Shikoku: how long till the inevitable?

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  • @dlroberts,

    Egags, that's a crazy shotgun approach!
  • @ayk - Money aside, it's the most effective approach right? :-)
  • edited November 2011
    Sorry, I was thinking more on the behavioral/temperament/work side. Like how the WPGCA use their bird trials etc.
  • Ah, well, that's a legit thing to think about. But, first I think you/we/whomever would need to clarify what the goal of the preservation is. My feeling is that it's a health preservation effort first and foremost, and that temperament will come along as a secondary goal with time. So I'd argue the right questions to start with are what health testing is appropriate to improve the breed. Working trails are later in the process IMO.
  • That makes sense.
  • aykayk
    edited November 2011
    @dlroberts

    Maybe I'm too cheapo in my thinking, but I just don't see the value of mapping a Shikoku's genome just blind.

    If the target is to locate disease genes found in the Shikoku, then you have to know what dogs are affected and what dogs are clear for what disease. (Not go in blind.) Then you focus on the genetic differences (which is still a huge number of genes to wade through since each dog is an individual.)

    Then you'll want to compare unrelated but affected dogs so you can narrow down the amount of genes that researchers have to look at. Since there probably isn't such a thing as a completely unrelated Shikoku, this means recruiting other breeds who share the disease. And if the other breeds happen to have powerful parent clubs with deep pockets, that's a huge bonus.

    But this search for genetic diseases wasn't what I was envisioning when I thought of recruiting research grad programs. If I had to create a project, I would focus on the Major Histocompatability Complex genes and not the entire dog genome. I would want to see what MHC haploids exist in the Shikoku (or even the Shiba with their severe allergy issues) and find out what can be done to preserve the greatest amount of variability in the MHC. Find out what lines has the most variability or unique variability that needs to be kept and passed on in the breed no matter if registrations drop. This may not cause a correlated diversity in the other parts of the dog genome, but it should keep the immune system nimble and robust.



  • In terms of genetics and the molecular aspect, like Dave, I am a proponent of deep sequencing for the following reasons:

    1. The canine genome has already been sequenced so we have a reference point

    2. How many MHC genes are there? If there are many, each sequencing reaction usually only covers 1kb so you would need multiple sequencing primers. So, keep in mind that you have to PCR your sample (with a High-fidelity enzyme, around $500 per 200uL and you would need 0.5 uL per reaction so for a population of 100 samples, it would cost $125 in enzyme costs alone, if you are lucky and don't have to repeat any). Then, you would have to clean up your PCR product (kit for 200 preps costs about $231). The cost (at UCR) for about 1kb of good reads is 6.80 per sample (if you sequence in bulk, it's about $471 for 96 sequencing reactions). Remember, if your gene is big, you will need multiple primers so your cost could get exponently high. In addition, this is just raw costs, I am not counting costs for tips/tubes/etc.

    3. Are the MHC genes representative of diversity? Have there been studies as to how diversity is measured? What is the acceptable rate of mutation per generation in canines/what do we expect to see? What will we use as our point of reference? For instance, Kais are very diverse so obviously we cannot use them. (in this case, we would use a species known highly to not be diverse and a highly diverse species as reference points). In addition, I don't think that we could just sequence MHC genes and use them to extrapolate how diverse our sample set is. Perhaps MHC genes are under strict regulation by other factors (methylation, chromatin modifications, etc.) so we may not get a representative view in terms of diversity.

    4. In terms of conservation, from previous threads, it seems that hip dysplasia is a major concern (healthwise). We know that hip dysplasia is a multiple trait loci and not all the contributing genes have been identified. By doing the whole genome sequencing, we could compare the xrays to the genomes of affected individuals, perform bioinformatic/statistical analysis identify correlations of mutations in genes of affected individuals...with hopes of identifying new genes involved in this pathway. This will be important below.

    5. Ayk when you state that, "I just don't see the value of mapping a Shikoku's genome just blind," I would have to disagree with you because whole genome sequencing provides a massive amount of data...the caveat is the you need a bioinformatician to process it. The information obtained from gathering this data is tremendous and I think that it would help in terms of perservation as a whole.

    The reason why I feel that this is the way to go is that, working in a molecular lab, we perform whole genome sequencing quite often (I come from a big lab). In addition, we have to keep in mind that whether we are sequencing several genes or what it is, molecular biology could get VERY expensive, especially when things don't work and to be quite honest, I don't think are pockets are that deep. Therefore, to help, we would need to apply for funding or get someone to apply for funding. Funding is very difficult in this environment, therefore, our funding idea must have a BROAD impact. Even though are main focus may be on Shikokus, we have to answer a question that have broader impacts--> i.e. identification of hip dysplasia genes finds some genes that are linked to osteoporosis in humans or hip dylasia target meds, etc. In addition, in the end, I think it is more cost effective to do whole genome sequencing...a single end experiment with 50 cycles (means 50 nucleotides per read) cost $1078; 100 cycles=$1393 per lane and if we have a lot of samples, we could do paired-end (2x100 cycles) for $2272. Plus, we could add more samples per lane by barcoding the samples (you get less reads (so less detailed)) but for preliminary data, that should be ok. In addition, we don't have to do all the samples at once, we could do a subset, by which we get preliminary data, publish in a good journal (if you want to publish in a high impact journal whole genome analysis are quite helpful), then we could apply for a larger amount of funding for a large-scale experiment. I mean, what we have here already is a thesis work for a graduate student.

    This is just my two-cents on molecular aspect of the project, I didn't mean to offend anyone...and Dave, the reason why it's so expensive for human genome is the number of reads needed is tremendous!!! There are large areas of the genome that are highly repetitive and cannot be read plus, the non-coding regions...

    Personally, I was planning to get several Shikoku (I love them) so perhaps we could set up an exchange breeding program? My biggest concern would probably be who we end up selling the puppies to, if they would adhere to what we impose. In addition, I have several family members who would be interested in co-owning (they only want single dogs) so I don't have a problem with importing non-puppies.
  • aykayk
    edited November 2011
    @ttddinh,

    Glad to see someone here who can keep me honest. My field is environmental chemistry so I haven't taken MCB classes for at least 11 years. In a different arena, I mentioned to Dave that I was just a layman and gave him some referrals to people whose profession is in population/evolutionary biology and genetics. So no offense taken at all. :-)


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    Edited in:

    In response to point 1, 4, and 5: I see that the complete canine genome of a Boxer was sequenced. I would think the best way to identify high profile, non-breed specific diseases like hip dysplasia would be through the already established Canine Health Foundation (http://www.akcchf.org/) which receives the support of the AKC, individuals from various breeds, and grants. I don't see the need to start-up a duplication of efforts from the Shikoku community.

    The Canine Health Foundation also explores breed-specific disease genes and it seems that they don't go in blindly. They utilize their knowledge of what samples are from affects and what are from clears, and determine what must be a "statistically significant association." Here's an example: http://www.akcchf.org/research/funded-research/1615.html.

    I don't know if I'm explaining myself clearly, but I hope the link helps in outlying my thought processes.


    In response to point 2 & 3: The kb and costs details are admittedly beyond the scope of my knowledge. However, there are already research papers on DLA genes in the Canine MHC which identify different haploids/alleles and note the distribution of those alleles across different dog breeds. Ex. http://www.ncbi.nlm.nih.gov/pubmed/16101828 (This particular article is weak though because of the assumption that dogs are unrelated if no common grandparents are found. That's far from the case.)

    The Tasmanian devil is a good case study about how low MHC diversity causes low diversity, but it seems that low MHC diversity and increased autoimmune issues are more indicators of low diversity...
  • If anyone's interested, I just heard of two adult females, 2 and 3 years old that might be available. Age is catching up to the gentleman who owns them, so he's trying to reduce the amount of dogs in his kennel. I can go take a look and scope out their temperament and take a few pictures.

    I'm kind of bummed out right now. I know the risks in the breed, and to be honest I've thought Rome could possibly be a little suspect in either hips/knees, something I figured we'd get tested in the future. Because of that I haven't really trained him that hard since he was a pup, just a bit here and there. He's 1 and a bit now, and I took him for some proper hunting last week. He was fine the whole time, running and whatnot. He was only out day2 and 3, and was running around without any visible signs of anything wrong, except at the end of day 3 walking back to the car I thought he was turning his back feet a bit when pushing off of them.

    Well come the next day, after a bit of running while on a walk, he started limping a bit. It came and went the next day, but after trying to rule out a whole slew of things, it looks like it's off to the vet. The one thing I do take from this is that I think that in lieu of testing of hips/elbows/knees here in Japan, getting a dog from a proven working line, from a working kennel is quite possibly the next best thing (or even better in some ways. I know a friend who also runs Shikoku out of show lines has found one of his males has this 'mystery leg pain', and that showed up one day after a run in the mountains.

    Anyway, I'm all up for an organized reconstruction/preservation effort of the breed. I'll do whatever I can on my end.
  • That is awesome!

  • edited November 2011
    It sounds like an expensive undertaking but count me in.


    I'm working on some preparations as well so that I can focus on importing for co-owns and even supporting a larger pack of Shikoku Ken at home. I'm suppose to be getting a puppy from a breeder who is having the same "problem" as most others...he is getting older, is going to retire soon but says young people aren't into Shikoku these days...he wants me to carry on his lines. Unfortunely he didn't do the breeding this year. So I'm waiting for a litter for next year.


    @ttddinh - Theresa, I would LOVE to see an "exchange breeding program" amongst Shikoku! For the most part, as far as planning and preparations goes, it would seem that it is already going in this direction. As for puppy homes adhering to the contract...well we do our best to screen and place puppies into proper and honest homes. I would like to believe that a majority of people are honest. That it is only those few people who ruin it for others...so I'd expect that we won't have much trouble with the homes we place our pups in (not so much with our breed). As always...leave a paper trail! Should something blow up later on...we can always make up a court case and deal with these situations as they arise. Thankfully, thus far though it would seem, everyone wants to help out with the preservation of the breed. So I don't see it being an immediate issue to concern ourselves too much about just yet. Our main priority at the moment is maintaining diversity. I do worry about breeding ourselves into a corner...we don't want that! I'd also like to work on breeding away from bad hips and allergies (also the stomach sensitivity if possible).


    @thewalrus - Shigeru, I'm not sure that I'll be ready to import any Shikoku this year but I want to import some young but older females for next year (in addition to the puppies). Shoushuu needs an experienced girlfriend, lol. If you can take a look at the females that would be great! I do have some homes interested in "adopting" an older Shikoku and those that are open to co-ownerships.
    By the way, I'm sorry to hear about Rome...I hope it was a fluke and that he will be okay. I mean...he is still young. / Oh, I would not be opposed to incorporate some of the working Shikoku bloodline. Infact, I've been thinking about having several different lines: show, working and "mixed show/working" lines for variety (more selection).
  • Sounds like for adult show line dogs, a day run in the mountains should be done before making the decision to import them.
  • I've had a couple conversations about the breed, its preservation, health issues etc with NIPPO members and judges over the past week or so, and wanted to share some info.

    Shikoku numbers: Obviously the number of Shikoku registered every year is very low (but over the past couple years I believe it is holding at around 300), and we need more pups being born. I think this is something everyone will agree on.

    Shikoku health (namely genetic hip/knee/elbow issues): From what I've been hearing, there were issues with hips etc, but they were more noticeable up till around 10 years ago. NIPPO members have been breeding away from dogs with suspect movement, obvious bad hips, temporary lameness (the nebulous 'nerve pain' as well), to where most people I've talked to can't remember the last time they saw a dog with joint problems.

    Just a bit of insight into the traditional world of NK in Japan, there's a lot of emphasis on tradition (obviously), so knowledge passed on from previous generations, and instinct based on personal experience is valued very highly. There is definitely not a feeling here that the breed is in danger as far as overall health goes. When I bring data to their attention like the recent x-rays of some of the Shikoku in the States, it's surprising to them and I think a natural reaction is to think it's environmental or find some other reasoning for it. There is a lack of knowledge about canine genetic issues, a lack of data of its occurrence in the breed, plus a feeling that these are animals, not machines, so there will be some imperfections.

    What I've taken away from this is that:
    1. We need to increase Shikoku numbers.
    2. There is a need for more data on the breed, not just the dogs in the States, but possibly more importantly, here in Japan.

    I think if there is enough data collected (and it includes data from dogs in Japan), and if that data were to show significant issues in the breed, that NIPPO and its members will take notice. At the moment all we have is scattered data from a very small sample of the breed, so it's probably too early to draw any conclusions.

    On my end, I'll keep trying to find good pups for people in Japan and overseas, get my kennel set up, test my dogs, and hopefully get other NIPPO members interested in testing theirs too. There's no money to be made in breeding the NK here in Japan, NIPPO and the other societies are strictly non-profit, and most of the kennels will place their dogs within Japan in homes that will help with preservation for free, or well under $1000. For someone who has a kennel of say 10 dogs, to get them all tested will be quite an expense, and the only way I can think of this being a feasible proposal is if I can find a way to finance the testing (selling pups from a litter or something maybe?).

    Anyway, just thought I'd add this to the discussion.
  • edited November 2011
    Edit: I moved my comment to the preservation thread...

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  • @thewalrus--I would be interested in the dogs...since they are older, is there any way we could get them tested first?

    Also, I am seriously thinking about writing a grant for whole genome sequencing of shikokus...I think it would be very interesting. Now, I just need a PI who works on canine hip dysplasia who would take me in :) oohh the joys of looking for a post-doc!
  • edited November 2011
    @ttddinh

    That would be really awesome!!
  • edited November 2011
    @ttddinh - No better way to get hired as a post-doc than to come with your own funding. You don't need a PI to take you in, you need a lab with the equipment you need to bring your project too. ;-)

    Also, I'm not in bioinformatics, but I know a lot about the techniques they use. And my father has been heavily involved in biomathematics institutes. If we can get people to do the biochemistry, I can certainly bring the computational/mathematical people to the table. :-)
  • COIs would be good to start organizing 1 generation is closer to 10 than none and at 10 maybe we'll see more. I am happy to help this effort. Are all the imports so far from a geographical area or wherever hunters are?
    I don't have a Shikoku yet, but I would like to ask, too, if we have a strong enough dedication to disclose ALL health problems to one another and share this through a central place with access to all breeders.
    I really enjoy some of the suggestions here.
    If breeders are not open and honest with one another, then the Shikoku is extinct. With organized data, I might ask a couple of friends involved in search institutions if they know.
    There was a primitive dog research project at UC Davis.
    I believe it was part of the canine genome early on.
    Let me talk with friends.
  • Brad too, knows Vladamir Beregevoy. Someone said here on the forum, that they were on the OrigCangen list. I am too, but as I have been involved with family problems recently I am waiting for the moment to read the last couple of hundred. That list is voluminous with all kinds incl scientist breeders, show people and it is always a lively discussion.

    Sounds above like there's a lot of support here for Shikokus.
    I will defer to others to lead, as I haven't been trained in science, though I am good at other things like photography.
    I was even thinking about hunting with them here in NH. I would be afraid of tangling with Lynx, (recently discovered here again), wildcat, raccoon and bear. I am NOT an experienced hunter but am familiar with sportsmanship.
    My distinct hope is that hunting, show or pet, that no one line will exclude themselves.
    Too much of that. Others may have different ideas and I have read some interesting things here.

    I hope that the science trained Skikoku breeders can help. I was more a fundraiser and artistic source for banners, awareness, PF for things like the DNA Bank.

    Forgive me if this is an old thread. I am catching up, bit by bit.

    Thanks.

    B




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