Unraveling the Mysteries of Merle All information from the book “Merle - SINE Insertion from Mc - Mh - The Incredible Story of Merle” 2018 Mary Langevin www.merle-sine-insertion-from-mc-mh.com Based on our published research paper – “Merle phenotypes in dogs - SILV SINE insertions from Mc to Mh” - “langevin et al” Mary Langevin, Helena Synkova, Tereza Jancuskova, Sona Pekova Published: September 20, 2018 https://doi.org/10.1371/journal.pone.0198536 If you have been breeding Aussies for any length of time, it is Original Merle Test Result – reported as Mc/M likely that at some point your dam has whelped a pup which has left you mystified by what you were seeing. Perhaps white body splashes that were not expected and are not to standard; dilute looking pups when d/d is not a possibility; brownish or off-shading on a black offspring; excessive white offspring and yet one parent is phenotypically solid; Merle offspring that were not expected as neither of the parents express a Merle pattern; a solid pup when one parent is M/M and all pups should be Merle; an unusual Tweed Merle pattern unlike that of the Merle parent and other littermates. The Tweed phenotype is described as a Merle pattern that expresses with random shaded-in or solid areas, usually with two or three distinguishable shades. Once thought to be a modifier of Merle and now shown to be created by several different Merle genotypes. Chromatogram High-Definition Test Result Including Base Pair - Mc/M - 224/265 There have always been questions surrounding Merle - a SINE insertion consisting of 3 parts - head, body and tail (poly-A Now the mission would be to assign the length of the base pair tail) which contains a long string of repeating base pairs. This numbers (the genotype) to the Merle pattern (the phenotype). The mutation impairs the ability of cells to produce normal pigment and lengths of each allele in the research paper (2018 “langevin et al”) leaves random areas of the coat which are diluted to a lighter were arrived at specifically to address the needs of breeders. Not only pigment. When Dr. LA Clark identified the Merle mutation in 2006 it how a particular dog would express but how this dog would breed was an exciting time! We thought we would get the answers we were when alleles were combined with those of a mate. In order to achieve searching for that had eluded breeders for generations. What came these precise results of genotype = phenotype, there must be a next was disappointing to say the least - solid dogs testing as M/M working knowledge of dogs in pedigrees; parents, offspring and that did not produce Merle offspring. How could that be? It left so related individuals through the generations. Although I was many questions unanswered that the testing was considered by many responsible for setting the boundary of each of the 6 M* alleles, I did to be flawed and in 2009 the only lab testing for Merle under patent not accomplish this on my own. Many owners and breeders from all removed the test from their color panel. breeds worldwide offered testing and pedigree information on hundreds of dogs. This is a prime example of how breeders who are In 2010 Thermo Fisher introduced the ABI 3500 Genetic on the frontlines of recognizing the colors and patterns from parent to Analyzer. We didn’t realize it at the time but this advanced analyzer offspring, have been instrumental in helping labs and researchers to would give us the answers we had been searching for and give develop new testing. breeders the information needed to make educated breeding choices. Note: I am often asked why every lab has not adopted the new testing The original Merle test could only accurately identify the body of method. Answer - the ABI 3500 Genetic Analyzer has an approximate the Merle mutation and the assumption was made that any reasonable cost of $200,000/US, a large sum of money for a lab to invest in if length of poly-A tail would produce some type of Merle pattern. they already have an older analyzer. Using the new technology available allowed for the “base pairs” of Merle’s tail to be counted. This is an extremely challenging process For this article I have specifically used examples of Australian when Merle’s long monotonous poly-A tail is involved. The Shepherds; however some cases may require the use of a different conditions and quality of products used for this process must be of the breed. Merle acts the same in all breeds. highest quality to give optimal results. NOTE - many Aussies pictured in the article are from Europe where there is a ban on tail docking 7 Alleles on the M Locus Setting the borders for the new Merle alleles was an immense task that came with an immense responsibility. Merle’s poly-A tail is a “continuum” from 200 - 280 base pairs. The word continuum is 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 The Scale of Merle - FOR BREEDERS - BY BREEDERS different from each other. It is without stop, without a break or interruption.” So how and why have the base pairs been broken down We can assume that Mh - Harlequin Merle is the original into different bins and named as separate alleles? ancestrally allele as it is the longest. So where have all the other allele lengths come from? This was done in order to address the three most important concerns that many breeders have. #1 - which combinations of alleles can express with a Merle pattern? Mosaicism #2 - how crisp and clear will that pattern be? Some of the most exciting test results which explained so many previously unexplained irregularities we often see in some Merle #3 - which combinations of alleles can delete pigment to white within patterns and differences in phenotype from parent to offspring, were the Merle pattern, and therefore come with the risk of hearing and/or the mosaic results. I remember very well my astonishment at seeing vision impairments? that first mosaic result, “WHAT IS THAT?!” And then that moment of profound revelation when suddenly it all came together and made These are important distinctions for a breeder who strives to sense. produce Merle patterns that fall within the given guidelines of many breed standards. There is also the issue of not wanting to produce Mosaicism - “Somatic Mosaicism” or “Somatic Mutation” is the “Double Merle” offspring in a resulting litter. Breeders not only need presence of two or more types of cells with different genotypes to know the most typical phenotype expression for a given allele and present in the body of one individual dog. Merle mosaicism results their combinations; but also how that dog will breed, what patterned from the shortening of the poly-A tail in one cell in the early stages of offspring this dog can produce when paired with the alleles from the embryo development. This mutation is then replicated during cell mate. These are the most essential questions and the alleles of m, Mc, division. The shortened length/allele will be present in only some of Mc+, Ma, Ma+, M and Mh provide those answers. the adult cells and in different parts of the body. From 308 Merle dogs tested, 56 are mosaics - an average of 18% or 1 out of every 5.5 The Merle trait is an “incomplete dominant”, one allele does not dogs having 3 or more different alleles on the M locus, indicating that completely dominate another. Depending on which 2 alleles are mosaic results are not uncommon. inherited, it can create an intermediate expression or a completely distinct pattern. When the base pair numbers were set for each allele NOTE: Merle’s poly-A tail is not unique in this sense of shortening. It it was not just a matter of looking at the phenotype of each allele as is common for the tail of all SINE’s to shorten. In this way heterozygous, each length had to be looked at as homozygous, as well researchers can estimate the age of the insertion - the longer the tail as when paired with a different length allele. There are 28 possible then the more recent the insertion. For example “at” - Tan Points on combinations from 7 alleles. 14 of those combinations can delete the A Locus is surmised to be an older SINE mutation as the poly-A pigment to white. In this article I cannot include examples of all tail length is very short and stable at 99 - 100 bp. However, judging combinations so will concentrate on the examples that provide the age of the Merle’s poly-A tail in this manner will not apply as the answers to unusual phenotypes. shortening has not been left to nature. As breeders we have artificially kept the longer visible lengths of M and Mh in play by intentionally breeding for the trait. If Merle had been left to nature • m Non-Merle Wild Type the visible pattern would likely be gone by now as every dog’s length became Mc. • Mc Cryptic Merle 200 - 230 bp • Mc+ Cryptic Merle + 231 - 246 bp Typically, only a small proportion of cells contain the shortened • Ma Atypical Merle 247 - 254 bp allele - this is referred to as the “minor” allele/s and represented in • Ma+ Atypical Merle + 255 - 264 bp test results with the use of [square] brackets. The two alleles with the higher peaks on the chromatogram having the larger fraction of cells • M Merle 265 - 268 bp are referred to as the “major” alleles.