Kevin Chase

A Soybean Transcript Map: Gene Distribution, Haplotype and Single-Nucleotide Polymorphism Analysis

The first genetic transcript map of the soybean genome was created by mapping one SNP in each of 1141 genes in one or more of three recombinant inbred line mapping populations, thus providing a picture of the distribution of genic sequences across the mapped portion of the genome. Single-nucleotide polymorphisms (SNPs) were discovered via the resequencing of sequence-tagged sites (STSs) developed from expressed sequence tag (EST) sequence. From an initial set of 9459 polymerase chain reaction primer sets designed to a diverse set of genes, 4240 STSs were amplified and sequenced in each of six diverse soybean genotypes. In the resulting 2.44 Mbp of aligned sequence, a total of 5551 SNPs were discovered, including 4712 single-base changes and 839 indels for an average nucleotide diversity of θ = 0.000997. The analysis of the observed genetic distances between adjacent genes vs. the theoretical distribution based upon the assumption of a random distribution of genes across the 20 soybean linkage groups clearly indicated that genes were clustered. Of the 1141 genes, 291 mapped to 72 of the 112 gaps of 5–10 cM in the preexisting simple sequence repeat (SSR)-based map, while 111 genes mapped in 19 of the 26 gaps >10 cM. The addition of 1141 sequence-based genic markers to the soybean genome map will provide an important resource to soybean geneticists for quantitative trait locus discovery and map-based cloning, as well as to soybean breeders who increasingly depend upon marker-assisted selection in cultivar improvement.

Coat Variation in the Domestic Dog Is Governed by Variants in Three Genes

Dog Coats Shed Genetic Secrets The coats of domestic dogs show great variation—long, short, straight, wavy, curly, wiry, or smooth. To investigate how this variation arises, Cadieu et al. (p. 150, published online 27 August) performed genome-wide association studies on 80 different dog breeds. The coat phenotype could be dissected into three simple traits of length, curl, and growth pattern or texture with each trait controlled by one major gene, FGF5 (fibroblast growth factor-5), KRT71 (keratin-71), and RSPO2 (R-spondin-2), respectively. In combination, variants in these three genes alone account for the vast majority of the coat phenotypes in purebred dogs in the United States. Thus, a small number of simply inherited traits can be remixed to create extraordinary phenotypic variation. Huge variations in the coats of purebred dogs can be explained by the combinatorial effects of only three genes. Coat color and type are essential characteristics of domestic dog breeds. Although the genetic basis of coat color has been well characterized, relatively little is known about the genes influencing coat growth pattern, length, and curl. We performed genome-wide association studies of more than 1000 dogs from 80 domestic breeds to identify genes associated with canine fur phenotypes. Taking advantage of both inter- and intrabreed variability, we identified distinct mutations in three genes, RSPO2, FGF5, and KRT71 (encoding R-spondin–2, fibroblast growth factor–5, and keratin-71, respectively), that together account for most coat phenotypes in purebred dogs in the United States. Thus, an array of varied and seemingly complex phenotypes can be reduced to the combinatorial effects of only a few genes.

Genetic basis for systems of skeletal quantitative traits: Principal component analysis of the canid skeleton

Evolution of mammalian skeletal structure can be rapid and the changes profound, as illustrated by the morphological diversity of the domestic dog. Here we use principal component analysis of skeletal variation in a population of Portuguese Water Dogs to reveal systems of traits defining skeletal structures. This analysis classifies phenotypic variation into independent components that can be used to dissect genetic networks regulating complex biological systems. We show that unlinked quantitative trait loci associated with these principal components individually promote both correlations within structures (e.g., within the skull or among the limb bones) and inverse correlations between structures (e.g., skull vs. limb bones). These quantitative trait loci are consistent with regulatory genes that inhibit growth of some bones while enhancing growth of others. These systems of traits could explain the skeletal differences between divergent breeds such as Greyhounds and Pit Bulls, and even some of the skeletal transformations that characterize the evolution of hominids.

Epistat : a computer program for identifying and testing interactions between pairs of quantitative trait loci

Abstract We describe a computer program, Epistat, which combines statistical methods and color-graphic displays to facilitate the analysis of interactions between pairs of quantitative trait loci (QTLs). Epistat organizes genetic-mapping data and quantitative-trait values into graphic displays which illustrate the individual effects of single loci as well as the interactions between any two loci. Keyboard commands allow the user to search the data set for individual QTLs and to test for interactions between QTLs. For a given trait, the program displays the effects of the alleles at each of two loci on the quantitative-trait value, as well as the effects of the interactions between these alleles. Loglikelihood ratios are used to compare the likelihood of explaining the effects by null, additive, or epistatic models. Examples of interactions in soybean are presented for near-infrared transmittance (NIT), seed number, and reproductive period. Epistat has been used to find numerous interactions between QTLs in soybean in which trait variation at one locus is conditional upon a specific allele at another.

Bilaterally asymmetric effects of quantitative trait loci (QTLs): QTLs that affect laxity in the right versus left coxofemoral (hip) joints of the dog (Canis familiaris)

In dogs hip joint laxity that can lead to degenerative joint disease (DJD) is frequent and heritable, providing a genetic model for some aspects of the human disease. We have used Portuguese water dogs (PWDs) to identify Quantitative trait loci (QTLs) that regulate laxity in the hip joint. A population of 286 PWDs, each characterized by ca. 500 molecular genetic markers, was analyzed for subluxation of the hip joint as measured by the Norberg angle, a quantitative radiographic measure of laxity. A significant directed asymmetry was observed, such that greater laxity was observed in the left than the right hip. This asymmetry was not heritable. However, the average Norberg angle was highly heritable as were the Norberg angles of either the right or left hips. After correction for pedigree effects, two QTLs were identified using the metrics of the left and right hips as separate data sets. Both are on canine chromosome 1 (CFA1), separated by about 95 Mb. One QTL, associated with the SSR marker FH2524 was significant for the left, but not the right hip. The other, associated with FH2598, was significant for the right but not the left hip. For both QTLs, some extreme phenotypes were best explained by specific interactions between haplotypes.

A genetic map of soybean (Glycine max L.) using an intraspecific cross of two cultivars : Minosy and Noir 1

Genetic markers were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: ‘Minsoy’ (PI 27.890) and ‘Noir 1’ (PI 290.136). A genetic linkage map was constructed (LOD ⩾ 3), consisting of 132 RFLP, isozyme, morphological, and biochemical markers. The map defined 1550cM of the soybean genome comprising 31 linkage groups. An additional 24 polymorphic markers remained unlinked. A family of RFLP markers, identified by a single probe (hybridizing to an interspersed repeated DNA sequence), extended the map, linking other markers and defining regions for which other markers were not available.

Mapping Loci for Fox Domestication: Deconstruction/Reconstruction of a Behavioral Phenotype

During the second part of the twentieth century, Belyaev selected tame and aggressive foxes (Vulpes vulpes), in an effort known as the “farm-fox experiment”, to recapitulate the process of animal domestication. Using these tame and aggressive foxes as founders of segregant backcross and intercross populations we have employed interval mapping to identify a locus for tame behavior on fox chromosome VVU12. This locus is orthologous to, and therefore validates, a genomic region recently implicated in canine domestication. The tame versus aggressive behavioral phenotype was characterized as the first principal component (PC) of a PC matrix made up of many distinct behavioral traits (e.g. wags tail; comes to the front of the cage; allows head to be touched; holds observer’s hand with its mouth; etc.). Mean values of this PC for F1, backcross and intercross populations defined a linear gradient of heritable behavior ranging from tame to aggressive. The second PC did not follow such a gradient, but also mapped to VVU12, and distinguished between active and passive behaviors. These data suggest that (1) there are at least two VVU12 loci associated with behavior; (2) expression of these loci is dependent on interactions with other parts of the genome (the genome context) and therefore varies from one crossbred population to another depending on the individual parents that participated in the cross.

Understanding the genetics of autoimmune disease: two loci that regulate late onset Addison's disease in Portuguese Water Dogs

Addisons disease, an immune‐mediated disorder caused by destruction of the adrenal glands, is a rare disorder of Western European populations. Studies indicate that the disorder is polygenic in nature, involving specific alleles of the CTLA‐4, DRB1*04 and DQ, Cyp27B1, VDR and MIC‐A and ‐B loci. A similar immune form of Addisons disease occurs in several breeds of domestic dog, with frequencies ranging from 1.5 to 9.0%. The high frequency of the disease in domestic dog breeds likely reflects the small number of founders associated with many breeds, subsequent inbreeding, and the frequent use of popular sires.

Genetic Regulation of Osteoarthritis: A QTL Regulating Cranial and Caudal Acetabular Osteophyte Formation in the Hip Joint of the Dog (Canis familiaris)

Dogs, Canis familiaris, share more than 200 genetic disease phenotypes with humans [Patterson et al., 1982; Patterson, 2000]. Genes for specific diseases are often concentrated in purebred dogs (genetic isolates) due to founder effects, inbreeding and/or frequent use of ‘‘popular sires.’’ As a result, recessive, interactive, or polygenic modes of inheritance are more readily investigated, leading to identification of Quantitative Trait Loci (QTLs) [Chase et al., 2002, 2004]. The recent completion of the sequence for the canine genome has facilitated the comparison of the dog genome with human and othermammalian genomes, allowing the further investigative analysis of canine QTLs in other mammalian systems, notably the human and mouse. Here we examine changes that involve pathological remodeling of bone, altered joint conformation, and osteophyte formation, visible radiographically as osteoarthritis (OA) [Olsson, 1971; Riser, 1973]. We have related such changes in the coxofemoral (hip) joint of Portuguese Water Dogs (PWDs) to genotypes defined by SSR markers and associated themwith a specific region (haplotype) of the canine genome (QTL). Radiographs and blood for DNA were collected from 431 PWDs through the Georgie Project [http://www.georgieproject. com, Karen Miller director; Chase et al., 2002]. The dogs, ranging in age from 1.7 to 17 years (median age, 6 years), included 171 males and 260 females and represented a crosssection of the entire PWD population in the USA. They trace their ancestry to 31 founders through ca. 25 generations and consanguinities range from 0 to 0.6 with a mean of 0.2 [Chase et al., 1999]. We have associated marker alleles with a few infrequently used founders using the consanguinity between that founder and all dogs known to carry the allele. Permutation tests are used to establish the significance of each association [Alroy et al., 2000]. DNA was isolated from blood and characterized by PCR amplification and electrophoretic identification of the alleles of simple sequence repeat geneticmarkers [Francisco et al., 1996; Mellersh et al., 2000; Chase et al., 2002, 2004]. Osteoarthritis (OA) was scored from ventrodorsal radiographs of the pelvis as illustrated in Figure 2 of Chase et al. [2002]. In all, 431 dogs were scored for subchondral sclerosis of the cranial acetabular margin, osteophytes of the caudal and cranial acetabular margins, and femoral head osteophytes (illustrated in Fig. 1). The severity of each of these phenotypes was scored on an ordinal scale from 0 (none) to 3 (most severe). Left and right hips were scored independently. Scores ranged from0 to 22 out of a possiblemaximumof 24 (12 leftþ 12 right). Fifty percent of the dogs had a score greater than 0. Methods for estimatingheritability (h), identifying theQTL and estimating its effect on the variation of specific phenotypes (R) have been described previously [Chase et al., 2004]. Estimation of QTL significance used permutations, as described in Chase et al. [2002]. In the PWD population OA is heritable (h1⁄4 30%). About half of the population show some degree of OA. OA is significantly correlated with the Norberg Angle (an indicator of joint laxity). However, whereas the Norberg Angle is significantly greater for the right than the left hip, there is no significant difference in the OA scores between the right and left joints. OA also is significantly correlated with the 4th Principal Component (PC) defined by variation in the skeletal metrics of the pelvis and limb bones. There is no significant correlation with other PCs. We had identified two QTLs on autosome 1 (CFA 1) associated with the Norberg Angle [Chase et al., 2004] and several QTLs associated with PC4 (unpublished data). In addition, we have identified two QTLs related to autoimmune Addison’s disease (unpublished data). We analyzed these QTLs (11 in all) for association with OA. One QTL identified by the SSR marker, FH2320 on CFA 3 and associated with PC4, also was significantly associated withOA (P 0.002, corrected for pedigree effects and number of QTLs tested [Chase et al., 2002]). Table I presents the relevant region of the canine genome and its syntenic counterparts on the human and mouse genomes. We have characterized the effects of this QTL in greater detail. It accounts for about 16% of the OA variation (R1⁄4 16.4%), and involves primarily cranial and caudal acetabular marginal osteophytes. This same QTL affects PC4 through its effect on the ischial tuberosity, the width of which is segregating in the PWD population (R1⁄4 7.5% for the trait residual after removing the effects of PCs 1, 2, and 3). This QTL also

Genetic Mapping of Fixed Phenotypes: Disease Frequency as a Breed Characteristic

Traits that have been stringently selected to conform to specific criteria in a closed population are phenotypic stereotypes. In dogs, Canis familiaris, such stereotypes have been produced by breeding for conformation, performance (behaviors), etc. We measured phenotypes on a representative sample to establish breed stereotypes. DNA samples from 147 dog breeds were used to characterize single nucleotide polymorphism allele frequencies for association mapping of breed stereotypes. We identified significant size loci (quantitative trait loci [QTLs]), implicating candidate genes appropriate to regulation of size (e.g., IGF1, IGF2BP2 SMAD2, etc.). Analysis of other morphological stereotypes, also under extreme selection, identified many additional significant loci. Behavioral loci for herding, pointing, and boldness implicated candidate genes appropriate to behavior (e.g., MC2R, DRD1, and PCDH9). Significant loci for longevity, a breed characteristic inversely correlated with breed size, were identified. The power of this approach to identify loci regulating the incidence of specific polygenic diseases is demonstrated by the association of a specific IGF1 haplotype with hip dysplasia, patella luxation, and pancreatitis.