuity (VA) and central subfield thickness (CST). MAIN OUTCOME MEASURES Mean change in VA from baseline to 36 months, injections, visits, completion, switching and suspensions of therapy >180 days at final review. RESULTS Overall (527 eyes) mean VA change (95% CI) was +10 (7, 12) letters, 37% had final VA ≥70 and 30% ≤35 letters, mean CST changed -306μm. Completers (257/527, 49%) had mean 36-month changes in VA and CST of +12 letters and -324μm with a median of 18 injections at 26 visits. The adjusted mean VA change was similar with each VEGF inhibitor (mean, +11.4 letters) despite a greater reduction in CST with aflibercept (-310μm) vs. ranibizumab (-258μm) vs. bevacizumab (-216μm; P 73 letters, 42/527, 8%) lost 7 letters. Switching (160/527, 30%) was most often to aflibercept (79 eyes). Using suspensions and discontinuation reasons we identified similar proportions had ceased therapy (154/527, 29%) as were still receiving it at 36 months (165/527, 31%). Only 62/527 eyes (12%) had resolution of macular edema without treatment for over 6 months. CONCLUSIONS Patients with CRVO that commenced VEGF inhibitors in routine care for whom follow-up was available had VA improvements of around 12 letters at three years, but with more than 50% lost to follow the VA outcome for the entire group is likely worse. The choice of VEGF inhibitor influenced CST but not VA outcomes. We estimate that around half of eyes were still receiving injections after 36 months

Dexamethasone Implant for Diabetic Macular Oedema: 1-Year Treatment Outcomes from the Fight Retinal Blindness! Registry

INTRODUCTION Phase III clinical trials of dexamethasone intravitreal implant for diabetic macular oedema (DMO) have reported significant improvements in visual acuity (VA). Studies evaluating the treatment of DMO in routine clinical practice provide data to identify areas that need improvement. This study evaluated 12-month treatment outcomes of dexamethasone implant for DMO in routine clinical practice. METHODS Retrospective data analysis of eyes that started dexamethasone implant for DMO from 1 June 2013 to 30 April 2019 in routine clinical practice tracked in the Fight Retinal Blindness! Registry. RESULTS Of the 4282 eyes (2518 patients) that started DMO treatment in the specified period, 267 (6%) eyes (204 patients) received 454 dexamethasone implant injections. Two-fifths (106 eyes) had received prior treatment for DMO. The mean (95% confidence interval [CI]) VA change at 12 months was 1.8 (- 0.5, 4.2) letters from the mean (standard deviation [SD]) VA of 56.5 (19.8) letters at baseline, with 41% eyes achieving at least 20/40. The mean (95% CI) change in central subfield thickness over 1 year was - 79 (- 104, - 54) µm from a mean (SD) of 459 (120) µm at baseline. Eyes that completed 1 year of follow-up received a median (Q1, Q3) of 2 (1, 2) dexamethasone implants. One-tenth of phakic eyes received cataract surgery while 2% had a pressure response requiring anti-glaucoma medications. CONCLUSIONS One-year treatment outcomes of dexamethasone intravitreal implant for DMO in routine clinical practice were inferior to those in the clinical trials perhaps because of fewer treatments in clinical practice