High-Resolution Mapping of Quantitative Trait Loci by Selective Recombinant Genotyping

Corresponding author: A. Korol, University of Haifa, Mount Carmel, 31095 Haifa, Israel., korol{at}esti.haifa.ac.il (E-mail)

Communicating editor: J. B. WALSH

	ABSTRACT

TOP ABSTRACT THEORY RESULTS AND DISCUSSION PRACTICAL FEASIBILITY AND... LITERATURE CITED

Selective recombinant genotyping (SRG) is a three-stage procedure for high-resolution mapping of a QTL that has previously been mapped to a known confidence interval (target C.I.). In stage 1, a large mapping population is accessed and phenotyped, and a proportion, P, of the high and low tails is selected. In stage 2, the selected individuals are genotyped for a pair of markers flanking the target C.I., and a group of R individuals carrying recombinant chromosomes in the target interval are identified. In stage 3, the recombinant individuals are genotyped for a set of M markers spanning the target C.I. Extensive simulations showed that: (1) Standard error of QTL location (SEQTL) decreased when QTL effect (d) or population size (N) increased, but was constant for given "power factor" (PF = d²N); (2) increasing the proportion selected in the tails beyond 0.25 had only a negligible effect on SEQTL; and (3) marker spacing in the target interval had a remarkably powerful effect on SEQTL, yielding a reduction of up to 10-fold in going from highest (24 cM) to lowest (0.29 cM) spacing at given population size and QTL effect. At the densest marker spacing, SEQTL of 1.0–0.06 cM were obtained at PF = 500–16,000. Two new genotyping procedures, the half-section algorithm and the golden section/half-section algorithm, allow the equivalent of complete haplotyping of the target C.I. in the recombinant individuals to be achieved with many fewer data points than would be required by complete individual genotyping.

LOW resolution of the estimated chromosomal location of quantitative trait loci (QTL) is a major obstacle in application of QTL linkage mapping results for marker-assisted selection and comparative positional cloning of the gene corresponding to the QTL. Up to a certain point, mapping resolution (defined as the standard deviation of estimated QTL location, or SEQTL) can be improved by increasing marker density (DARVASI et al. 1993 ). However, for given sample size and standardized QTL substitution effect, ultimate map resolution is fixed and cannot be further improved even with infinite marker density (DARVASI et al. 1993 ; DARVASI and SOLLER 1997 ). Consequently, approaches to improving QTL map resolution primarily involve increasing the standardized QTL substitution effect, e.g., by using replicated progenies (SOLLER and BECKMANN 1990 ; WELLER et al. 1990 ), by including the effects of cosegregating QTL as regression cofactors (ZENG 1994 ; JANSEN and STAM 1994 ), or by employing multiple-trait analysis (JIANG and ZENG 1995 ; KOROL et al. 1995 , KOROL et al. 2001 ). More complex approaches, termed "genetic chromosome dissection," involve producing or identifying recombinants in the chromosomal intervals shown to carry significant QTL and evaluating the recombinant chromosomes through progeny testing (DARVASI 1997A , DARVASI 1998 ; HILL 1998 ; SOLLER and ANDERSSON 1998 ). Effective sample size can also be increased by accumulating recombinants in advanced generations (DARVASI and SOLLER 1995 ).

The most straightforward method for increasing mapping resolution, however, is simply to increase the size of the mapping population, in this way accumulating recombinants in the interval of interest. When this strategy is employed, a useful tactic for reducing genotyping costs has been to produce a mapping population with easily scorable morphological markers flanking the interval containing the target locus. A few hundred recombinant individuals for these markers are identified, and only these individuals are genotyped for the set of closely spaced molecular markers spanning the target interval (e.g., KLEIN-LANKHORST et al. 1991 ; see also RHODES et al. 1998 and review in DARVASI 1998 ).

Here we propose a similar procedure, selective recombinant genotyping (SRG), to be applied when the target locus is a QTL with a moderate or even small substitution effect. In this case, mapping resolution is expressed as the C.I. or as the SEQTL. SRG would ordinarily be implemented following an initial total or partial genome scan that has detected a QTL in a backcross (BC), F₂, or half-sib sire-family design. It presupposes the possibility of forming or accessing a very large mapping population. In addition, we present two new genotyping procedures, the half-section algorithm and the golden section/half-section algorithm, which allow the equivalent of complete haplotyping of the target C.I. in the recombinant individuals to be achieved with many fewer data points than would be required by complete individual genotyping.

The procedure described here is similar in conception to the "contrast mapping" procedure of THALLER and HOESCHELE 2000 . The present study generalizes and extends their results by considering BC and F₂ populations and the effects of selective genotyping and marker spacing on the accuracy of QTL location. The results are also presented in a form somewhat different from that used by THALLER and HOESCHELE 2000 , namely, as SEQTL rather than as the proportion of QTL located to the true QTL interval. However, the present study amply supports the bottom line conclusion of THALLER and HOESCHELE 2000 , namely, with large family sizes "it is feasible to map a QTL to a region of 2 to 4 cM" (p. 103).

	THEORY

TOP ABSTRACT THEORY RESULTS AND DISCUSSION PRACTICAL FEASIBILITY AND... LITERATURE CITED

Selective recombinant genotyping:
We assume a situation in which QTL mapping by any of the customary procedures (complete individual genotyping, selective genotyping, selective DNA pooling) has detected a QTL in a confidence interval defined by a pair of flanking markers, M₁ and M_k. It is further assumed that a set of additional evenly spaced ordered markers (denoted M₂, ... , M_i, ... M_k-1) spanning the interval from M₁ to M_k are available and that haplotypes of the parental lines or individual sires have been determined with respect to the entire set of markers. In the proposed scheme, high-resolution mapping is based on genotyping the markers M₂–M_k-1 only for those individuals from the high and low population tails that are recombinant for the flanking markers. Thus, if the parental F₁ or sire chromosomes are M₁M_k/m₁m_k, the progeny individuals chosen for further genotyping will be those that carry recombinant parental chromosomes M₁m_k and m₁M_k. The main question concerns the degree to which the SEQTL depends on the standardized QTL allele substitution effect d, on the total size of the mapping population (N), and on marker spacing (c in centimorgans) in the interval M₁–M_k. In addition, DARVASI 1997B has shown that most of the information for QTL map location is found in the high and low tails of the mapping population. To explore this possibility of reducing genotyping costs, we also studied the effect of genotyping only the high and low proportions (P) of the population for the initial recombinants.

To address these questions, a Monte Carlo analysis was employed. Standard interval maximum-likelihood (ML) analysis was used combined with selective genotyping that uses trait values of both genotyped and nongenotyped individuals to provide ML estimates of the QTL effect and position (LANDER and BOTSTEIN 1989 ; RONIN et al. 1998 ). The interval analysis was unconditional, with no prior assumption of the QTL location. The simulated QTL was located at the center of a chromosome of a total length of 480 cM, so that end effects did not limit the SEQTL. Each of the tails was composed of t individuals, so that P = t/N. Then, for each marker subinterval M_i - M_i+1 (i = 1, ... , k - 1) from the interval M₁–M_k, the conditional LOD score was calculated, assuming that the QTL resides in this subinterval. The estimates of the QTL effect and residual variance obtained in the initial analysis for the entire M₁–M_k interval were used as coordinates of the starting point in the optimization procedure for each subinterval. It was assumed that all individuals in the high and low selected groups had been genotyped for markers M₁ and M_k and that the M₁–m_k and m₁–M_k recombinants had been identified.

Width of the M₁–M_k interval was taken as 24 cM; QTL location was at the midpoint of the interval. It was assumed that mapping takes place within a backcross or half-sib population, so that contrast values in SD units are d (or , in the case of a half-sib population). The following parameter combinations were investigated in the main body of the simulations: d = 0.25, 0.50, 1.00; N = 1000, 2000, 4000, 8000, 16,000; P = 0.05, 0.10, 0.20, 0.25, 0.50; c = 24, 8, 2.66, 0.88, 0.29 (marker spacing was chosen to ensure that in no instance did a marker position coincide with a QTL position). For a BC population, d = 0.25, and 0.50 and 1.00 correspond to QTL variances of 0.015, 0.0625, and 0.25, respectively. For each combination of parameters, 1000 Monte Carlo runs were conducted. The direct empirical value of the SEQTL was calculated on the basis of the estimated values of QTL location.

Genotyping requirements:
Genotyping requirements will differ somewhat depending on whether the SRG procedure is implemented in a BC, F₂, or half-sib family (half-sib) designs. For clarity, a complete analysis is provided first for the BC design, and modifications required by F₂ and half-sib designs are then discussed. It is convenient to organize the genotyping requirements according to the three steps of the SRG fine-mapping procedure. Table 1 provides a summary of genotyping requirements for the three designs, according to these steps, and for total genotyping.

BC design

• Step I. Identifying recombinant offspring: The proposed procedure is based upon individual genotyping of the entire selected sample to identify recombinant individuals in the region M₁–M_k. This will involve 4NP data points = 2NP individuals x 2 data points/individual (data point: the genotype of a single individual with respect to a single marker) and will identify R = r(2NP) recombinants, where r is the proportion of recombination between markers M₁ and M_k. For small target intervals of length L cM, r L/100. Parental haplotypes for the flanking markers are obtained in the course of identifying recombinant individuals.

• Step II. Determining the parental haplotypes with respect to the internal markers: This step is needed to identify the complete marker genotype for each individual as required for the interval mapping procedure. Given a segment of length L, the number of additional markers needed within the segment to provide marker spacing c, is given by M_L = (L/c) - 1. Determining parental haplotypes for F₂ or BC designs is simply achieved by genotyping the parental lines. Thus, the number of genotyping data points required in this case will be 2M_L.

• Step III. Genotyping recombinant individuals for the markers within the target segment: Once parental haplotypes are known, each recombinant individual is genotyped for all internal markers. The total genotyping data points for the recombinant individuals will thus equal LNPM_L/100.

F₂ design
Since an F₂ individual can receive a recombinant haplotype from either of the two parents, the proportion of F₂ recombinant individuals is twice that of a comparable BC population. Most F₂ progeny will carry only a single recombinant chromosome. For these individuals, analysis is the same as for a BC design. Some of the F₂ progeny will be double recombinants. These will be of two sorts: (1) double recombinants involving opposite-phase haplotypes (i.e., M₁ ... m_k/m₁ ... M_k), which will not be recognized as recombinants in the initial screen for recombinant progeny and will not be included among the recombinant progeny and (2) double recombinants involving same-phase haplotypes (i.e., M₁ ... m_i ... m_j ... m_k/M₁ ... M_i ... m_j ... m_k). These will be included among the recombinant progeny and will carry twice as much information as a single recombinant. Thus, in an F₂ design overall, the total number of progeny genotyped, and hence requiring genotyping data points to identify the recombinant individuals, will be half that for a BC design. Once the recombinant individuals are identified, however, genotyping requirements are more or less the same as for the BC design, although double recombinants will require some additional data points to establish both points of recombination.

Half-sib design
In principle, a half-sib design is the exact equivalent of a BC design, in that any individual progeny will receive a recombinant chromosome from only one parent (the sire). However, they differ in that, in a BC design, all markers are fully informative, because the allele derived from the F₁ parent can be identified unequivocally, and hence the recombinant individuals and their haplotypes at each marker are determined by genotyping that marker. This is not the case for the half-sib design, because of the incomplete informativity of the individual markers in an outcrossing population. That is, when an individual has the same (heterozygous) genotype as its sire, it is not possible to determine the marker allele transmitted to the individual from its sire. In this case, the genotyping data point will not be informative for determining recombinant status of the haplotype transmitted from the sire to the individual. The same will hold when the dam is genotyped, if individual sire and dam all share the same (heterozygous) genotype. This applies both to the initial step of identifying progeny that received recombinant haplotypes from their sire and to the step of identifying the full haplotype of the recombinant individual. The easiest way around this is to genotype additional markers close to the initially chosen marker. Assuming conservatively that only 50% of genotypings are informative, it is easy to see that the total number of genotypings required to identify and haplotype the recombinant progeny is double that required for the BC or the F₂ situation.

In addition to the above, obtaining the sire haplotype is also affected by incomplete informativity of the markers. In this case, haplotype of the sire for the flanking markers will be obtained from the many progeny that are genotyped in the screen for recombinant progeny. With respect to the internal markers, DNA will often be available for one or both parents of the sire. In this case, genotyping the sire, his sire, and his dam for the internal markers, i.e., 3 M_L genotyping data points, will provide the sire haplotypes for all markers except those for which the sire and his parent(s) are heterozygous for the same pair of alleles. For these markers, it will be necessary to genotype progeny of the sire. For this, it will be efficient to use the nonrecombinant progeny, already identified as described in the preceding screen for recombinants. Because nonrecombinant progeny are used and the phase of the flanking markers is known, a single individual will provide a sire haplotype for all sire markers, except those for which sire and progeny are heterozygous for the same pair of alleles. Since maximum heterozygosity for the same pair of alleles is 0.5, 10 nonrecombinant individuals should easily be sufficient for haplotyping a sire. These individuals will need to be genotyped only for M_L internal markers. Thus, haplotyping a sire will require 3–13 M_L genotyping data points.

The half-section algorithm
The total number of genotyping data points can be reduced greatly by assuming that all M₁–m_k, m₁–M_k recombinants represent single recombination events in the interval M₁–M_k. This is plausible since double recombinants are not included among the observed M₁–m_k, m₁–M_k recombinants, and triple recombinants are exceedingly rare. Consequently, the marker genotype of each recombinant individual is determined completely by the single point of recombination within the target segment for that individual. The location of the point of recombination within the target segment can be progressively narrowed by noting further that, once a subinterval spanning several markers within the segment is shown to be nonrecombinant, it is no longer necessary to further genotype any of the markers in this subinterval. Clearly, by genotyping a single marker in the center of the recombinant subinterval, the size of the subinterval containing the point of recombination is progressively reduced by one-half. Thus, if a total of M markers are taken to span the target segment (including the two flanking markers), the number of markers genotyped per individual that are required to identify the point of recombination will be between n and n + 1, where n = integer part of log₂M. A small number of worked examples show that the average n is closely approximated by n = log₂M.

Application of this principle leads to a procedure that we term the "half-section (HS) algorithm," illustrated in Fig 1. For the HS algorithm, the genotype of each individual is determined independently of all others. Thus, the total number of genotyping points for the entire set of recombinants, T, is simply the average number of genotyping points per individual multiplied by the total number of recombinant individuals, R, i.e., T = Rn.

View larger version (41K): In this window In a new window Download PPT slide   Figure 1. Illustration of the "half-section" algorithm for determining marker genotype of a recombinant individual. (A) In the example, the point of recombination is between M6 and M7, so that in the initial scan for recombinants the individual was identified as an M1–m25 recombinant. Notation: standard letters, e.g., M16, denote a marker; italicized letters, e.g., M16 and m16, denote alleles at the marker. Step 1 (B): Genotype M13, the central marker in the interval M1–M25. The allele found is m13, showing that the point of recombination is between M1 and M13. From M13 to M25, the genotype of the individual is now known to be m13 ... m25. Step 2 (C): Genotype M6. The allele found is M6, showing that the point of recombination is in the interval M6–M13. From M1 to M6, the genotype of the individual is now known to be M1 ... M6. Step 3 (D): Genotype M9: The allele found is m9, showing that the point of recombination is in the interval M6–M9. From M9 to M13, the genotype of the individual is now known to be m9 ... m13. Step 4: There are two options: i. Genotype M7. In this case, the allele found is m7, showing that the point of recombination is between M6 and M7. The complete genotype of the individual is known. ii. Genotype M8. In this case, the allele found is m8, showing that the point of recombination is between M6 and M8. It is still necessary to genotype M7. The allele found is m7, showing that the point of recombination is between M6 and M7, and the complete genotype of the individual is now known. Total genotyping points for this individual will be 4 if option (i) is chosen and 5 if option (ii) is chosen.

Application of the HS algorithm involves sequential splitting of the recombinant progeny into progressively smaller subgroups. Each subgroup is genotyped for a different marker and split further. Thus, the early markers are used on subgroups with many members, the later markers on subgroups with only a few members. As the genotyping progressed, more and more markers were used in each round, but each marker was set up and used only once on a specific subgroup. For example, consider genotyping 400 recombinants for 31 markers. In complete genotyping, each individual is genotyped for all 31 markers: a total of 12,400 genotyping data points. When using the HS algorithm, all individuals are genotyped for marker 1; 200 individuals are genotyped for markers 2 and 3; 100 individuals each are genotyped for markers 4–7; 50 individuals are genotyped for markers 8–15; and 25 individuals are genotyped for markers 16–31—overall, a total of 1600 genotyping data points. Only four rounds are required for the entire HS genotyping procedure. With the negligible exception of three-point recombination within the target interval, the genotyping results given by the HS algorithm are exactly equivalent to those given by complete genotyping. Set-up costs for markers are the same as for complete genotyping; the only additional cost is for sorting the progeny for genotyping, according to the results of the previous round.

The golden section algorithm
The number of required genotyping data points can be reduced even more by noting that, within the target segment, the complete genotype of all individuals is required only across the subinterval that contains the QTL. If mapping analysis is carried out concurrently with genotyping, it is possible to progressively narrow the interval within the segment within which the QTL is found. It is then necessary to genotype only recombinants in this QTL-containing interval to further narrow the QTL location. Recombinants outside of this interval do not contribute information for QTL map location within the interval. Since we consider a situation with a single QTL in the target chromosomal region, it can be assumed that the expected LOD function (ELOD) will be a unimodal function (HYNE and KEARSEY 1995 ; RONIN et al. 1999 ). This is so, even though other data sets of a comparable mapping population will have a LOD score function whose maximum is at a different location. Therefore, in applying this principle, we can use the golden section (GS) algorithm (GILL et al. 1981 ) to choose the markers for genotyping to progressively narrow the subinterval within which the QTL is found. The GS algorithm is commonly used in numerical analysis for efficiently finding the maximum of a function with a single maximum (or minimum) measured without errors. As applied to QTL mapping, the GS algorithm basically involves identifying two flanking points between which the maximum of the mapping criterion (LOD function) is known to reside and evaluating the LOD function at these two points. The chosen points are, respectively, F and 1 - F of the distance between the two flanking points [where F is the golden section parameter equal to the Fibonacchi constant, F = 1/(1 + ) 0.38]. The point for which the value of the LOD function is less defines a new flanking point. The process is now reiterated. It can be shown that the number of individuals genotyped at each successive step will be R, (1 - F)R, (1 - F)²R, etc. Thus, application of this procedure will require only [1 + (1 - F) + (1 - F)² +...]R 2.62R genotyping data points, irrespective of the number of markers in the target segment. Fig 2 illustrates the application of the GS algorithm.

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. Illustration of the "golden section" algorithm for determining marker genotype of a recombinant individual. (A) As in Fig 1, the point of recombination of the tracked individual is between M6 and M7, so that in the initial scan for recombinants the individual was identified as an M1–m25 recombinant. The maximum LOD score (MLS) for the given data set is assumed to be located near M14. Step 1: Genotype M16, located 0.62 of the distance from M1 to M25. Since all recombinant individuals are genotyped, the number of genotyping points for this step is R. We assume that points of recombination are distributed uniformly throughout the interval M1–M25. Consequently, of the total number of recombinant individuals, 0.62R will have allele m16, and 0.38R will have allele M16. For the individuals with allele m16, the genotype at all markers in the interval M16–M25 is known: m16 ... m25. For the individuals with allele M16, the genotype at all markers in the interval M1–M16 is known: M1 ... M16. Step 2 (B): Genotype M10, located 0.38 of the distance from M1 to M25. Only the 0.62 of recombinant individuals that were m16 need to be genotyped. Individuals that were M16 will not be genotyped further. Thus, the total number of genotyping points for this step is 0.62R. Of the individuals genotyped for marker M10, 0.62 will have allele M10, and 0.38 will have allele m10. Mapping analysis is now carried out with markers M1, M10, M16, and M25. According to the assumed location of the MLS, M16 will have a higher LOD score than M10. Thus, M10 now becomes the new left flank marker. At this point markers M2–M9 are no longer of interest from the point of view of high-resolution mapping and will not be genotyped in any individuals. Step 3 (C): Genotype M19, located 0.62 of the distance from M10 to M25. For the 0.62 of individuals that were m16, genotype at M19 is known to be m19. Thus, genotyping needs to be done only for the 0.38R = (0.62)2R individuals that were M16. Note that the individual with point of recombination between M6 and M7 is included in the group that need not be genotyped for M19. Mapping analysis is now carried out with added marker M19. M16 will have a higher LOD score than M19. Thus, M19 now becomes the new right flank marker. Markers M21–M25 are no longer of interest. Step 4 (D): Genotype M13, located 0.62 of the distance from M19 to M10. It is necessary to genotype, only for M13, those individuals that were m16 and m13 = 0.24R = (0.62)3R individuals. Mapping analysis is now carried out with added marker M13. M13 will have a higher LOD score than M16. Thus, M16 becomes the new right hand flanking marker, and the QTL has been located between M10 and M16. Markers M17 and M18 are no longer of interest. Step 5: At this point it is convenient to determine genotypes for all markers in the interval M10–M16 (hence the entire algorithm can be referred to as GS-HS rather than as GS). For M11 and M12 genotypes need to be determined only for the 0.125 of individuals that were M10–m13–m16 (0.25R genotyping points), and for M14 and M15, for the 0.125 of individuals that were M10–M13–m16 (0.25R genotyping points). For all other individuals, genotypes for markers M11, M12, M14, and M15 can be inferred as above from the previously determined genotypes for M10, M13, and M16. For example, the individual we are tracking would have been m10–m16, so that genotypes for markers M11, M12, M14, and M15 must be m11, m12, m14, and m15.

In practice, due to finite population size, the LOD values will deviate slightly from the ELOD values. That is, there always will be some small fluctuations from monotonic behavior of the LOD function to both sides of the final estimate of QTL position on the chromosome implicit in the given data set (see HYNE and KEARSEY 1995 ). Consequently, there is a nonzero (albeit a very small) probability of placing the QTL in a wrong subinterval (and of following up the wrong recombinant individuals) using the GS criterion. Under such a situation, the final steps in the application of the GS method (which is an efficient tool for optimization of deterministic unimodal functions) become inefficient. Therefore, we propose employing the optimal properties of GS in producing the first 2.62R data points. Then, using an internal pair of already genotyped markers, M_i and M_j, which flank the last location of the maximum LOD, we continue with complete genotyping of all remaining markers (i.e., residing between M_i and M_j) for individuals that are recombinants M_im_j and m_iM_j. This complete genotyping is conducted on the basis of the high-saving HS algorithm. Total genotyping data points for the internal segment under this combined GS-HS procedure will be 3M or less. Clearly, 3R < R log₂M, for M > 8. In principle, therefore, the GS algorithm will generally require fewer data points than the HS algorithm. However, both represent major savings relative to complete genotyping. If, in the data set obtained in an actual experiment, the ELOD function was bimodal, the GS algorithm would not be applicable, and the HS algorithm would be used.

	RESULTS AND DISCUSSION

TOP ABSTRACT THEORY RESULTS AND DISCUSSION PRACTICAL FEASIBILITY AND... LITERATURE CITED

The complete set of simulation results (data not shown) gave the SEQTL according to proportion selected in each tail (P), allele substitution effect at the QTL (d), size of mapping population (N), and marker spacing (c). A very wide spectrum of SEQTL values was obtained, ranging from 77.7 cM for the least powerful parameter combination (P = 0.05, d = 0.25, N = 1000, c = 24) to 0.05 cM for the most powerful combination (P = 0.50, d = 1.00, N = 16,000, c = 0.29). In an attempt to condense and simplify the total data set, nonlinear regression analysis was used to express the SEQTL as a power function of the simulation parameters. While the prediction equation obtained in this way explained much of the variation in SEQTL, many individual points were quite far from their predicted values. Consequently, the regression equation could not be used as a substitute for the tabulated values. However, the regression analysis did show a tight relationship between effects of N and d on SEQTL. This accorded with the well-known fact that test statistics for determining linkage between markers and QTL stand in proportion to d²N (SONG et al. 1999 ). Indeed, within a given combination of P and c, SEQTL were more or less the same for parameter combinations of d and N, for which d²N was the same. For example, within the parameter combination P = 0.05, c = 0.29; SEQTL for d = 0.25, N = 16,000; d = 0.5, N = 4000 and d = 1.0, N = 1000 (d²N = 1000 in each case) were 0.54, 0.51, and 0.59, respectively. Because of its powerful effect on SEQTL, the parameter d²N is termed the "power factor" or PF. Examination of Table 5 of THALLER and HOESCHELE 2000 shows the same dependence of accuracy of inferring QTL location on d²N; compare, e.g., in their Table 5, the "power" values for QTL effect 0.5, N = 100, 500, 2500 to those for QTL effect 0.25 and N = 400, 2000, and 10,000.

On the basis of the above relationship, a second table was prepared, giving SEQTL according to P, c, and d²N (data not shown). Where there were two or more combinations of d and N with the same value of d²N, these were averaged. The effect of proportion selected, P, was now examined. When this was done, with increase in P there was a consistent reduction in SEQTL at given PF and c, with the exception of the transition from P = 0.25 to P = 0.50, which was accompanied by only a very slight overall reduction in SEQTL (SEQTL at P = 0.50 was on average 0.96 of SEQTL at P = 0.25). This is expected, since virtually all of the information for QTL map location is found in the high and low 25% of the population (DARVASI 1997B ). When the reduction in SEQTL in going from P_j = 0.05, 0.10, and 0.20 to P = 0.25 was calculated for given PF and c, there was much fluctuation within the individual cells of the table, but for given P_j, overall trends were not found, and the reduction in SEQTL appeared to be consistent across the entire table of values (data not shown). The average reduction in SEQTL relative to P = 0.25 in going from P_j = 0.05, 0.10, and 0.20 to P = 0.25 was 0.46, 0.69, and 0.94, respectively. SEQTL for P = 0.05, 0.10, 0.20, and 0.50 were therefore transformed to a P = 0.25 basis by multiplying by the appropriate average factor (0.46, 0.69, 0.94, and 1.04, respectively). The results were averaged and are given in Table 2. It is of interest that the factors for P = 0.05, 0.10, and 0.20 appear to stand in close proportion to (P/0.5)^0.5, indicating a massive reduction in information content of the marginal data points in each case.

Examining the effect of marker spacing in Table 2 shows that the phenomenon of maximum achievable resolution for given PF noted by DARVASI et al. 1993 is found only for the lowest power factor, PF = 62.5. At all other power factors, with each step decrease in c there was a consistent, albeit often small, reduction in SEQTL. The reduction in SEQTL with successive step decreases in c (i.e., from c = 24 to c = 8, c = 8 to c = 2.66, c = 2.66 to c = 0.88, and c = 0.88 to c = 0.29) differed in a nonlinear manner depending on the power factor and on the specific step. In general, the reduction in SEQTL per step decrease in c was greater for the initial steps and smaller for the final steps and was greater for large PF and smaller for small PF (Table 1). It is noteworthy that an increase in marker spacing alone can increase map resolution by as much as eightfold, depending on the power factor. This finding is potentially of major importance. It tells us that when PF is high, saturation of the genomic interval carrying the detected QTL by additional markers is justified. Furthermore, in many cases, by the use of multiple-trait analysis (KOROL et al. 2001 ) the scaled multiple-trait allele substitution effect of a QTL (D) is much greater than the single trait effect (d). Since the PF stands in proportion to D², this will markedly increase the PF at the same N. This increase in PF, in turn, will enable a further major decrease in SEQTL by adding even more markers to the genomic interval carrying the detected QTL. Thus by combining multiple-trait analysis with marker saturation, map resolution for given N can be increased manifold. The possibility of multiple-trait interval mapping analysis for selective genotyping design was already shown by RONIN et al. 1998 .

Along similar lines, there was a consistent reduction in SEQTL with an increase in PF at all levels of c. However, the reduction did not stand in simple proportion either to the PF itself or to the square root of the PF. Thus, a further simple reduction of Table 1 with respect to c or PF was not possible. Table 2 can therefore be taken as the final condensed representation of the data.

The actual SEQTL for given d, N, P, and c can be approximated closely by going to the corresponding value of PF and c in Table 2 and multiplying by the inverse of the P_j to P = 0.25 reduction factor. For example, the SEQTL for d = 0.5, N = 4000 (PF = 1000), c = 2.66, P = 0.2 in the initial data simulation was 1.14. To reconstruct this value from Table 1, go to PF = 1000, c = 2.66 in Table 2 to find the value 0.785. This is multiplied by the factor 1/0.69 to give SEQTL = 1.14, which in this case happens to equal exactly the value found by simulation (data not shown). Not all equivalents were this exact, but most were very close.

DARVASI and SOLLER 1997 showed by simulation that the 95% confidence interval of QTL map location with a backcross or half-sib design, using a completely saturated map, can be closely approximated by the expression 95% C.I. = 3000/d²N. On this approximation, the expected SEQTL with a fully saturated map can be approximated as SEQTL = 95% C.I./4 = 750/PF. These values are also shown in Table 2 and should be compared to those obtained for c = 0.29, which are the limit values of the present simulation. The values obtained in the present study for PF = 62.5 and PF = 125 were much greater than the Darvasi and Soller (DS; DARVASI and SOLLER 1997 ) values. This is due to the fact that the DS simulation assumed that the QTL was within the simulated target region and hence gives smaller values than the present simulation gives when the SEQTL is large and when some estimated QTL positions are outside the target region. The values obtained in the present simulation for PF = 250–4000 were somewhat less than the DS values. The reason for this is not clear. Finally, the present study gave values equivalent to those of the DS approximation for PF = 8000 and 16,000. In general, therefore, the values given by the DS approximation are consistent with those of the present simulation.

Fig 1 and Fig 2 illustrate the HS and GS genotyping procedures. An example of the relative efficiency of the HS, GS, and combined GS-HS algorithms alone on mapping resolution is given in Table 3, which explores these relationships by simulation for the cases d = 1; N = 4000, 8000; P = 0.10, 0.20; c = 0.125; and an initial interval of 24 cM, so that total number of markers = (24/0.125) + 1 = 193. Total genotyping data points required by HS, GS, and GS-HS algorithms are 7.58R, 2.62R, and 3R, respectively. At this very dense spacing, SEQTL obtained by use of the GS algorithm alone are two- to threefold greater than SEQTL obtained by use of the HS algorithm. SEQTL obtained by the use of the combined GS-HS algorithm, however, are essentially equal to those obtained by the HS algorithm. Since genotyping results obtained by the HS algorithm are exactly the same as those provided by complete genotyping, the latter procedure was not simulated separately.

Clearly, the need for a small additional genotyping "investment" caused by moving from the GS to complete genotyping (2.62R 3R) is due to fluctuations caused by finite sample size. The estimates in Table 3 demonstrate that this small investment provides the same resolution as given by HS at a higher cost (note the close results for HS and GS-HS obtained by 7.58R and 3R genotyping data points, respectively).

	PRACTICAL FEASIBILITY AND IMPLEMENTATION

TOP ABSTRACT THEORY RESULTS AND DISCUSSION PRACTICAL FEASIBILITY AND... LITERATURE CITED

The results of this study show that when large mapping populations are available, SEQTL can be reduced to subcentimorgan levels, even for QTL of moderate effect (d = 0.25). This gives 95% confidence intervals of QTL location in the range of 1–5 cM. Confidence intervals of this magnitude provide tightly linked markers for marker-assisted selection, a strong basis for a search for population-wide linkage disequilibrium in outcrossing populations, and a platform for a search for the actual gene corresponding to the QTL.

By careful consideration of Table 2, the trade-off between population size, proportion selected, and marker spacing can be calculated, so as to obtain maximum return for the research investment. If large families are available and samples can easily be accessed, it will be more cost effective to use a small P with largest possible family size and wider marker spacing. If families are relatively small, or if it is difficult to access samples, it will be more cost effective to use a large P and closer marker spacing.

The major requirement for application of these procedures is availability of a population of required size and sufficient density of informative markers. The common dinucleotide microsatellite markers are generally not available at a spacing of <1–2 cM. However, with the introduction of single nucleotide polymorphism markers an increase by one or two orders of magnitude in the number of markers and a decrease of an order of magnitude in costs of genotyping are confidently expected for the near future.

With respect to population size, F₂ and BC populations of 10,000 or more can readily be produced in many species of agricultural plants. Thus, these species are excellent candidates for SRG. In agricultural animal species, the enormous sire half-sib families, consisting of 10,000 or more daughters that are routinely produced through artificial insemination in dairy and in some beef cattle populations, have the requisite family structure for QTL mapping, and phenotypic information is available on each individual. For application of SRG to poultry and swine breeding nuclei, progeny can be collected across a number of sires heterozygous for the same QTL to provide the desired total number of progeny for high-resolution mapping. This would require a preliminary step in which many sires are analyzed to identify sires heterozygous at the QTL. To reduce genotyping costs, screening of sires for heterozygosity could be achieved by selective DNA pooling (DARVASI and SOLLER 1994 ; LIPKIN et al. 1998 ).

Given the required population size, the genotyping load is not overly great when the GS-HS or HS algorithms are used. For example, for a QTL mapped to a target interval of 20 cM, and with a mapping population of N = 10,000 for BC or half-sib designs or of 5000 for F₂ designs, application of SRG at P = 0.20 and c = 1 cM would require 7500, 11,500, or 23,000 genotyping data points for F₂, BC, or half-sib designs, respectively, plus a small number for haplotyping the parents. This comes out to only a little more than one or two data points per daughter!

Although a given SRG mapping population will allow high-resolution mapping of all QTL segregating in the population, each QTL will have to be analyzed separately. Thus, high-resolution mapping of 10 QTL in the above mapping population would require a total of 100,000–200,000 data points. However, this is still only 10–20 data points per individual, 250-fold less than would be required for high-resolution mapping of the entire genome at a marker spacing of c = 1.0 cM.

An important aspect of the considered procedure is the assumption that the target QTL was correctly assigned to the segment bounded by the flanking markers M₁–M_k. Depending on the choice of C.I. stringency, the possibility will always exist that the true QTL position is not within the target segment, but in the adjacent segment, to the right or left. Thus, if SRG analysis indicates that the QTL is located to the extreme end of the target segment, one would go on to identify recombinants in the adjacent segment (at a cost of 2NP data points) and conduct an SRG analysis across both segments. Setting the initial target interval with much wider limits than the 95% C.I. would not be as useful, because in most instances the QTL will map within its 95% C.I. so that the additional effort is not needed, and, with a very wide target interval, double and triple recombinants will play more of a spoiling role.

The present results relate to expected SEQTL under various assumed design and parameter combinations. The question arises as to the relevance of the SEQTL of the present study, obtained across many simulations, to the C.I. of map location as it might be estimated from the one-time data of an actual experiment. In this case, bootstrap and information matrix methods are available to obtain approximate confidence intervals for QTL map location. However, it would also be possible to use the estimate of QTL effect obtained from the actual experiment to obtain a SEQTL estimate by interpolation in Table 2. We believe that C.I. obtained by the two approaches will be similar, but this remains to be explored in detail.

In addition, once an estimate of QTL effect has been obtained, the results of this study are relevant to deciding whether and to what degree further marker density in the C.I. could reduce the SEQTL and C.I. of map location.

	ACKNOWLEDGMENTS

Constructive comments of two referees are acknowledged with thanks. This study was supported by the Israeli Ministry of Absorption and German-Israel Cooperation project (DIP project founded by the Inernationales Deutsch-Israelische des BMBF Projektkooperation), by the Framework 5 Program of the E.U., and by the U.S.-Israel Binational Science Foundation.

Manuscript received November 10, 2002; Accepted for publication April 21, 2003.

	LITERATURE CITED

TOP ABSTRACT THEORY RESULTS AND DISCUSSION PRACTICAL FEASIBILITY AND... LITERATURE CITED

DARVASI, A., 1997a  Interval specific congenic strains (ISCS): an experimental design for mapping a QTL into a 1-centimorgan interval. Mamm. Genome 8:163-167.[Medline]

DARVASI, A., 1997b  The effect of selective genotyping on QTL mapping accuracy. Mamm. Genome 8:67-68.[Medline]

DARVASI, A., 1998  Experimental strategies for the genetic dissection of complex traits in animal models. Nat. Genet. 18(1):19-24.[Medline]

DARVASI, A. and M. SOLLER, 1994  Selective genotyping for determination of linkage between a marker locus and a quantitative trait locus. Genetics 138:1365-1373.[Abstract]

DARVASI, A. and M. SOLLER, 1995  Advanced intercross lines, an experimental population for fine genetic mapping. Genetics 141:1199-1207.[Abstract]

DARVASI, A. and M. SOLLER, 1997  A simple method to calculate resolving power and confidence interval of QTL map location. Behav. Genet. 27(2):125-132.[Medline]

DARVASI, A., A. VINREB, V. MINKE, J. I. WELLER, and M. SOLLER, 1993  Detecting marker-QTL linkage and estimating QTL gene effect and map location using a saturated genetic map. Genetics 134:943-951.[Abstract]

GILL, P. E., W. MURRAY and M. H. WRIGHT, 1981 Practical Optimization. Academic Press, New York.

HILL, W. G., 1998  Selection with recurrent backcrossing to develop congenic lines for quantitative trait loci analysis. Genetics 148:1341-1352.[Abstract/Free Full Text]

HYNE, V. and M. J. KEARSEY, 1995  QTL analysis: further uses of ‘marker regression’. Theor. Appl. Genet. 91:471-476.

JANSEN, R. C. and P. STAM, 1994  High resolution of quantitative traits into multiple loci via interval mapping. Genetics 136:1447-1455.[Abstract]

JIANG, C. and Z-B. ZENG, 1995  Multiple trait analysis and genetic mapping for quantitative trait loci. Genetics 140:1111-1127.[Abstract]

KLEIN-LANKHORST, R. M., A. VERMUN, R. WEIDE, T. LIHARSKA, and P. ZABEL, 1991  Isolation of molecular markers for tomato (L. esculantum) using random amplified polymorphic DNA (RAPD). Theor. Appl. Genet. 83:108-114.

KOROL, A. B., Y. I. RONIN, and V. M. KIRZHNER, 1995  Interval mapping of quantitative trait loci employing correlated trait complexes. Genetics 140:1137-1147.[Abstract]

KOROL, A., Y. RONIN, A. ITZCOVICH, and E. NEVO, 2001  Enhanced efficiency of QTL mapping analysis based on multivariate complexes of quantitative traits. Genetics 157:1789-1803.[Abstract/Free Full Text]

LANDER, E. S. and D. BOTSTEIN, 1989  Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121:185-199.[Abstract/Free Full Text]

LIPKIN, E., M. O. MOSIG, A. DARVASI, E. EZRA, and A. SHALOM et al., 1998  Mapping loci controlling milk protein percentage in dairy cattle by means of selective milk DNA pooling using dinucleotide microsatellite markers. Genetics 149:1557-1567.[Abstract/Free Full Text]

RHODES, M., R. STRAW, S. FERNANDO, A. EVANS, and T. LACEY et al., 1998  A high-resolution microsatellite map of the mouse genome. Genome Res. 8:531-542.[Abstract/Free Full Text]

RONIN, Y. I., A. B. KOROL, and J. I. WELLER, 1998  Selective genotyping to detect quantitative trait loci affecting multiple traits: interval mapping analysis. Theor. Appl. Genet. 97:1169-1178.

RONIN, Y., A. KOROL, and E. NEVO, 1999  Single- and multiple-trait analysis of linked QTLs: some asymptotic analytical approximation. Genetics 151:387-396.[Abstract/Free Full Text]

SOLLER, M. and J. S. BECKMANN, 1990  Marker-based mapping of quantitative trait loci using replicated progeny. Theor. Appl. Genet. 80:205-208.

SOLLER, M. and L. ANDERSSON, 1998  Genomic approaches to improvement of disease resistance in farm animals. Rev. Sci. Tech. 17:329-345.[Medline]

SONG, J. Z., M. SOLLER, and A. GENIZI, 1999  The full-sib intercross line (FSIL) design: a QTL mapping design for outcrossing species. Genet. Res. 73:61-73.

THALLER, G. and I. HOESCHELE, 2000  Fine-mapping of quantitative trait loci in half-sib families using current recombinations. Genet. Res. 76(1):87-104.[Medline]

WELLER, J. I., Y. KASHI, and M. SOLLER, 1990  Power of daughter and granddaughter designs for determining linkage between marker loci and quantitative trait loci in dairy cattle. J. Dairy Sci. 73:2525-2537.[Abstract]

ZENG, Z-B., 1994  Precise mapping of quantitative trait loci. Genetics 136:1457-1468.[Abstract]

This article has been cited by other articles:

				A. Korol, Z. Frenkel, L. Cohen, E. Lipkin, and M. Soller Fractioned DNA Pooling: A New Cost-Effective Strategy for Fine Mapping of Quantitative Trait Loci Genetics, August 1, 2007; 176(4): 2611 - 2623. [Abstract] [Full Text] [PDF]

				D. J. de Koning, C. P. Cabrera, and C. S. Haley Genetical Genomics: Combining Gene Expression with Marker Genotypes in Poultry Poult. Sci., July 1, 2007; 86(7): 1501 - 1509. [Abstract] [Full Text] [PDF]

				C. Granhall, H.-B. Park, H. Fakhrai-Rad, and H. Luthman High-Resolution Quantitative Trait Locus Analysis Reveals Multiple Diabetes Susceptibility Loci Mapped to Intervals <800 kb in the Species-Conserved Niddm1i of the GK Rat Genetics, November 1, 2006; 174(3): 1565 - 1572. [Abstract] [Full Text] [PDF]

				V. Orgogozo, K. W. Broman, and D. L. Stern High-Resolution Quantitative Trait Locus Mapping Reveals Sign Epistasis Controlling Ovariole Number Between Two Drosophila Species Genetics, May 1, 2006; 173(1): 197 - 205. [Abstract] [Full Text] [PDF]

				C. Yagil, M. Sapojnikov, A. Wechsler, A. Korol, and Y. Yagil Genetic dissection of proteinuria in the Sabra rat Physiol Genomics, March 13, 2006; 25(1): 121 - 133. [Abstract] [Full Text] [PDF]

				C. Lee Selection Bias in Quantitative Trait Loci Mapping J. Hered., July 1, 2005; 96(4): 363 - 367. [Abstract] [Full Text] [PDF]

				S. Sen, J. M. Satagopan, and G. A. Churchill Quantitative Trait Locus Study Design From an Information Perspective Genetics, May 1, 2005; 170(1): 447 - 464. [Abstract] [Full Text] [PDF]

				Z. Xu, F. Zou, and T. J. Vision Improving Quantitative Trait Loci Mapping Resolution in Experimental Crosses by the Use of Genotypically Selected Samples Genetics, May 1, 2005; 170(1): 401 - 408. [Abstract] [Full Text] [PDF]

				L. Varga, G. Muller, G. Szabo, O. Pinke, E. Korom, B. Kovacs, L. Patthy, and M. Soller Mapping Modifiers Affecting Muscularity of the Myostatin Mutant (MstnCmpt-dl1Abc) Compact Mouse Genetics, September 1, 2003; 165(1): 257 - 267. [Abstract] [Full Text] [PDF]