New Technology for Next-gen DNA Sequencing Validated by the VitisGen2 Project

Illumina DNA processing cell, which looks similar to a computer chip, being held up for the camera.A new technology called rhAmpSeq™ is allowing grape
geneticists and breeders to rapidly find and validate 2000 markers in diverse grape varieties and species.  These ‘core genome markers’ provide a new set of mileposts that were used during a pilot study by the USDA-funded VitisGen2 project to map traits in six unrelated ‘mapping populations’ representing the diversity of U.S. grape breeding programs, including numerous Vitis species.

The rhAmpSeq technology, developed by Integrated DNA Technologies (IDT) and commercialized in March 2019, allows researchers to mix and amplify DNA from up to 4,000 individual samples and simultaneously sequence thousands of different markers of each sample.  This dramatically reduces the cost of DNA sequencing across the 19 grape chromosomes – and offers a detailed map of the entire genome for each of the 4000 individuals in one batch.

Complicated workflow schematic for rhAmpSeq process: activation of primers, amplification, and indexing.

The rhAmpSeq technology uses normal DNA primers followed by an RNA base and a PCR blocker in PCR 1. After these primers bind specifically to the target DNA, the RNA and blocker are removed and PCR amplification continues. This improved specificity enables thousands of markers to be amplified simultaneously for a given sample. Then ‘barcoding’ primers are used in PCR 2 to label each sample uniquely prior to pooling all samples for sequencing. This allows amplification and sequencing of 2000 DNA markers in up to 4000 samples.

 

The VitisGen2 project team, a multi-institutional research collaboration funded by the USDA-National Institute of Food and Agriculture Specialty Crops Research Initiative, played a key role in adapting the rhAmpSeq process to plant breeding platforms. The VitisGen2 approach provided two key improvements to the AmpSeq technology developed during the first VitisGen project: 1) improved marker design after sequencing 7 new genomes, which drastically improved marker transferability across diverse breeding lines, and 2) the rhAmpSeq marker chemistry itself, which improves the specificity of AmpSeq, enabling more markers to be multiplexed in a single PCR (from 400 markers with AmpSeq to 2000 markers with rhAmpSeq). Unlike single nucleotide markers that provide binary information (reference allele or alternative allele), the larger 250 base pair sequences employed by VitisGen2’s rhAmpSeq strategy generates high information content per marker.

Professor Bruce Reisch commented, “As early testers of the rhAmpSeq system from IDT, we were really pleased to see how we were able to rapidly accelerate the VitisGen2 program by using a nearly 2,000 marker rhAmpSeq panel to analyze 19 Vitis linkage groups. The core marker set was useful across six unrelated populations representing the diversity of the genus, and the workflow was very easy to use and allowed high-throughput processing.”

“We sought to develop an amplicon sequencing technology that addressed the unmet needs of researchers—increased specificity of PCR amplification, while minimizing primer dimer formation,” said Yongming Sun, IDT’s bioinformatics senior staff scientist. “Through collaboration with our diverse group of beta testers, including Cornell University’s College of Agriculture and Life Sciences, we developed the rhAmpSeq Amplicon Sequencing System, which allows researchers to achieve a high degree of multiplexing capability with thousands of assays in a single tube.”

Although the VitisGen2 project team helped pioneer this use of the technology, other scientists and plant breeders see the technology as being broadly applicable to a wide variety of crops.

For more information, you can read the March 6 news release from IDT: https://idtb.io/38tvsg

The VitisGen2 project is supported by the USDA-NIFA Specialty Crop Research Initiative (Award No. 2017- 51181-26829).

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Upcoming VitisGen webinar on computer vision

The VitisGen2 team is excited to announce that our 2019 webinar series will be kicking off next month!

Headshot of Dani MartinezOur first webinar will be with panelist Dani Martinez, talking about “Advanced computer vision techniques: New technologies to streamline grape breeding”.

This webinar will present a brief but thorough overview to the general public of what computer vision is, including its different techniques and the technology behind it. In addition, it will detail the methodology implemented in the powdery mildew phenotyping robots of the VitisGen2 project, as a case study.

Learn more and register for the webinar!

 

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Staff spotlight on Janet van Zoeren

Janet van Zoeren joined the VitisGen2 extension and outreach team a few months ago, when she started as viticulture extension support specialist at Cornell University. Her role with the project involves updating the website, writing articles about project developments and publications, and generally focusing on presenting the work of the VitisGen team to a broader audience.

We asked her to talk about her background, role with VitisGen2, what excites her about the project, and how she would describe her job to a kindergardener. Read the full article at: http://www.vitisgen2.org/team-members/janet-van-zoeren.

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“The Phenotyping Bottleneck” – new Wines and Vines article

Images of grape leaves inoculated with powdery mildew. Resistant cultivar is compared to a susceptible cultivar. It is difficult to tell the difference visually, but easy using computer vision.

Comparing grape cultivars susceptible (left) vs resistant (right) to powdery mildew, with human microscope imaging above vs. using computer vision below.

A new article by Tim Martinson and Lance Cadle-Davidson was published in the December issue of Wines and Vines.

The full pdf of the article is available at “The Phenotyping Bottleneck: How grape breeders link desired traits to DNA markers“.

 

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Staff spotlight on Surya Sapkota

Surya Sapkota and David Gadoury watching like phenotyping robot Blackbird images grape leaf disks.Surya Sapkota, post-doctoral researcher at Cornell University and long-time member of the VitisGen projects, works with preparing, inoculating, and processing grape leaf tissue samples for powdery mildew resistance assessments.

We asked him some questions about his background (he learned about growing crops at a young age from his parent), his role with VitisGen2 (“I work closely with experts in robotics, machine vision, optics, microscopy, and image analysis to facilitate high-throughput phenotyping”), and what he looks forward to when he gets home (his seven month old son).

Read the full article at: http://www.vitisgen2.org/staff-spotlight-surya-sapkota!

 

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Alumni Update – It’s a Boy!

Shanshan Yang, a former postdoc on the VitisGen1 genetics team, is proud to announce the birth of her baby boy, Ryan, on June 5th!  Shanshan is currently the Bioinformatics Core Manager at the Biodesign Institute of Arizona State University.  Congratulations, Shanshan!

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Why the World’s Most Popular Wine Grapes Are Vulnerable to a Pandemic

An article by Jen Pinkowski in Mental Floss discusses the lack of genetic diversity in the most commonly cultivated V. vinifera grapes.

“The vast majority of wine produced across the world derives from a single grapevine species: Vitis vinifera. The domesticated grape has thousands of varieties, and quite a lot of genetic diversity among them, according to a 2010 paper in PNAS that analyzed genome-wide genetic variation of more than 1000 samples of V. vinifera subsp. vinifera and its wild relative, V. vinifera subsp. sylvestris. But that’s not true for all grapes: Nearly 75 percent of cultivars had a first-degree relationship to at least one other. They were either parents or children.”

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The Quest to Grow the First Great American Wine Grape

An article by Kevin Begos in the Smithsonian Magazine features the VitisGen2 project.

VitisGen is a project that aims to do for wine what the Human Genome Project did for humans. That is: use the vast power and rapidly declining cost of DNA research to pinpoint the precise chromosomal locations in American grapes that drive flavors, aromas, grape size and other important attributes.”

Grape breeding PhD student Laise Moreira collects flower tissue for analyzing sex trait in grapevine as part of the VitisGen2 project at the University of Minnesota Horticultural Research Center in Excelsior, MN. (Matthew Clark / VitisGen)

 

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Molecular Changes in Vitis vinifera Associated with the Onset of Pierce’s Disease

A new publication out of UC Davis, co-authored by VitisGen2 genetics team member, Dario Cantu, investigated molecular changes in vines infected by Pierce’s disease.  The bacterial disease is of major concern because it is vectored by a ubiquitous insect, the glassy winged sharpshooter, and it kills vines within 3-5 years by attacking the xylem.  The study outlined a list of molecular markers for further investigation and possible use in breeding programs.

Symptoms of Pierce’s disease on a grapevine leaf. (Jack Kelly Clark / UC ANR)

 

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Webinar – Automated Evaluation of Grape Breeding Progeny to Reduce the Phenotyping Bottleneck

April 19th 2018, 2pm EST – Automated Evaluation of Grape Breeding Progeny to Reduce the Phenotyping Bottleneck – watch recording

While genetic information is becoming inexpensive, measuring attributes of interest such as disease resistance or cluster architecture has been a laborious, manual process. VitisGen2 researchers are developing methods of more rapidly and objectively screening ‘mapping populations’. Their goal: to streamline the process of associating plant traits with genetic markers.

 

 

 

Panelists from the VitisGen2 Breeding and Local Phenotyping Team (left to right):
Lance Cadle-Davidson, VitisGen2 Project Co-Leader and USDA Research Plant Pathologist
Rachel Naegele, USDA Research Horticulturist
Anna Underhill, MSc student at University of Minnesota

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