Tuesday, September 23, 2014

Fingerprinting Raspberries and Blackberries

This past summer Team Rubus presented a poster, "Who’s your raspberry daddy?" at two Research Station field days in June. And in August, we presented another poster titled "Comparative diversity analysis of Southeastern Rubus germplasm through molecular and pedigree techniques" at the International Horticultural Congress. Although the titles of the presentations were different, the presentations were essentially the same. Both were discussing how our breeding program has incorporated fingerprinting technology into our program. Below is a brief synopsis of this work.

Why do we need fingerprints?
- Can sort mix ups in research and grower fields and nurseries
- Distinguish between closely related varieties
- Potential patent protection (not used yet for Rubus)
- Determine how diverse germplasm is in a breeding program

How do we produce fingerprints?
- Collect young leaf samples in spring or summer
- Extract DNA from leaves, and tag each plant using 6 DNA markers that are specific to red raspberry (Bassil et al.)

- Each genotype/variety/cultivar will have a unique profile  or "fingerprint" for the 6 DNA markers
- The "fingerprint" of each sample is matched to a reference profile in our database, to ensure it is true to type (actually the genotype/cultivar/variety which it is labelled as).

This is a slide from one of the presentations. The first picture shows some of the leaf tissue​, ​mortar​ and pestle​ (old fashion way to grind the tissue), the second picture is of extracted DNA in ​test tubes being prepared for analyses. ​T​he 3rd image is of a PCR printout, and the green bands and blue bands represent ​the DNA fragments, separated by size.  Green and blue are two different markers, which are analyzed simultaneously.​

Here is an example what the data looks like from our selection NC 654 which is a cross between Mandarin (mom) and Willamette (dad). For the DNA marker Meek19, Mandarin, peaks at 170/186, and Willamette, peaks at 170/174.  NC 654 (child) peaks at 170/174.  We can verify that Mandarin and Willamette are indeed the parents of NC 654 using Meek 19, whereby the 170 peaks comes from Mandarin and the 174 peak comes from Willamette. This combination of peaks is unique to NC 654. 

In addition, through some modifications of the established protocol for red raspberry, we were able to determine that we could use these same markers for black raspberry and blackberry.

Bassil NV, Nyberg A, Hummer KE, Graham J, Dosset M, Finn CE. A Universal Fingerprinting Set for Red Raspberry. Proc. Xth Intl. Rubus and Ribes Symp. Acta Hort. 946. ISHS: 2012.

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