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The Chicken Genetic Map and Beyond Hans H. Cheng USDA, AgriculturalResearch Service Avian Diseaseand OncologyLaboratory 3606 East MountHope Road East Lansing,MI 48823 Introduction Under the Chinese calendar, 1993 is the year of the chicken which symbolizes a new beginningor rebirth. It is fittingthat the official start of the National Animal Genome Research Program (NAGRP) and its commitment to developing molecular genetic maps in several species including the chicken should begin in 1993. With funding specifically designated for the chicken genetic map, we will see the beginning of a new era that promises great advances in science and technologywith the chicken. In this discussion,I will describe the new types of markers being placed on the genetic map, compare the relative advantagesand disadvantagesof each type of marker, and present howthe genetic map can be utilizedto identifygenes involvedin complextraits. The Chicken Genetic Map A genetic map is simply an ordered array of genetic markers showing genetic distances between each locus. The first classicalgenetic map of the chicken was publishedin 1936 by Hutt and regular revisionsof the map have appeared since then (for review, see Bitgoodand Somes, 1990). The majority of the genetic markers placedon the classicalmap were morphologicalmarkers suchas yellowskinor pea comb. The reliance on morphologicalmarkers has restricted the utility of the classical genetic map. Due to the limited number of morphological markers available, few linkage groups have been established resulting in incomplete coverage of the genome. In addition,chickensused in crosses are unlikelyto carry more than a few of the morphologicalmarkerswhich restrictsthe number of loci beingfollowedeven more. Finally,morphologicalmarkers are subjectto environmental or developmental influences and are frequently limited by epistasis and pleiotrophy. Due to the limitationof the classicalmap, advances in genetic mapping of whole genomes, and the initiationof the NAGRP, a program was established in the United States at East Lansingto make a molecular genetic map of the chicken. Concurrently,programsin other countrieshad begun with the same goal. The enormoussize of the projecthas led to internationalcollaborationsto developthe chickengeneticmap. In constructingthe moleculargenetic map, it was apparent to the East Lansinggroupthat utilityto boththe scientistand the breeder be a high priority. To achieve maximum usefulness of the genetic map, the genetic markers should satisfy several criteria and have certain characteristics. First, the markers should be highly abundant and evenly distributed throughout the genome which ensuresthat all regionsof the genome can be marked. Second, the markers should be highly polymorphicwhichenables them to be useful in almost any cross. Third, the markers must be easily scored to permit a large 39 number of loci or progeny to be typed. Finally, the markers should be in a form that can be easily disseminated among laboratories. The _ew molecular genetic map will rely on DNA-based markers which will increase the usefulness of the map. DNA-based markers can satisfy all the requirements of an ideal genetic marker. First, there is an almost limitless number of potential markers available. Second, the markers can be sensitive to single-base changes making them highly polymorphic between individuals. Third, through the use of the polymerase chain reaction (PCR), the markers can be easily typed. Fourth, distribution of the markers is easily achieved through DNA sequence information or probes cloned into bacteria. Finally, DNA is easily obtained from the blood of chickens and is generally not affected by the environment or the developmental stage of the chick. A variety of DNA-based markers known by their acronyms have been and are being developed for molecular genetic maps. Each type of marker has its own relative advantages and disadvantages. Since many types of markers are being placed on the chicken genetic map, a short discussion of how these markers are developed and their relative strengths and weaknesses is merited. Restriction fragment length polymorphisms or RFLPs are the oldest type of DNA-based marker. First proposed as a genetic marker in 1980 by Botstein et al., RFLPs have fueled the drive towards developing saturated linkage maps in many species. RFLPs are developed as depicted in Figure 1. DNA isolated from each individual is cut using a restriction enzyme that typically recognizes a unique 6 base-pair sequence. Differences in the DNA sequences between individuals result in differences in DNA fragment lengths after enzyme treatment. These differences are detected by separating the DNA fragments by size on an agarose gel and visualization of the appropriate fragment by Southern blot hybridization. RFLPs have the major advantage of detecting related or conserved genes from other species, thus, enabling comparative mapping between species (see Table 1). However, the technology is very labor intensive, relatively slow, and requires the use of radioisotopes. Furthermore, only single- or low-copy DNA can be assayed resulting in the inability to follow a large portion of the genome that contains high-copy DNA. Although a useful scientific tool, it is unlikely that RFLPs will be routinely used commercially. Random amplified polymorphic DNA or RAPD markers are based on PCR (Williams et al., 1990). In PCR (Saiki et al., 1988; see Figure 2), specific regions of the genome are exponentially amplified in 30-40 repetitive cycles of three steps per cycle. In the first step, the DNA strands are denatured by heating to 94°C. In the next step, short stretches of synthetic DNA known as primers pair to complementary sequences of the template DNA when the reaction is cooled to the appropriate temperature. Finally, DNA polymerase replicates DNA starting from these primers as the temperature is elevated to 72°C. Through repetitive cycles, PCR is able to selectively amplify the region between the primers. The amplified product can be quickly visualized by gel electrophoresis and staining. RAPD markers are generated by using a single primer of 10 bases in length in the PCR (see Figure 3). Since the primers are very short, they are able to bind to numerous sites in the genome and many products are formed. If the individuals being compared are identical at the amplified locus, then the DNA of each individual will direct the production of a band of identical size. 40 Differences between individuals at several loci are easily detected by the presence or absence of similar-sized bands in stained agarose gels. RAPD markers offer many advantages (see Table 1). First, the procedure is very quick with results obtained in one day and without the need for radioisotopes. Second, inexpensive commercial sets of primers are available (Operon Technologies, Alameda, CA) enabling the potential detection of many hundreds of loci. Third, multiple loci are assayed simultaneously in one test and these loci can be in both high- or low-copy DNA. Finally, unique to RAPD markers is the ability to target specific loci or phenotypes which makes tagging genes of simple traits relatively easy. The main disadvantages are that RAPD markers are dominant markers, thus, they cannot distinguish between a homozygous or heterozygous individual.possessing the allele. Also, RAPD markers are very sensitive to PCR conditions resulting in problems with reproducibility. The third type of marker is the simple sequence repeats or SSRs, also known as microsatellites (Litt and Luty, 1989; Weber and May, 1989). These markers are based on the observation that there are numerous tandem repeats of 1 to 6 base-pairs sequences throughout the genome, e.g. (CA)12. Using PCR with primers that flank these tandem repeats, amplified products are generated (see Figure 4). Polymorphisms in the number of repeats occur frequently and can be detected by visualization on acrylamide or agarose gels. These types of markers are becoming very popular since they are very easy to use, give high reproducibility, and are highly polymorphic between individuals (see Table I). The main disadvantages are that marker development is labor intensive and radioisotopes are normally used in detection of polymorphisms. Another marker that is specific for the chicken is based on the chicken repeat element, CR1. It also relies on PCR to produce the DNA polymorphisms which enhances its ease of use. More information on this type of marker will be published soon by Levin, Crittenden, and Dodgson. All types of DNA-based markers have been developed for the molecular genetic map. Two backcross populations have been internationally recognized as the reference populations for which all the markers will be scored upon. Both reference populations have been previously described (Bumstead and Palyga, 1992; Crittenden et al., 1992). Currently on the East Lansing map, 188 markers have been scored. Of these, 158 of the markers have been resolved into 26 linkage groups with an average distance of 8 cM per marker (see Figure 5). The preliminary map contains about 1253 cM within linkage groups; it is estimated that the entire chicken genome contains 2000-3000 cM. The saturation of markers on the map is fairly good as 85-90% of randomly selected markers fall into one of the existing groups. The Compton map (United Kingdom) is very similar in the number of markers scored, coverage, and saturation..Efforts are underway to consolidate the two maps into one. A major advantage of the molecular genetic map is that additional markers can be placed on the map using the same reference population. Since the DNA from each individual has been isolated and stored, there is no need to produce another population to score more DNA polymorphisms. Instead, the molecular genetic map will be continually expanded with more genetic markers 41 screened on the existing reference populations which will result in a more complete and saturated map. Strategy for Identifying Quantitative Trait Loci (QTLs) The main purpose of the chicken genetic map is to identify and map genes involved in traits of economic importance. The vast majority of these traits are multigenicand complex in nature. The chicken genetic map will allow for the systematic analysis of the entire genome to see where genes that contribute to the trait of interest are located and how much these genes contributeto the trait. Successful identificationof these QTLs will lead to the developmentof linkedmarkers for possibleuse in marker-assistedselection. Several procedures have been .developed to use genetic maps to identifyQTLs. Lander and Botstein(1989) have developed one popularlyused method called interval mapping. In this procedure,crosses are made between individualsor groupsthat differgreatlywithrespectto the trait of interest. These resource populations produce segregating populations for the trait. All the individuals are scored for the phenotype of the trait. The more extreme individualsare then screened with 50-150 evenly-spacedpolymorphicmarkers. Each positionin the genome is hypothesizedto containa QTL for the trait. Next, the most likely phenotypic effect of the putative QTL is determined for each genotype at that locus and the odds ratio calculated (the chance that the data would resultfrom a QTL dividedby the chance that it wouldresultfrom no QTL). For convenience, the IOglO of the odds ratio is reported and called the LOD score which is proportional to the likelihoodof the existence of a QTL at that position. A threshold LOD score determines when an,actual QTL is present. This methodologyhas been successfullyapplied in the identificationof genes involved in agronomic traits of tomato (Paterson et al., 1988), hypertensionin the rat (Jacob et al., 1991), heterosisof corn (Stuberet al., 1992) and plant and plant pathogeninteractionsin the commonbean (Nodoriet al., 1993). A similar methodology has identified quantitative traits involved in the growth and reproductiverates of soybean (Keim et al., 1990), and low-phosphorusstress in corn (Reiter et al., 1991). We hope to apply this method towards identification of genes that confer resistance to Marek's disease (MD). It is known that certain alleles of the major histocompatibility complex (MHC) or B complex confer genetic resistance to MD (Briles et al., 1977; Bacon, 1987; Bacon and Witter, 1992) which has resulted in some use of haplotyping to indirectly select for favorable birds. This markerassisted selection overcomes many obstacles associated with dealing with hazardous pathogens which results in substantial time and money savings to the commercial breeder. Besides the MHC, other genetic factors are known to exist that have a major influence on resistance to MD. For example, line 6 and line 7 chickens share the same B 2 haplotype yet differ greatly with respect to resistance to MD. Recent results suggest that the contribution of resistance to MD by non-MHC genes may play a larger role than the MHC. Using three commercial White Leghorn lines, sires that were heterozygous for MHC haplotype from a susceptible or resistant line were mated to dams that were homozygous for MHC haplotype from a moderately resistant line (Groot and Albers, 1992). 1359 chicks were produced, scored for MHC haplotype, and infected with Marek's 42 disease virus (MDV) at one day of age. Measuring mortality, it was determined that sire line had a larger effect than either maternal or paternal MHC haplotype suggesting that non-MHC genes play a more prominent role in disease resistance than the MHC. This data is consistent with what is known in the mouse where breeding experiments have estimated that the MHC accounts for only 12-26% of the interline genetic differences in antibody response (Biozzi et al., 1980). Our strategy to detect non-MHC genes that confer resistance to MD is to make an F2 population using the resistant line 6 and the susceptible line 7 as the parents. Each chick will be inoculated with MDV and the level of resistance measured using a variety of traits. Evenly-spaced markers from the chicken genetic map will be used to genotype each chick and interval mapping will declare when a QTL is found and where is the location. Each putative QTL will be further confirmed in other populations that segregate for resistance to MD. Once markers have been confirmed to be linked to genes conferring resistance, efforts will be made to test these markers in commercial populations. If the markers are found to be tightly linked in commercial lines, then markerassisted selection can be applied. In the case of MD, introgression of genes conferring resistance by marker-assisted selection would complement existing vaccinal efforts for control (Gavora and Spencer, 1979). Although of limited application in chicken breeding, marker-assisted selection has had a long history of success in plant breeding. Prior to the discovery of DNA markers, plant breeders were able to use linked isozyme markers, which detect difference in enzymes, in marker-assisted selection. A classic example is the use of the alkaline phosphatase marker to detect nematode resistance in tomato. The use of this marker eliminated the need to directly screen the plants with the pathogen. Tomato breeders quickly realized that the marker was more reliable, quicker, less expensive, and easier to use than the traditional test. In addition, the marker eliminated the need to work with a pathogenic organism. The use of marker-assisted selection in plants has been extended with the development of many DNA-based markers that are linked to various genes of interest. Summary Molecular biologyhas recentlygiven us the tools to generate a limitless number of genetic markers. With a large number of markers in hand, it is possibleto develop highly-saturatedgenetic maps. A moleculargenetic map of the chicken is rapidlybeing completed. As a consequence of this genetic map, it will be possibleto identifyand map genes involvedin complex traits such as production and disease resistance. Once identified, markers linked to the genes of interest can be developed for commercial use in marker-assisted selection whichwill result in substantialsavingsof time and money. 43 Figure 1. Restriction Fragment Length Polymorphism (RFLP). Double-strandedDNA is illustratedby the two horizontallines with arrows;the arrow indicates the 3' end. The vertical lines represents the pairing of complementarybases between strands. DNA isolatedfrom individualsA and B is cut using a restrictionenzyme. Recognitionsites for the restrictionenzyme are indicated by the short arrows pointingdown. Agarose electrophoresis separates the DNA fragments by size. The open box indicateswhere the DNA was loaded. Smaller fragmentsmigratefaster than the larger fragmentsto the bottom of the gel. The appropriate fragment illustrated by the filled box is detected by a process knownas Southern blothybridization. In this process, a radiolabeledpiece of DNA knownas the probe is used to detect complementary DNA. As shown,individualA has an extra recognitionsite for the enzyme when compared to individualB resultingin a smallerfragment beingdetected by the probe. Figure 2. Polymerase Chain Reaction (PCR). The symbols are the same as in Figure 1. DNA is denatured by elevatingthe temperatureto 94°C. The reaction is cooled and the primers represented by the short horizontal arrows bind to complementary sites. Elevation of the temperature to 72°C allowsthe DNA polymeraseto replicatethe DNA startingfrom the 3' end of the primer. These three steps are repeated for 25-40 cycles. The region between the primersis selectiveamplifiedover 100,000-fold. Figure 3. Random Amplified Polymorphic DNA (RAPD). The symbols are the same as in Figure 1. DNA from individualsA and B is used as template in separate PCRs with a single primer of 10 bases in length. The relative shortness of the primer allows it to bind to numeroussites in the genome; the bindingof only four sites by a primer in each PCR is depicted. If the two sites are within 5 kilobases of each other and oriented withthe 3' ends facing each other, then an amplified DNA product is made. This can be visualized by agarose gel electrophoresisand staining. Differences in the banding patterns of the individualsindicates genetic differences; each band of a particular size represents a different locus. As illustrated, individualA amplifies a band of smaller size not observed in individual B. On the other hand, individual B amplifies a band of larger size not observed in individualA. The other three bands are common between the two individualsindicating that the two are identicalat those loci. Figure 4. Simple Sequence Repeats or Microsatellites. The symbols are the same as in Figure 1. DNA from individualsA and B is used separately as template in a PCR with primersthat flank a regioncontaining a CA-repeat. Gel electrophoresis of the PCR product indicatesthat individualB directs the synthesisof a larger DNA productwithone more CA-repeatthan individualA. 44 Figure 5. Autosomal Genetic Linkage Map of the Chicken. The current autosomal genetic linkage map of the chickenis based on a panel of 52 backcross progeny from the East Lansing reference population. Loci are in italics and the type of locus or probe is given in parentheses. The numbers on the left are approximate map distances given in centiMorgans (cM). (C)=classical locus, (F)=RFLP, (A)=RAPD, (R)=CR1 repeat, (M)=microsatellite, (V)=single-locus minisatellite or VNTR. ADL stands for anonymous loci typed at the Avian Disease and Oncology Laboratory, and MSU stands for loci typed at Michigan State University, Department of Microbiology. Other loci are named for the gene represented. Loci that are marked by the probe type only were typed in other laboratories and the data is not yet published. Table 1. Properties of DNA-Based Markers. 45 _ m •_ o_ o d _ "1 I 46 Figure 2: Polymerase Chain Reaction (PCR) ANNEAL PRIMERS DENATURE AND re=..= PRIMER EXTENSION AND EXTEND DENATURE, ANNEAL, 25-40 CYCLES REPEAT FOR DNA AMPLIFIED >100,000 FOLD 47 02) c) 0 E_ O. o < _ ._ "0 ,mm c-" m cO mm "I mm m m mm 48 IIII 49 / = _T "_ _ T .iJ I I ..T I .¥ T I I I _- " 50 I i Table 1: Properties of DNA-Based Markers RFLPs RAPDs _SSRs Distribution Ubiquitous Ubiquitous Ubiquitous Part of genome surveyed Low copy All All Level of polymorphism Moderate to High Moderate to High High Type of genetic marker Codominant multiple alleles Dominant two alleles Codominant multiple alleles # of loci detected 1-3 1-10 1 Detection of homologous loci Yes. No ? Technical difficulty High Low Low Reliability High Low-High High Speed Slow Very fast Fast DNA quantity 1000+ ng 10-50 ng 10-50 ng 50+ kb Pure 10+ kb Relatively pure 5+ kb Crude Use of radioisotopes Yes No Yes Cost High Moderate Moderate Ease of dissemination Difficult Easy Easy - DNA quality 51 References Bacon, L.D. 1987. Influenceof the major histocompatibility complexon disease resistance and productivity. PoultrySci. 66:802-811. Bacon, L.D., and Witter, R. 1992. Influenceof turkeyherpesvirusvaccinationon the B-haplotypeeffect on Marek's disease resistance in 15.B-congenic chickens. Avian Dis. 36:378-385. Bitgood, J.J., and Somes, R.G. 1990. Linkage relationships and gene mapping. In: Poultry Breeding and Genetics, R.D. Crawford (ed.), Elsevier,New York,pp. 469-495. Biozzi,G, Siquera, M., Stiffle,C., Ibanez, O.M., Mouton,D., and Ferreira, V.C.A. 1980. Genetic selections for relevant immunological functions. In: Immunology 80, Progress in Immunology IV. M. Fougereau and J. Dausset (eds.), Academic Press,New York, pp. 432-457. Botstein,D., White, R.L, Skolnick,M, and Davis, R.W. 1980. Constructionof a genetic linkage map in man using restriction fragment length polymorphisms.Am. J. Hum. Genet. 32:314-331. Briles, W.E., Stone, H.A., and Cole, R.K. 1977. Marek's disease: Effects of B histocompatibilityalloalleles in resistant and susceptible chicken lines. Science 195:193-195. Bumstead, hi., and Palyga,J. 1992. A preliminary linkagemap of the chicken genome. Genomics 13:690-697. Crittenden,L.B., Provencher, L., Santangelo,L., Levin, I., Abplanalp, H., Briles, R.W., BrilesW.E., and Dodgson,J.B. 1992. Characterizationof a Red Jungle Fowl by White Leghorn backcross reference population for molecularmappingof the Chickengenome. PoultrySci. 72:334-348. Groot, A.J.C., and Albers, G.A.A. 1992. The effect of MHC on resistance to Marek's disease by White Leghorncrosses. Proc. XlX World's Poultry Congress,Amsterdam,The Netherlands, pp. 185-168. Gavora, J.S., and Spencer, J.L. 1979. Studies on genetic resistance of chickens to Marek's disease - a review. Comp. Immunol. Microbiol. Infect. Dis.2:359-371. Hutt, F.B. 1936. Geneticsof the fowl. VI. A tentativechromosomemap. hieue Forsch. TierzuchtAbstam.(DuerstFestschrift).pp. 105-112. Jacob, H.J., Lindpainter,K., Lincoln,S.E., Kusumi, K., Bunker, R.K., Mao, Y.P., Ganten, D., Dzau, V.J., and Lander, E. S. 1991. Genetic mappingof a gene causing hypertension in the stroke-prone spontaneously hypertensiverat. Cell 67:213-224. 52 Keim, P., Diers, B.W., Olson,T.C., and Shoemaker, R.C. 1990. RFLP mapping in soybean: association between marker loci and variation in quantitative traits. Genetics 126:735-742. Lander, E.S., and Botstein, D. 1988. Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121:185-199. Litt, M., and Luty, J.A. 1989. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J. Hum. Genet. 44:397-401. Nodari, R.O., Tsai, S.M., Guzman, P., Gilbertson, R.L., and Gepts, P. 1993. Toward an integrated linkage map of common bean. III. Mapping genetic factors controlling host-bacteria interactions. Genetics 134:341350. Paterson, A.H., Lander, E.S., Hewitt, J.D., Peterson, S., Lincoln, S.E., and Tanksley, S.D. 1988. Resolution of quantitative traits into Mendelian factors by using a complete RFLP linkage map. Nature 335:721-726. Reiter, R.S., Coors, J.G., Sussman, M.R., and Gabelman, W.H. 1991. Genetic analysis of tolerance to low-phosphorus stress in maize using restriction fragment length polymorphisms. Theor. Appl. Genet. 82:561-568. Saiki, R.K., Gefand, D.H., Stoffe, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Stuber, C.W., Lincoln, S.E., Wolff, D.W., Helentjaris, T., and Lander, E.S. 1992. Identification of genetic factors contributing to heterosis in a hybrid from two elite maize inbred lines using molecular markers. Genetics 132:823839. Weber, J.L., and May, P.E. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44:388-396. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., and Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acid Res. 18:6531-6535. 53 Questions to Dr. Hans H. Cheng , Question (Dr. Bob Gowe): I understood your slide to indicate that there would be no epistasis associated with quantitative trait loci (QTLs) identified by DNA markers? Answer: I think that there is a slight misunderstanding here. Procedures such as interval mapping used to identify QTLs with a molecular genetic map do not place any judgments with regards to the gene interactions of identified loci. Where I think the confusion arises is that the DNA markers themselves are not subject to epistatic or environmental effects since they do not change unless rare mutations occur. Thus, DNA markers are considered "neutral" and for this reason can be used at any time. In contrast, morphological markers often show epistasis or development effects, e.g., slow-feathering is observed only early in feather development. Question (Dr. Milton Boyle): Could you explain what you meant by targeting your probe to a specific gene or trait using RAPD markers? Answer: RAPD markers are the only DNA markers available that one can quickly use to generate linked markers to simple traits, phenotypes, or genetic locations. Using a procedure called bulked segregant analysis (BSA; see Michelmore et al., PNAS 88:9828, 1991), a population that is segregating for your trait, gene, or genetic locus is generated and scored. The characteristic being scored then allows you to divide your population into 2 groups. Then you simply screen various RAPD markers to see which ones are polymorphic. Any polymorphic marker observed must be linked to your trait, gene, or genetic locus. For example, let's assume that you would like a marker linked to the dominant white gene. Since the white feather color is dominant, an F2 population would yield 3 white chicks for every non-white chick. The white phenotype would separate the chicks into 2 groups, the white chicks and the non-white chicks. RAPD markers would be used to screen for DNA polymorphisms. Since all the other genetic loci that are not linked to the white gene are segregating independently, these unlinked loci will be present in both groups. Thus any DNA polymorphism observed will be linked to the white gene. This procedure is extremely fast and simply, taking less than 2 weeks to identify and confirm linkage of a genetic marker. I have used this procedure numerous times to target markers to simple traits in plants. 54