US Dept of Agriculture dives into blueberry and cranberry genotyping

Every year over 1.5 million tonnes of blueberries and over half a million tonnes of cranberries are produced. This is expected to rise significantly over the coming years to meet ever-increasing global demand.^1,2 In 2021, the USA was the world’s second-largest blueberry producer and the largest global cranberry producer.^2,3 Both berries are well documented for their health benefits, but the DNA resources that would assist genetic studies and breeding improvements for these crops are limited.

The Vaccinium Coordinated Agricultural Project (VacCAP), which is funded by the United States Department of Agriculture’s National Institute of Food and Agriculture Speciality Crop Research Initiative (USDA-NIFA-SCRI) programme, is focused on addressing major bottlenecks limiting the growth of the US blueberry and cranberry industries by developing marker assisted selection socie(MAS) capacity in breeding programmes. The project is transdisciplinary in studying fruit quality traits by integrating genetics, genomics, plant physiology, socioeconomics and consumer sensory studies.

1. Establish genomic resources to enable effective association mapping studies in blueberry and cranberry.
2. Discover fruit characteristics and DNA markers that can help maximise and match industry profitability with consumer preferences in blueberry and cranberry.
3. Deliver molecular and genetic resources to improve blueberry and cranberry fruit quality traits that maximise and match industry profitability with consumer preferences.
4. Assess the potential socioeconomic impact of blueberry and cranberry fruit quality improvements on market demand.
5. Engage US Vaccinium breeders and stakeholder groups to accept and make use of advanced phenomics and genomics tools to build a more coordinated and efficient cultivar development system.

 

In the early 1920s blueberry cultivation started in the United States and they are now the second largest US berry crop after strawberries.^4,5 The majority of blueberry plants now grown commercially are highbush (Northern and Southern) and rabbiteye. Lowbush blueberries (sometimes referred to as wild blueberries) are mostly harvested from the wild. Vaccinium plants are perennial and their life span in the production setting can be short or long, with some cultivars productive for only 1-5 years or as long as 40-60 years.⁴

Blueberry cultivars belong to the genus Vaccinium and section Cyanococcus. Vaccinium plants are geographically widespread and different species have a high density in different locations across North and South America, Southeast Asia, and the Andes. There are genera related to Vaccinium outside of Cyanococcus, including those found in remote parts of the South Pacific. However, the current level of science does not yet easily allow combining genetics of Cyanococcus with non-Cyanococcus plants.⁴ Blueberries have a highly heterozygous complex genome.

Cranberries

Canada, Chile and the United States account for 90% of the cranberries produced worldwide.² The commonly farmed cranberry is Vaccinium macrocarpon Ait., which is a low-growing, vining woody perennial plant.⁶

Vaccinium macrocarpon is a diploid and one of the few crops that can be found growing in the wild in a wide variety of wetlands, mineral soils and moss lawns.⁶ Its wild relative Vaccinium oxycoccos L. (small cranberry) can be found as a diploid, tetraploid or, rarely, hexaploid, which only grows in peatlands.⁷ The small cranberry has a northern circumboreal distribution making it an interesting source of traits, including cold hardiness.⁶ Increasing global temperatures are likely to have a negative impact on cranberry yields, fruit quality and disease resistance, as these traits are known to be affected by heat stress.⁷

The USDA VacCAP project team includes geneticist Dr Nahla Bassil, Dr Shaun Clare and project director Dr Massimo Iorizzo (North Carolina State University). We spoke to them about the project: Why cranberries and blueberries? “Blueberry and cranberry are perennial crops, and breeding cultivars using traditional methods is a time-consuming process. Marker assisted selection (MAS) represents an opportunity to accelerate the process and combine multiple traits. However, DNA-based resources for these two crops are limited.” How did you select the lines to sequence to cover the diversity? “We used Principal Component Analysis of diversity data and pedigree information to identify representative accessions to sequence.” With SNP markers in hand, what traits are being looked at as beneficial, advantageous and desirable?  “In the VacCAP project we are targeting desirable levels of many traits including: texture/firmness and chemistry (volatiles and non-volatiles), which affect sensory perception like sweetness and acidity but can also extend shelf life and reduce fruit damage during mechanical harvest.” What unique challenges do cranberry and blueberry have in terms of their genome genetics? “The majority of cultivated blueberries are tetraploid, and some are hexaploid. As a diploid, cranberry has a less complex genome, but is highly heterozygous like blueberry, which brings a different level of genome complexity.” What criteria were used to compare different platforms and how did Flex-Seq meet those?  “Flex-Seq was selected because it is flexible and the content of each panel can be modified without a large initial investment. Dosage can be estimated for the cultivated tetraploid blueberry. SNP, as well as haplotype data, can be generated. The same panel can be used across different programs. It is relatively cost-efficient for genome wide association studies.”

Choosing Flex-Seq as a high-throughput genotyping platform

The VacCAP study set out to provide a high-throughput genotyping platform for cranberry and blueberry that could:
Interrogate the SNP catalogue

• Deliver high SNP recovery
• Provide accurate genotyping
• Be flexible
• Offer reproducible genotype calling
• Be cost-effective
• Generate dosage and haplotype in the tetraploid cultivated blueberry

The cranberry SNP catalogue was developed by Dr Ping Zhang at Washington State University. This included de novo SNPs from the 12 cranberry accessions that were sequenced and 8 SNPs that were associated with traits of interest from the association mapping done by Dr Juan Zalapa (co-PI on the project). Core genes, associated genes and trait-associated SNPs were then prioritised.

The blueberry catalogue included more sequences. Mandie Driskill of Fall Creek Nursery performed the SNP calling. The catalogue included de novo SNPs identified from 47 diverse accessions and transcriptome sequences of 16 cultivars. An additional 491,000 SNPs were provided by VacCAP community members from 8 association studies and 5 biparental maps. Like cranberry, core genes, associated genes and trait-associated SNPs were prioritised.

The Flex-Seq platform was chosen for several reasons:

• It is a two probe hybridization based approach
• It generates 200-300 base pairs sequences, including target group and variations in that region.
• The process is easy: The research team collects the leaf samples and LGC Biosearch Technologies performs the DNA extraction as well as the genotyping and sends the data back to the team.
• The volume ordering is cost-effective.

The diversity set tested on the platform:

Blueberries

Northern Highbush tetraploid: 72 samples 4x

Southern Highbush tetraploid: 72 samples 4x

Rabbiteye hexaploid: 21 samples 6x

Others, including samples from active breeding programmes: 27 samples mainly 6x
 

Cranberries

American Cranberry diploid: 184 samples 2x

Common Cranberry: 3 samples

Hybrids: 5 samples

An initial Flex-Seq probe set of 50,000 loci was designed and developed. From this, the best performing 22,000 blueberry and 15,000 cranberry loci were selected for downstream analyses and in future genotyping projects.

Biosearch Technologies provided output data in FASTQ format, similar to a FASTA file but with the addition of a built-in quality score for every nucleotide along that read, together with a variant call format file (VCF) and their own custom haplotype file. The haplotype file combines all of the aligned reads, FASTQ files processed into BAM files, then uses the VCF file to assemble haplotypes for each probe. This file format includes the probe ID, every single variant in the VCF for that probe, a list of all the genomic positions and the reference genome alignment and long haplotype sequences with the read depth of the haplotype.

Study conclusions

• Flex-Seq has excellent recovery in blueberry and cranberry, including distantly related accessions.

• The platform provided uniform coverage across the genome and SNP classes for genetic studies.

• Flex-Seq can use higher depth to obtain more variants across more loci or more accurate dosage calls in higher ploidy (6x, for example).

Flex-Seq platform

The  Flex-Seq platform enables breeders to cost-effectively screen tens of thousands of markers. Flex-Seq uses probe hybridisation which ensures consistent and repeatable results. Up to 98% of the markers are recovered. The panels are easily customisable with the addition or removal of markers. This flexibility results in effective flexibility when compared to chip-based array. The low cost per sample facilitates more screening, increasing yield and genetic gain and getting complex traits to market sooner.

Talk to one of our scientists

Whatever stage of the genotyping journey you are on, Biosearch Technologies have the technology portfolio, the scientific expertise and the partnership commitment to support you to reach your goals. Our KASP genotyping technology has been transforming agriculture for 21 years. We can address challenges and help you accelerate your genotyping journey with our in-house NGS and PCR services.

Our scientific team is available to collaborate with you to achieve your genotyping goals. You can email us at techsupport@biosearchtech.com.

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