it is limited to a certain time frame. Please see the website for the scientists that are offering this:
I wish there was more information on the website in regards to who is doing the testing, etc. I hate to be a Negative Nelly, but it’s asking people to pay $40 per test with little information in regards to what is being done and who is doing it. 
the site explains everything and who is doing it
[QUOTE=Daventry;7491218]
I wish there was more information on the website in regards to who is doing the testing, etc. I hate to be a Negative Nelly, but it’s asking people to pay $40 per test with little information in regards to what is being done and who is doing it. ;)[/QUOTE]
I don’t think there is any reason to believe that this research group is lacking in knowledge or competence: the information that may come out of participation in this sort of study extends much farther than the yes/no answer typically offered in a commercial gene test.
English questions please address to G. Gregorio
gestuet.falkenhorst@t-online.de
If I had a suitable horse, I’d be sending off samples :yes:
Mutations in MITF and PAX3 Cause “Splashed White” and Other White Spotting Phenotypes in Horses
Methods/techniques
Linkage and association mapping
We isolated genomic DNA from either EDTA blood or hair root samples from all horses. We genotyped 31 horses with the illumina equine 50K SNP beadchip containing 54,602 SNPs. We used the PLINK v1.07 software for pruning of the genotype data set [30]. We removed 28,381 SNPs that did not have genotype calls in every animal and 10,753 SNPs that had minor allele frequencies below 5%. For the final analysis 31 horses and 19,319 SNPs remained. We used the Merlin software [31] and a fully dominant model of inheritance to analyze the data for parametric linkage. We used the PLINK software for genome-wide association analyses. Empirical genome-wide significance levels were determined by performing 100,000 random permutations of the assigned phenotypes.
DNA sequencing and mutation analysis
We amplified exons, flanking intron regions, and proximal promoter sequences of the KIT, MITF, and PAX3 genes as well as exon 2 of the EDNRB gene (Table S4). We subsequently sequenced the PCR products on an ABI 3730 capillary sequencer (Life Technologies). We analyzed sequencing data with the Sequencher 4.9 software for polymorphisms (Gene Codes). In the macchiato horse and its parents we sequenced all exons of the KIT, MITF, MLPH, PMEL, SLC36A1, and SLC45A2 genes.
Electrophoretic mobility shift assay (EMSA)
We expressed recombinant wild-type and N310S MITF from in E. coli Rosetta 2 cells in LB medium supplemented with 1% glucose (amino acid residues 112–207 from uniprot acc. Q95MD1). We lysed the cells by sonication in 20 mM Tris, 500 mM NaCl, 50 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% (v/v) ?-mercaptoethanol, pH 9. We applied the soluble fraction to Ni-NTA resin and eluted MITF with an imidazole step gradient (0.2–0.6 M) and further purified the proteins by reverse phase HPLC on a C18 column, using a gradient of 5–95% acetonitrile (0.1% TFA) over 20 ml at 1 ml/min. We lyophilised the protein-containing fractions and stored them at ?20°C. The protein was refolded by resuspension in 20 mM Tris (pH 7.4), and we confirmed the correct folding by circular dichroism (CD) spectropolarimetry.
We performed the EMSA using an M-Box containing oligonucleotide (5?-GGAAAGTTAGTCATGTGCTTTTCAGAAGA-3?) as previously described [32]. The EMSA reactions contained 20 mM Tris, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 33 µg/ml BSA at pH 7.4. After incubation on ice for 30 min, we separated the samples on a 6% (w/v) non-denaturing polyacrylamide gel, in 0.5× TBE (45 mM Tris, 45 mM boric acid, 2.5 mM EDTA, pH 8.3) and analyzed the gels using a PhosphorImager (Molecular Dynamics). We quantified the bands from three repeated experiments and calculated average values and standard deviations.
it is the same group that does this testing that has found all the different W types last year. Two names are given in the site, Dr. Rony Jude and Professor Leeb. There are many more in that group, but if you search the research from last year you will finde these names among the scientists that found all the new W forms!
My only problem with it was that the webpage and domain name itself are not directly linked to a website for a testing lab, University or anything else…or providing links to any recent work or research. It’s just a free webpage through a local website design company like Weebly. So if the viewer doesn’t recognize the two searchers names posted, they will just have to trust the validity of the webpage and send off their money. I’m not denying that it isn’t legitimate work…just that some Horse Owners might shy away from participating because the webpage is just an entity of it’s own and not a subpage of a larger organization.
Leeb in the article is a respectable researcher. I have been in contact with him over the years about my white TBs.
They have offered this possibility but they are scientists and researchers and have no idea how to go public with such and since I am friends with Dr Jude I have offered to do them a website for this. It cost me weeks of time to have it set up and translate and do the forms. Since this is a time limited offer I thought a free website would do this good. We would never have thought someone would have thought this not to be honest.
And since there was no time to ask many for allowances to use their pictures, you only see pictures of my W20 horses. Sorry if this got a wrong idea.
This is a research not the final offer of a test which will then take place later over the labs once this research has set all finally in stone.
I am excited about this. I have two mares that I am going to test… thank you so much for posting the link!
[QUOTE=aurum;7492760]
They have offered this possibility but they are scientists and researchers and have no idea how to go public with such and since I am friends with Dr Jude I have offered to do them a website for this. It cost me weeks of time to have it set up and translate and do the forms. Since this is a time limited offer I thought a free website would do this good. We would never have thought someone would have thought this not to be honest.
And since there was no time to ask many for allowances to use their pictures, you only see pictures of my W20 horses. Sorry if this got a wrong idea.
This is a research not the final offer of a test which will then take place later over the labs once this research has set all finally in stone.[/QUOTE]
Gotcha! Hopefully owners will read this and send in their samples.
So you send in hair samples to find out if your horse carries a splashy color gene and then you breed to another horse that may suppress all that fancy color without you knowing it… and then what do you have?? I guess I just don’t get this stuff…
The more we know about color genetics, the more we have the ability to see if there are lethal or at least non-lethal but still detrimental consequences associated with those genetics.
Thankfully genetic testing linked the (sometimes) colorful Frame genetics to the LWO problem, and we can avoid that 
It’s theorized there are lethal homozygous DWs (or now, really, it’s being called just “White”) gene, so it would be nice for MOs to know that so they don’t potentially start wasting breedings that never take If we can get a strong correlation between the genotype and the phenotype, that’s moving along in trying to avoid those combinations if necessary. Given the increasing number of known W mutations, it’s not likely they’ll all be available for public testing - too $$. But through all the W testing being done, some patterns/phenotypes are starting to emerge as being associated with different ones, as well as their combinations.
Hmmm, if I do decide to breed my filly I tentatively have a stallion picked out who looks to me to be a bit of a bling killer. If my filly who does have quite a bit of white has that W20 would that make her less likely throw a foal whose bling is killed?
It’s not just W20 which you have to “worry” about though But yes, a white killer is better option if you don’t want more white than she has. Even if it’s proven that W20 doesn’t do anything more than loud but still “normal” white markings, if you don’t want white that comes to the stifles and elbows and belly splotches, then a white killer will help keep things more normal. Theoretically anyway LOL
The bigger issue would be if you ended up with a W5 x W20 horse - those tend to be all/mostly white
I just don’t want a foal with no white. I mean it wouldn’t be the end of the world of course but would prefer that not happen. Would you test JB? At this point I don’t even know if I will breed her at all so it may be a moot point.
I would love to see Juliet tested. I think she is Splash for sure, possibly with a DW in there as well. It would be a great help to the overall project to have one like her tested, especially if you also do the SW test (1 test looks at all 3 variations).
It’s very unlikely she’d produce a foal without any white, not given who her sire is, not given how much white she has despite being black-based. Who’s the potential sire?
I’ll PM you, I don’t know if the stallion owner would like me calling him a bling killer especially since he has thrown foals with white.
Ok, I looked at the stallion’s offspring gallery, and thankfully there are lots of pics of the foals with their dams, to see how much white the dams brought to the picture.
I wouldn’t call this one a white killer, but he certainly doesn’t appear to do much to increase white. I saw a lot of foals with white very similar to to the dams - low socks begat low socks, a star begat a star, etc. I saw 1-2 with more white than either sire or dam, and that was likely just the right combination of 2 different white genes for an additive effect.
I wouldn’t worry at all about no white with this stallion and Juliet. Not all the foals listed showed the dams, so I don’t know if any of the dams had a ton of white yet produced something so little to un-marked, but with the rest I did see, I wouldn’t be concerned.
Thanks! Things are very up in the air with the ownership of my barn and I don’t want to be scrambling for a place to move a pregnant mare so am holding off for now. It is good to know just in case though.
From talking to Leeb before since there isn’t a test for splash/ it’s the one they know the least about its probably on the table for research…
