Report on the Horse Genome Mapping Workshop, ISAG, Tokyo, September, 2004.
Chairman and Committee
Following elections within the group, ISAG members voted unanimously that the new Chairman of the Horse Mapping Committee would be Dr Telhisa Hasegawa (Japan), supported by a committee comprising Dr Ernie Bailey (Kentucky, US), Dr Knut Roed (Norway), Dr Alan Guthrie (South Africa) and Dr Imke Tammen (Australia).
A series of presentations took place describing various aspects of the progress being made in horse gene mapping.
Dr Tozaki (Japan)
Dr Tozaki and his collaborators have isolated approximately 1000 equine microsatellite markers from enriched libraries, using a technique they have previously published. These markers have been utilised for mapping on the AHT, the International genetic linkage maps and on the "Texas" radiation hybrid map. This significant number of markers has added greatly to the quality of all three maps and about 700 of the 1000 markers have been assigned to specific chromosomes. At this stage of map development, for most chromosomes the number of markers mapped is roughly proportional to the chromosome length. Dr Tozaki noted that for ~20 markers the chromosome assignment was not consistent between all three maps, highlighting possible discrepancies that need to be resolved. He noted that even on the third generation map there were still 17 regions where the interval between markers was still 18-30cM. They are continuing to develop microsatellite markers with a view to map expansion, an effort that is much appreciated by all in the horse mapping community.
E. Ahrens (Switzerland)
Elmer Ahrens described work that he is undertaking in his PhD studies. It involves an examination of the chromosomal relationship between E.caballus and E.przewalski in relation to the variation in chromosome number between these two species. His approach involved utilising FISH techniques with equine BAC clones from ECA5. One neat aspect of the work is that he is using a caballus x przewalski hybrid individual for the FISH analysis, so that both horse and przewalski chromosomes are present on the same metaphase, the individual's chromosome content being 2n = 65. No official nomenclature exists for the E.przewalski chromosomes. It is thought that the meta-centric horse chromosome ECA5 is represented by two chromosomes in E.przewalski, where ECA5p become przewalski chromosome "23" and ECA5q has become "24". Four equine BAC clones, two from each arm of ECA5 were used to investigate the relationship, with the BAC clones from the p arm hybridising as expected. In contrast, the BACs from the q arm (NF1A and IGLd) hybridised to ECA7 and przewalski 6. The BAC clones had originally been mapped to ECA5 by FISH, not by linkage or RH. Discussion took place as to the best way to resolve the ambiguity and several individuals are collaborating to this end.
Dr Horin (Czech Republic)
Dr Horin has been characterising genes involved in the equine immune system, and pointed out that up to 5% of mammalian genes have been implicated in host defence, and that these genes are frequently clustered (e.g. some interleukins), possibly indicating co-evolution of functional proteins. The genes he has studied include, CD14, COX2, MYD88, TFRC, IFN-gamma, IL4, IL6, and TNF. Most of the genes map to regions of conserved synteny as expected. He has gone on to develop SNPs in many of these genes that are useful for association studies. These SNPs have been deposited in the database. He proposes that the Equidae are a good system to look at the conservation of clustering of immune system genes because of the rapid karyotype evolution that has taken place in the genus.
A. Santani (Texas A&M, USA)
Avril Santani described the work that she has been undertaking on the characterisation of the horse Y chromosome for her PhD studies. The Y chromosome is involved in a number of inherited disease and developmental problems that make it important for study including aging, tumourogenesis, graft rejection and most importantly, infertility. It is only recently that David Page and colleagues have described the human Y chromosome in detail, work that took approximately 10 years for mapping and sequencing. The work on the Y chromosome is complicated by the presence of large palindromes, which are thought to be involved in gene conversion events. Other mammalian Y chromosomes remain relatively poorly characterised, given the preference of sequencing laboratories to sequence female individuals. A BAC contig map was built of the euchromatic part of the equine Y chromosome, initially utilising probes derived from genes and STS markers that had been placed on the equine RH map. Contigs were assembled using fingerprinting and extended utilising BAC end sequences. It is hoped that the contig map will form a prelude to determining the DNA sequence of the horse Y euchromatic region and the work has led to the development of a high resolution STS map that can be used to look for deletions involved in stallion infertility. In a comparative analysis, the horse Y chromosome was most similar to the pig Y chromosome.
Much of the work is described in her excellent recent PNAS paper.
Dr Raudsepp (Texas A&M, USA)
Dr Raudsepp provided an update on the equine RH map and indicated areas where the second generation RH map would improve on the first generation map. The marker distribution in the first map was not particularly even and the aim is to get a 1Mb resolution amp with a good balance between type I and type II markers. There is a Web based interface for individuals to place markers onto the map, but sometimes no linkage is found. They are using a combination of RH, FISH and linkage information to generate the best map possible. On the first map 13 RH groups were not anchored to chromosomes and another 22 groups had a single anchor, and several inconsistencies might be present. The second generation map aims to resolve these issues and will include many more microsatellite markers (~1200 TKY and UMNE markers), along with many new gene assignments. Have been working through individual chromosomes and about 14 ECAs are completed. The information generated, in combination with the reciprocal Zoo-FISH work recently published by Yang et al, provide a greatly improved resolution of conserved synteny in the horse.
They currently have ~2900 markers typed and plan to do ~900 more to reach their target of a 1Mb map, taking into account non-assigned markers.
Dr Swinburne (AHT UK)
Described the testing and analysis of a hypothesis that the proportion of grandpaternal DNA present in Thoroughbreds might be correlated with their ability. The basis for the hypothesis is the discrepancy in selection pressure applied in the Thoroughbred industry, where only ~1% of males, selected as elite individuals, enter the breeding population, whilst approximately 50% of females, of broad ability, are required to produce the number of progeny required by the industry. A whole genome scanning panel of 290 markers, optimised for Thoroughbred horses was used to genotype pairs of full-siblings where one was an elite individual and the other was an average or poor performer, together with their parents and available grandparents. The genotypes of several grandparents had to be regenerated from their offspring, as they were deceased. When the data was analysed there was no statistical support for the hypothesis.
Dr Penedo (VGL Davis, USA)
Dr Penedo described the third phase of the International Horse Mapping effort that was based around trying to combine the data available from the previous three, different, genetic linkage maps. To this end, data from Sweden, the AHT in Newmarket and the Second Generation International map had been combined together with new Japanese (TKY) markers recently typed in Davis to generate a file with 839 markers. These comprised 825 autosomal markers and 14 X-linked markers. The data was analysed using Crimap and 766 (93%) markers were placed into 31 linkage groups. 626 (82%) markers could be placed in a linear order with an additional 140 assigned to regions.
59 (7%) markers remained unassigned, these markers generally being of low polymorphism with low average numbers of informative meioses. The 626 ordered markers had a map length of 3739 cM, with an average spacing of 6.3cM. She mentioned that the group at Davis, including Lee Millon, had developed a new Horse Chromosome Viewer programme available on the Web that lets linkage, RH and FISH data be compared. They plan to continue mapping additional markers onto the International and AHT reference families to achieve a map resolution capable of mapping the complex inherited defects present in the horse.
