USDA ARS Genotyping Laboratory

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08/14/2020 Added

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[00:00:03.095]I'm Guihua Bai,
[00:00:04.570]the Director of USDA Central Small Grain Genotyping Lab.
[00:00:09.620]Our lab is part
[00:00:11.410]of a USDA Hard Winter Wheat Genetic Research Unit.
[00:00:15.440]This belong to the Center of Grain
[00:00:18.290]and Animal Health Research.
[00:00:21.390]Our mission are:
[00:00:23.050]to provide a genotyping service
[00:00:25.330]to hard winter wheat breeding programs in the Great Plains
[00:00:30.000]and develop novel genetic resources and genomic tools
[00:00:35.610]to support wheat research in the region.
[00:00:39.320]Our lab was the first USDA genotyping lab
[00:00:43.060]established in 2002.
[00:00:47.240]The lab has three USDA scientists
[00:00:50.270]with Doctors Paul St. Amand and Amy Bernardo
[00:00:55.020]mainly responsible for genotyping service.
[00:00:58.470]And several postdocs graduate students,
[00:01:03.210]innovative scientists hired through Kansas State University
[00:01:07.880]to conduct research.
[00:01:09.610]Our research facility has four rooms
[00:01:14.090]of lab space, five greenhouses,
[00:01:18.630]and one seeds prep room.
[00:01:22.060]We also have access to experimental farms
[00:01:26.789]at the Rocky Ford and Ashley Burtons.
[00:01:30.400]Our research focuses on the trade of regional importance.
[00:01:36.970]In the past 18 years,
[00:01:39.460]we identified numerous QTL
[00:01:43.100]for more than 15 plants stress and yield related traits
[00:01:49.180]and developed linked markers for marker-assisted selection.
[00:01:55.060]Our work produced one patent,
[00:01:59.470]more than 200 peer-reviewed publications
[00:02:02.990]and the co-release of more than 50 cultivars.
[00:02:06.890]Recently we cloned
[00:02:09.920]two important wheat stress-resistant genes.
[00:02:14.570]One's PHS1 for preharvest sprouting resistance
[00:02:20.080]and another one, Fhb1 for Fusarium head blight resistance.
[00:02:25.390]We also contributed to the cloning of three sorghum genes,
[00:02:29.640]three Medicago genes
[00:02:32.910]and Fhb7, that's another FHB resistant genes.
[00:02:38.450]With financial support from U.S. Wheat
[00:02:42.060]and Barley Scab Initiative,
[00:02:45.494]we transferred the major FHB resistant gene,
[00:02:48.691]Fhb1 into 17 local adaptive backcross
[00:02:54.070]to develop new resource for breeding
[00:02:56.540]using marker-assisted backcrossing.
[00:02:58.287]The selected line showed a high level of FHB resistance
[00:03:04.380]and have been released to breeding program in the region.
[00:03:08.180]Currently, we are developing functional marker
[00:03:12.970]for wheat resistance gene to Hessian fly, curl mite, rusts
[00:03:18.960]and the Fusarium head blight
[00:03:21.070]use fine mapping and candidate gene approaches.
[00:03:24.290]Now several scientists from our lab
[00:03:27.350]will give a brief introduction of their work.
[00:03:31.080]I am Dr. Amy Bernardo
[00:03:32.760]and I have been working in the lab since 2004.
[00:03:35.900]My major responsibilities are
[00:03:37.730]to develop next generation sequencing-based genotyping tools
[00:03:41.980]for high-throughput screening
[00:03:43.730]and to provide service on NGS-based marker analysis.
[00:03:49.480]I manage the routine operation of an ABI 3730 DNA Analyzer.
[00:03:54.870]This sequencer has a stacker that can load up to 16 plates.
[00:03:59.850]The autosampler automatically picks up the plate
[00:04:02.500]and runs it,
[00:04:03.790]and it drains new plate in the stack.
[00:04:10.679]This sequencer uses capillary electrophoresis
[00:04:14.500]to separate DNA fragments with one base pair difference.
[00:04:18.010]We have used it for SSR and SDS marker analysis
[00:04:21.970]and DNA sequencing since 2006.
[00:04:26.420]For marker analysis,
[00:04:27.890]each run can combine up to four different PCRs
[00:04:31.340]labeled with four different dyes.
[00:04:33.740]The machine can run up to 13 plates of 384 samples a day.
[00:04:40.230]Sanger type sequencing can run six plates
[00:04:43.030]of 96 samples daily.
[00:04:45.690]We use an Ion Proton Next-Generation Sequencer
[00:04:48.650]for high-throughput SNeP genotyping.
[00:04:51.290]We make GBS, MRASeq and GBMAS libraries
[00:04:55.340]and sequence the libraries
[00:04:56.700]and sends SNeP data to our customers.
[00:05:00.010]We make GBS libraries by DNA digestions
[00:05:03.190]with restriction enzymes, barcoded adapter ligation and PCR.
[00:05:08.450]We developed MRASeq to analyze genome-wide random SNePs.
[00:05:13.080]Compared to GBS, MRASeq uses PCR only
[00:05:17.300]without need of enzyme digestion and ligation steps.
[00:05:20.850]Thus, it is simpler, cheaper and has no license requirement.
[00:05:25.610]We develop GBMAS to multiplex markers with known sequences.
[00:05:29.790]GBMAS using barcodes and multiplex PCR can pull hundreds
[00:05:34.720]of SNePs for different important traits
[00:05:37.300]from several hundreds of samples in one sequencing run.
[00:05:41.810]In order to prepare chips for sequencing,
[00:05:44.470]we load libraries, chips and re-agents on the Ion Chef.
[00:05:51.830]Each chip contains 165 million wells
[00:05:55.310]and each well is loaded with one DNA molecule.
[00:05:58.690]It takes 13 hours to complete a chef-run.
[00:06:02.520]After the chef-run,
[00:06:04.100]we load the chip on the proton sequencer.
[00:06:07.070]It takes 2.5 hours to sequence one chip
[00:06:10.050]and first queue files
[00:06:11.080]for both runs are available the morning after sequencing.
[00:06:14.790]Each chip sequencing run
[00:06:16.840]can generate up to 100 million reads or about 20GB of data.
[00:06:21.770]Two chip runs take less than two days
[00:06:24.480]from sample prep to get data.
[00:06:27.230]We are purchasing an illumina NextSeq 2000.
[00:06:30.730]The new sequencer can generate up to 1 billion reads
[00:06:34.160]or 300GB per run in 48 hours.
[00:06:39.170]I'm Hui Chen,
[00:06:40.003]a Research Assistant Professor at Kansas State University.
[00:06:44.180]My assignment in the lab is to study personal genomics
[00:06:48.760]and develop new breeding technologies
[00:06:52.210]in particular to develop new gene editing methods
[00:06:56.420]for validating gene function
[00:06:59.130]and molecular breeding in wheat.
[00:07:02.278]Traditional CRISPR/Cas9 gene-editing
[00:07:05.450]relies on genetic transformation and plant regeneration.
[00:07:09.967]And moreso wheat colitis have a low leg of callus induction
[00:07:15.830]and regeneration in tissue culture process.
[00:07:19.750]Upgrading only Bobwhite and Fielder are useful
[00:07:25.280]with transformation.
[00:07:27.324]CRISPR/Cas9 limit the application of gene editing
[00:07:32.630]in rooting with breeding and genetical research.
[00:07:36.870]Recently, we developed
[00:07:38.480]a new barley stripe mosaic virus-mediated
[00:07:42.240]CRISPR/Cas9 gene-editing system.
[00:07:45.350]In this new system, we only transfer Cas9 gene
[00:07:48.440]into Bobwhite once.
[00:07:50.640]Then editing different genes
[00:07:52.890]by integrating the Cas9-OE Bobwhite seedlings
[00:07:57.230]with the viral inoculum.
[00:07:59.688]To prepare the viral inoculum,
[00:08:02.222]tobacco leaves are infected by BSMV, RNA the known vectors.
[00:08:07.830]The CRISPR/Cas9 is Fusarium Cas9 for targeted gene.
[00:08:12.800]The infected tobacco leaves sap is used
[00:08:16.020]to inoculate Cas9-OE Bobwhite seedlings.
[00:08:20.800]The edited plants are identified
[00:08:23.520]by testing M1 seedlings using Sanger sequencing.
[00:08:29.380]We use this system both in editing
[00:08:32.200]in different wheat cultivars
[00:08:35.523]by fostering multicross to transform Cas9 gene
[00:08:39.960]from the Cas9-OE Bobwhite to targeted cultivars.
[00:08:45.800]The Cas9-OE F2 progenies are selected by PCR.
[00:08:52.150]Then inoculate selected Cas9-OE F2 plants
[00:08:56.980]with the viral sap
[00:08:59.790]containing target Cas9-OE to start editing.
[00:09:04.030]This will bypass wheat transformation process.
[00:09:07.460]Viral octol applications is to edit FHB susceptible gene
[00:09:13.490]to improve FHB resistance.
[00:09:17.200]Our lab has recently coordinate TaHRC
[00:09:22.500]as Fhb1 will knockout
[00:09:26.100]TaHRC susceptible gene in three lines
[00:09:31.280]using this new gene-editing system.
[00:09:34.420]And this edited mutants showed enhanced FHB resistance
[00:09:40.827]comparing to the non editing control plants.
[00:09:44.530]This project is supported
[00:09:46.480]by the U.S. Wheat and Barley Scab Initiative.
[00:09:50.810]Currently, we're also using this new gene-editing system
[00:09:55.810]to study the functions of the other genes,
[00:09:59.580]ODG and insect resistance
[00:10:02.820]as well as yield trait in wheat.
[00:10:06.780]Hi, I'm Paul St. Amand,
[00:10:08.380]USDA Research Geneticist at the Genotyping Lab.
[00:10:13.290]My main responsibilities are genotyping service,
[00:10:16.000]which we provide for the wheat research
[00:10:18.130]and breeding community.
[00:10:19.510]Every year, we work closely with programs
[00:10:22.110]in the Great Plains region and worldwide.
[00:10:25.220]The data we generate are used to produce new wheat cultivars
[00:10:29.440]in germplasm for our gene mapping, gene discovery,
[00:10:33.450]fingerprinting, and other research purposes.
[00:10:36.560]Since 2004, more than 120 released US wheat cultivars
[00:10:41.360]have used our marker data during their development.
[00:10:44.260]We work with over 100 scientists
[00:10:46.380]from more than 20 US universities and breeding companies.
[00:10:50.170]We also collaborate with scientists in other countries.
[00:10:54.600]Every year we test thousands of wheat lines
[00:10:56.780]with more than 100 trait related markers
[00:11:00.090]and thousands of GBS markers,
[00:11:02.670]millions of data points and billions of sequencing reads.
[00:11:07.620]Now I'd like to give you a brief lab tour
[00:11:09.480]on how we do genotyping.
[00:11:11.740]Genotyping usually consists of five steps:
[00:11:14.370]sample collection and treatment,
[00:11:16.360]DNA isolation and normalization, marker amplification,
[00:11:21.290]marker detection or analysis and data reporting.
[00:11:25.770]We collect tissue samples into 96-well plates
[00:11:28.630]and freeze-dry them to remove all of the water.
[00:11:35.320]The samples are ground with steel beads
[00:11:37.010]to break up the cell walls.
[00:11:41.353](machine whirring)
[00:11:48.110]Then we extract the DNA in a 96-well format
[00:11:51.060]using our Biomek Robot.
[00:11:53.300]We use the CTAB protocol for DNA extraction.
[00:11:58.025](machine whirring)
[00:12:13.770]We heat the samples in an oven to assist with the reaction
[00:12:17.090]and centrifuge the plates
[00:12:18.360]to separate the organic and aqueous phases.
[00:12:21.710]We use our Biomek to transfer the upper aqueous phase
[00:12:25.120]and perform other liquid handling work.
[00:12:27.800]A fluorescence reader is used to quantify DNA
[00:12:30.400]in a 384-well plate.
[00:12:32.691](machine whirring)
[00:12:36.260]We use a single channel robot
[00:12:38.040]to normalize individual samples
[00:12:39.790]to the same concentration before use.
[00:12:42.730]Most frequently used assays are KASP, SSR and GBS.
[00:12:47.360]All of our assays are PCR-based.
[00:12:53.430]KASP assays are detected using fluorescent dyes
[00:12:56.520]in our plate reader.
[00:12:57.910]Typically two alleles are displayed
[00:13:00.160]using different colored dyes.
[00:13:01.960]SSR assays are detected using our ABI 3730 DNA analyzer.
[00:13:08.310]For GBS, we use several bioinformatics pipelines
[00:13:12.010]to extract marker data from sequencing reads.
[00:13:14.780]Finally, we deliver the genotyping data
[00:13:17.350]to our breeders and geneticists.
[00:13:20.770]In addition to genotyping, we also do training related
[00:13:23.460]to genotyping marker analysis and bioinformatics.
[00:13:26.780]If you'd like to know more about what we do,
[00:13:28.620]contact us or check out our website.

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USDA ARS Genotyping Laboratory

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