Introduction And History Of Merle

There have been many questions regarding Merle since Dr. LA Clark first idenfied the mutation in 2006. Since Merle affects so many breeds I am sharing this excerpt from my book in order to make this important information more accessible for everyone in all breeds.

MERLE - a SINE insertion (Short Interspersed Element) on the SILV gene.

Merle - a SINE insertion (Short Interspersed Element) on the SILV gene. Think of a piece of foreign DNA (viral-like) that has "inserted" itself into the chromosomes and has changed the number of base pairs by adding a piece of DNA. This SINE insertion lives off the host much like a parasite and can change the way cells function.Both SINEs and LINEs (Long Interspersed Element) are common in canines and all mammals, including ourselves. They can be a form of evolution, sometimes destructive and sometimes a useful incorporation into the genome. Most SINE insertions occur in areas of the genome where they cause no damage or change to the host. However, some occasionally insert in regions where they can disrupt and alter expression.

There is some thought that it is these SINE and LINE insertions in canines that have helped to "create" (with our outside influence) our different dog breeds.

In the case of the Merle mutation, a SINE insertion impairs the ability of cells to produce normal pigment - a defective or faulty design. Merle refers to the pattern on the coat, not the color. Random areas of the coat are diluted to a lighter pigment, creating a combination of areas consisting of diluted hairs mixed with areas of full pigmentation. Merle is an "incomplete dominant", one allele does not completely dominate another and depending on which 2 alleles are inherited in the individual dog, it creates an intermediate expression or a completely distinct pattern.The Merle SINE mutation consists of 3 parts - a head, a body and a tail (poly-A tail). The tail contains a long string of repeating base pairs. Think of the poly-A tail as a section of a lariat-like thread of DNA that "catches and ties" into the dog's DNA, the head then attaches itself - "bites down" - so the whole invader can work its way into the DNA.

This is not very scientific language, but gives a good visual of the process.The original Merle test only identified the body of the Merle mutation and the assumption was made that any reasonable length of tail produced some kind of Merle pattern; the longer the tail then the more Merle. No distinction was originally made between the tail length (base pairs) and Merle phenotype.

When Merle testing was first available any length of Merle was reported as a full M - Merle. Numerous owners had their dogs tested and were receiving results that their solid looking dog was in fact M/M.

How could a solid dog be a "double Merle"!?
The Merle test results were just not making sense and were considered by many people to be "flawed."

We now understand that these dogs were most likely "Mc/Mc" - or even possibly "Ma/Mc" - having 2 cryptic Merle mutations - a length of Merle so shortened or truncated that it can no longer cause a change to the coat pattern.
Eventually the test results were given using "Mc", indicating that the mutation was not a full "M" but a shortened one and genotype results seemed to make more sense with the phenotype of the dog. 

As the years have passed more Catahoula owners, especially in Europe, were having their Catahoulas tested through a lab in Germany, Biofocus.

During this time Dr. Helena Synková, was the breed advisor for the Catahoula Club EU in the Czech Republic, as well as being a Catahoula owner and breeder herself. By observation alone of litters produced by dogs that appeared at first glance to be non-Merle, Helena suspected that there was more to Merle then just "M" and "Mc". These non-Merle, solid looking dogs had a certain quality to their coat, some with even very faint Merle patterning that the eye could see, while maybe not visible in photos. These dogs were Merle testing as "Mc/m" or "M/m", but did not show the typical Merle expression and did not breed as the tests concluded.

Helena approached Biofocus with this information and working closely with Dr. Beatrix Böckmann, they started testing dogs whose phenotype did not match their genotype. By paying special attention to the length of the base pairs (the length of the poly-A tail), the findings concluded that there was also a third length of Merle - a length longer than cryptic Merle "Mc", but shorter than a true full Merle "M".

Biofocus started reporting this length on lab results but continued to identify this shortened Merle as "M" - a full Merle. Helena noted that there was a very clear phenotype when this shortened Merle was inherited with either "M" or "m".

In 2011, she wrote an article for several dog magazines in Europe and for the Catahoula Club EU in which she gave this shortened Merle length the working name of "Atypical Merle" - "Ma".

In May 2015, Biofocus officially recognized this shortened Merle as an allele on the M Locus and adopted the working name that Helena had assigned to it in her article - Atypical Merle - "Ma".

Merle testing by Biofocus gave us 4 separate alleles on the M Locus:

• Full Merle "M" - a length of 459 - 462 bp
• Atypical Merle "Ma" - a length of 443 - 449 bp
• Cryptic Merle "Mc" - a length of 400 - 425 bp - at this point the tail has been so shortened that Merle can no longer express.
• Non-Merle - wild type "m"- no SINE insertion length of 199 - 200 bp

Note - The M, Ma and Mc lengths given are a combination of "m" length 200 + the length of the SINE insertion.

Example "M" = length of "m" 200 + SINE length of 259 - 262 = 459 - 462.

With all 4 alleles identified on the M Locus, breeding results are finally making sense.

This is a prime example of how breeders of all breeds, who are on the frontlines of recognizing the colors and patterns from parent to offspring, have been instrumental in helping labs and researchers to develop new testing.

With the inclusion of the "Mc" allele, the differences in Hope's Merle test results from 2 different labs are not as noticeably "flawed" as the example of Leo above. 

However, if Hope was bred to an "M/m" male, she would be able to produce "M/Ma" - Patchwork offspring. This would be confusing to the breeder since she tested as "Mc/Mc" at a lab who has not yet included the "Ma" allele in their test results.

Once the different lengths of the Merle alleles started to become known, a lab in the UK - Laboklin and partner lab Labogen, started testing for the different base pair lengths of M, Ma and Mc. They also included Mh - Harlequin Merle in this testing.
Mh being a length of Merle longer then M. In the past, Harlequin Merle has been referred to as "Herding Harlequin", as it is often seen in herding breeds such as the Border Collie and Australian Shepherd. This pattern has been most typically recognized as having a similar type of expression as H/h - Harlequin as is present in the Great Dane breed but these breeds all tested as h/h - Non-Harlequin.
It was theorized that there could be another modifier of Merle causing this pattern, as these dogs routinely tested as M/m on the M Locus and S/S - Non-Piebald.

Several Catahoulas already tested through Biofocus were sent to Laboklin to ensure that the lab's base pair numbers for M, Ma and Mc were correct. As well, Catahoulas with unexplained white patterns were tested. These dogs were confirmed as Mh - Harlequin Merle - a length of base pairs longer then M, which can cause areas to delete to white.

It was exciting to have a test available that could report on all 5 alleles on the M Locus -
Mh, M, Ma, Mc and m - and finally explain dogs in all breeds with the "Herding Harlequin" pattern.

However Laboklin/Labogen has declined to share the base pair numbers for Mh - Harlequin Merle, as well as the number range they are using for the M, Ma and Mc alleles, citing "legal issues of confidentiality."

Their analyzer machine is different from the one that Biofocus uses (although still the same method of testing) and the base pair numbers slightly different and allowing for the Mh allele having higher base pairs then M. This will be explained in more detail in the following section.

Mh - Harlequin Merle

Vemodia - Laboratory of Molecular Veterinary Diagnostics

In March 2016, a representative from Vemodia Labs in the Czech Republic purchased the first release of "Catahoula Coat Color Genetics"and expressed great interest in developing their Merle test for the different alleles.
In early fall 2016, RNDr. Tereza Jančušková, Ph.D. and MUDr. Soňa Peková, PhD. contacted Dr. Helena Synková to arrange for the testing of 20 Catahoulas with already known genotypes.

Vemodia's data collecting method uses "Fluorescent Fragmentation Analysis" on an "ABI (Applied Biosystems) 3500 Capillary Sequencer". This genetic analyzer automatically counts the base pairs and produces a "chromatogram" - a "picture" showing the peaks of the base pairs and where they fall within the M Locus alleles.

Compared to Biofocus' analyzer, a Capillary Sequencer 310, the base pair numbers for the alleles are slightly smaller (tighter together on the number scale), resulting in higher base pairs numbers for each allele.The difference in numbers from both labs could be explained by several reasons -

Vemodia's analyzer is a newer and more advanced machine.
As well the kits of chemicals and enzymes used for the testing process are produced by many suppliers worldwide, each giving a possibly slighter difference.

How Merle Testing Is Done
DNA is isolated from samples - either blood, buccal swab or in some cases sperm. This sample is then processed using a technique called PCR - "Polymerase Chain Reaction" - this procedure amplifies (expands) a small segment of DNA into thousands or millions of copies with the identical sequence so that the sample can be studied in more detail. This is an extremely challenging process when Merle's long monotonous Poly-A tail is involved. This amplification is an artificial method; (unlike the DNA replication in living cells) thus the enzymes used for the in vitro (in a tube) amplification can make more errors then the in vivo system (the living cell) does. The conditions and quality of products used for this process must be of the highest quality to give optimal results.
A fluorescent dye is incorporated into one of the amplification primers which, in turn, "stains" the resulting PCR amplicon. This is referred to as Fluorescent "Labeling" or "Tagging". The obtained fluorescently labelled PCR products are then separated in a high-resolution polyacrylamide gel in a capillary sequencer, according to their size - the base pair numbers that can be read in the resulting chromatogram.

Testing with the cooperation of Vemodia has given us very clear phenotypes in relationship to genotype and the number of base pairs within each allele. Results have also shown dogs with more than two M locus alleles being "mosaics" with three or more M alleles. These findings will be covered in detail in the Mosaic chapter starting on page 33.

Merle testing by Vemodia has now given us 7 alleles on the M Locus

• m - Non-Merle - Wild Type
• Mc - Cryptic Merle          200 - 230 bp
• Mc+                                        231 - 246 bp
• Ma - Atypical Merle        247 - 254 bp
• Ma+                                        255 - 264 bp
• M - Merle                             265 - 268 bp
• Mh - Harlequin Merle    269 - 280 bp

Vemodia has been a wonderful lab to work with, very helpful and forthcoming with all their testing information.
They were also receptive to input from Helena and I when it came to genotype and phenotype.
I would like to thank RNDr. Tereza Jančušková, Ph.D. and MUDr. Soňa Peková, PhD. for all their work.
Examples of dogs tested for their trial and the corresponding chromatograms results will be shown starting on page ***

Example of a Merle test result Chromatogram from Vemodia Ma/M - 251/266 bp

There have also been "Mosaic" results with a dog having 3 (or 4) alleles on the M Locus.
This dog tested as Mc/Ma/M. By parents this dog should have been Ma/M - either of these alleles has mutated/shortened early on in the embryo development and created some cells with the Mc allele. If this shortened allele is present in the area of reproduction - semen or egg then it can be passed along to offspring.
"MOSAICISM" will be covered in detail starting on page "**"

M Locus

7 alleles on the M Locus "Merle phenotypes in dogs - SILV SINE insertions from Mc to Mh" [i]

• m Non-Merle - Wild Type
• Mc Cryptic Merle 200 - 230 bp
• Mc+ Cryptic Merle + 231 - 246 bp
• Ma Atypical Merle 247 - 254 bp
• Ma+ Atypical Merle + 255 - 264 bp
• M Merle 265 - 268 bp
• Mh Harlequin Merle 269 - 280 bp

[i] "Langevin et at" - Langevin M, Synkova H, Jancuskova T, Pekova S (2018) Merle phenotypes in dogs - SILV SINE insertions from Mc to Mh. PLoS ONE 13(9): e0198536. https://doi.org/10.1371/journal.pone.0198536

This scale demonstrates Merle's poly-A tail as a "continuum" from 200 - 280 base pairs.
The word continuum can be defined as a "whole with no part of which is noticeably different from its adjacent parts, although the ends or extremes of it are very different from each other."

A continuum is without stop, without a break or interruption; a continuance without intervening spaces; not interrupted.

This is a good description of the different Merle phenotypes. For example a dog who is m/Ma - 253 bp can have an expression somewhat like m/Ma+ - 256 bp. A dog who is m/Ma+ - 263 bp can have an expression somewhat like M - 265 bp.
So why then have the base pairs been broken down into different bins and named as separate alleles?

This was done in order to address the three most important questions that many breeders have.
#1 - which combinations of alleles can express with a Merle pattern?

#2 - how crisp and clear will that pattern be?
#3 - which combinations of alleles can delete pigment to white within the Merle pattern?

These are important distinctions for a breeder who strives to produce Merle patterns that fall within the given guidelines of many breed standards. There is also the issue of a breeder not wanting to produce "Double Merle" offspring in a resulting litter. The terminology of Double Merle will be covered starting on page 81.The following are example wordings from various breed standards -

• Disqualifications - White body splashes.
• Faults - Washed-out or degenerate colors.
• Blue Merle: predominantly clear.
• All colors are strong, clear and rich.
• The hairline of a white collar does not exceed the point at the withers.
• Fault - Conspicuous white body spots.
• Disqualifications - White body splashes, any conspicuous, isolated spot or patch of white on the area between withers and tail, on back, or sides between elbows and back of hindquarters.
• Merle pattern is expressed as lighter-colored areas contrasting with the darker base color, which may be any acceptable color. Neither the light nor the dark color should predominate.
• Faults - Rustiness in a black or a blue coat.

These breed standards do not allow for the muted, indistinct pattern that Ma+ often expresses as; the amount of diluted grey area of Ma/Ma; the grey or brownish "off-color" from Mc/Mc, Ma or Ma+; the white that is often expressed from Mh and any number of the 13 different allele combinations that can create white "splashes" on the body; the Tweed pattern created by many allele combinations and by Mh.
The base pairs numbers of each allele were arrived at specifically in order to address these important concerns for breeders.Test results given as 245 bp/265 bp are not informative enough. Would you know by these given numbers the most typical phenotype expressed for this dog? Or these results of 249 bp/249 bp? What would this dog look like?
What about breeding these two dogs together - 249/249 x 245/265 with a resulting litter of 245/249 and 249/265?
What Merle phenotypes would these offspring express? Would these length combinations be able to produce white in the Merle pattern? Would these parents and their offspring adhere to patterns specified in some breed standards?
These parents and their resulting litter are shown on page 92. (Pup #5 is bilaterally deaf.)
Neither parent nor any of the resulting offspring would adhere to many breed standards.Breeders need to know the most typical phenotype expression for a given combination; how that dog will breed, what patterned offspring can this dog can produce. These are the biggest questions and the alleles of Mc, Mc+, Ma, Ma+, M and Mh provide those answers. When the base pair numbers were set for each allele it was not just a matter of looking at the phenotype of each allele as heterozygous, each length had to be looked at as homozygous.

Phenotype examples given on page 13 of the book.