Springer 2005
Molecular Breeding (2005) 16: 261–270 DOI 10.1007/s11032-005-3424-7
0
Efficient genetic mapping of single nucleotide polymorphisms based upon DNA mismatch digestion Dainis Rungis1,2, Britta Hamberger1,2, Yanik Be´rube´1,2, Jennifer Wilkin1,2, Jo¨rg Bohlmann2 and Kermit Ritland1,* 1
Department of Forest Sciences, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; 2Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; *Author for correspondence (e-mail:
[email protected]; phone: +1-604-822-8101; fax: +1-604-822-9102)
Received 8 March 2005; accepted in revised form 16 September 2005
Key words: CJE, Heteroduplex digestion, Linkage mapping, SNPs
Abstract A single-strand specific (sss) nuclease, found in extracts of celery juice, can be used to digest heteroduplex DNA and hence identify heterozygous single nucleotide polymorphism (SNP) sites in PCR products. Here we show this method can be used to map specific genes with relative simplicity and low cost. A particular nucleotide substitution does not need to be identified, and in fact, a priori knowledge of the presence of a SNP is not required, as the entire length of the PCR product is interrogated for the presence of SNPs. This characteristic enables application of this technique to genomes that are not well characterized with regard to SNP polymorphism, and for rapidly placing particular genes onto linkage maps. While this technique is best suited for mapping markers in a backcross configuration, we show that in an F2 configuration, where alternative homozygotes cannot be discerned by this technique, data are still relatively informative about linkage.
Introduction Recently, a single-strand specific (sss) nuclease (CEL I or CJE), found in extracts of celery juice, was proposed as a simple and rapid method to assay induced mutations in genomes (TILLING, Oleykowski et al. 1998; Till et al. 2004) and to assay single nucleotide polymorphism in natural populations (ecoTILLING, Comai et al. 2004). The function of this nuclease is similar to the S1 family of sss nucleases, and works with a variety of co-factors to digest heteroduplex DNA immediately 3¢ of a mismatch site, or at sites of short insertions or deletions (indels) (Oleykowski et al. 1998). CEL I/CJE digestion has now been used in a variety of species, both plant and animal (for review see Gilchrist and Haughn 2005).
Molecular marker maps can be constructed with one or more of a variety of DNA markers, of which each have their advantages and disadvantages (Ritland and Ritland 2000). For genetic mapping, four criteria are especially important: (1) large numbers of loci need to be accessible in order to saturate the map with markers; this implies that marker development should be easy and rapid, (2) loci should be sufficiently polymorphic such that they usually segregate in a pedigree, (3) markers should be transferable between pedigrees and ideally between species, e.g., orthology of loci between species should be identifiable, and (4) ideally, coding DNA should be mapped as these can be used in ‘quantitative trait nucleotide’ (QTN) studies (Mackay 2001). Dominance vs. co-dominance is perhaps a 5th criterion although
262 in many cases, such as in backcross mapping, it is not an issue as only heterozygotes and homozygous recessives occur. None of the current DNA marker techniques satisfies all of these criteria. Simple sequence repeats (SSRs), are highly prized as molecular markers due to co-dominance and high levels of polymorphism, but a large effort is required to develop these markers (Squirrell et al. 2003) and SSRs are also specific to species or genera, restricting the transfer of map information. Amplified fragment length polymorphisms (AFLPs) and its predecessor, randomly amplified polymorphic DNA (RAPD), are more easily developed and yield many more loci, but their transferability is even more limited, sometimes not even between pedigrees within a species (Chagne et al. 2003). In principle, single nucleotide polymorphisms (SNPs) should meet most of these criteria. They are numerous (for example, approximately 10 million have been identified in humans), can be quite polymorphic when defined on the haplotype level (500 – 1000 bp length of DNA), should be highly transferable if assayed for conserved orthologous set (COS) genes (Fulton et al. 2002), can be restricted to coding DNA thus allowing QTN studies, and can be co-dominant (although this depends upon the technique used to assay the SNPs). Here we describe the application, utility and potential pitfalls of using celery juice extract (CJE) to digest and visualize segregating SNPs in a mapping population. In this technique, a PCR product from single individuals is denatured then re-annealed, resulting in detectable heteroduplex mismatches at heterozygous sites. There are several immediate advantages of using CJE digestion for genetic mapping. It does not require knowledge of target SNPs; instead, a region of the genome (a PCR product 500 – 1500 bp long) is scanned for the presence of heterozygous SNP sites. Hence, marker discovery is easy and rapid, with the major requirement being knowledge of DNA sequences for primer design, which can be obtained from expressed sequence tag (EST) databases. The CJE nuclease assay is inexpensive, and digestion products can be visualized on standard genotyping gel electrophoresis systems such as LiCor or ABI. CJE digestion is also highly sensitive, and will cleave at all heteroduplex sites regardless of sequence context (Oleykowski et al.
1998). As well, by using large EST databases from different species, orthologous genes across species can be identified and used in CJE developed maps, allowing cross-species comparisons. While CJE only detects the presence of heterozygotes (in the absence of a reference DNA), in backcross mapping the data consists of only heterozygotes vs. a single homozygote type, so this is not an issue. Interestingly, we also show that even in F2 crosses, where alternative homozygotes are not distinguishable, CJE data are still relatively informative about linkage.
Methods SNP genotyping techniques can be divided into two categories, according to whether the nucleotide sequence of the SNP site is known or not. When there is no knowledge of target SNP identity, single-strand conformational polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) are commonly used, but these techniques often require optimization and do not always detect all polymorphisms (Plomion et al. 1999; Pelgas et al. 2004). If the target SNP is known, there are a plethora of techniques available to genotype SNPs, but many of these are expensive or are economical only with large numbers of genotypes (far larger than the typical 100 – 200 member mapping population), and require specialized and/or dedicated equipment (Gut 2001; Jenkins and Gibson 2002). Here we describe the CJE procedure for genetic mapping, as implemented in our Genome BC spruce genetic mapping project, and discuss some problems particular to this technique. The digestion reaction conditions used are such that only partial cleavage of the DNA strand occurs, and so multiple polymorphisms can be identified within one PCR product (Oleykowski et al. 1998; Comai et al. 2004; Gilchrist and Haughn 2005). This is an accurate method of detecting polymorphism as both ends of the PCR product can be labeled, and any SNPs present will be detected using both dyes, with the size of the digestion products adding up to the size of the full length PCR products (+1 nucleotide, as strand cleavage occurs 3¢ of the heteroduplex on both strands) (Figure 1). Interestingly, crude extracts of CJE have been shown to perform as well as more highly purified nuclease preparations (Till et al. 2004).
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Figure 1. Diagram of CJE digestion of a PCR product. Note that the sum of each digested fragment and its complement in the other channel will equal the full length product +1 nucleotide as the enzyme cuts either DNA strand 3¢ of the heteroduplex site. As digestion is partial, multiple SNPs can be identified, and the presence of perfectly matched PCR products ensures that the full length PCR product is also visualized.
CJE protocol in the spruce genome project Like many SNP projects, the procedure involves two stages, first a ‘validation’ stage where primers amplifying correct PCR products and containing informative SNPs are identified in the parents of the mapping populations, followed by a high throughput ‘production’ stage where the actual mapping populations are assayed. Two mapping populations were genotyped using CJE digestion in this study – 68 · 780 and 68 · 1425. These are F1 populations of the crosses between an artificial white-Sitka spruce hybrid (68), and plus trees from the white spruce breeding program (780 and 1425). Each population contained 88 individuals. As these trees are outbred, they contain a mixture of heterozygous and homozygous loci, some of which will be segregating in the mapping populations. The spruce EST database utilized has been described previously (Rungis et al. 2004), and pine
ESTs were obtained from GenBank and the ‘The Genomics of Wood Formation in Loblolly Pine’ project (NSF award DBI 9975806: http://pinetree.ccgb.umn.edu/). For the first ‘validation’ stage, specific primers are designed to amplify genomic DNA using the Primer 3 software (Rozen and Skaletsky 2000), and primers are designed to all have a similar annealing temperature to allow for uniform PCR cycling conditions. The length of PCR product should be approximately 500 – 1500 bp. Theoretically, there is no minimum required length for PCR products; however the terminal 80 – 100 nucleotides of a PCR product migrate in a noisy area of the gel, such that polymorphisms are difficult to score in these regions. Therefore PCR products under 500 bp realistically only have a 300 bp window in which to detect polymorphisms, which reduces the probability of detecting polymorphisms in a given PCR product. Conversely,
264 PCR products over 1500 bp are difficult to run and visualize on polyacrylamide gels and give faint signals on the LiCor sequencers. PCR is performed with 25 ng genomic DNA, 0.2 lM of each forward and reverse primer, 0.2 mM dNTPs, 2.5 mM MgCl2 and 0.5 unit of AmpliTaq DNA polymerase in a 20 ll volume. PCR cycling conditions consist of an initial denaturation step of 95 C for 2 min, 30 cycles of 95 C for 20 s, 58 C for 20 s and 72 C for 30 s followed by a final extension step of 72 C for 3 min. These specific PCR products are run on 1% agarose gels to ensure that amplification was successful, and to identify primers that amplify multiple fragments of sufficient length difference to be resolved on an agarose gel. Both forward and reverse primers are tailed with a M13 sequence to facilitate visualization of PCR products on a LiCor 4200 (LiCor Inc. Lincoln, NB) after a second round of PCR amplification using infra-red dye (IRD) labelled M13 primers (Oetting et al. 1995). This round of PCR is performed using 1 ll of the specific PCR reaction, 0.05 lM each of the forward and reverse M13 IRD labelled primer, 0.2 mM dNTPs, 1.5 mM MgCl2 and 0.5 unit of AmpliTaq DNA polymerase in a 10 ll volume. PCR cycling conditions consist of an initial denaturation step of 95 C for 2 min, 30 cycles of 95 C for 20 s, 58 C for 20 s and 72 C for 30 s followed by a final extension step of 72 C for 3 min. The labelled fragments are then denatured at 95 C for 10 min, followed by a gradual return to room temperature to facilitate re-annealing. These re-annealed heteroduplexes are then digested with CJE enzyme (Colbert et al. 2001) and run on a LiCor 4200. Both strands are labeled at this ‘validation’ stage to enable any polymorphic fragments to be confirmed using the complementary fragment from the opposite strand. A fragment visualized using the forward primer should have a complementary fragment in the reverse primer channel such that the sum of the size of both fragments is equal to the length of the undigested PCR product (+1 nucleotide) (see Figures 1 and 2 and Colbert et al. 2001). In addition, primers amplifying multiple fragments that were too faint, or too close in size to be resolved on an agarose gel can be identified. At this point, SNPs heterozygous in one parent are flagged for scoring in the mapping population. Heterozygous SNP loci in a backcross configura-
tion will be segregating 1:1 in a mapping population. Note that a 1:1 ratio of segregating markers will also result with CJE genotyping if both parents are heterozygous at the same SNP locus (F2 configuration), as the individuals homozygous for alternate SNP alleles will not be distinguished by CJE digestion. Therefore, the genotype of both parents is needed to ensure that a segregating SNP is heterozygous in only one parent. Although we discuss below that linkage can be estimated using CJE digestion when both parents are heterozygous at a particular SNP, we excluded these cases, as existing genetic mapping software cannot handle this type of data. Now, at the second, ‘production’ stage, the entire mapping population is amplified using the 2-step PCR process described above. However, as SNP loci have already been characterized and confirmed in the parental testing stage, only one DNA strand needs to be labeled. This enables a doubling of throughput on two-channel LiCor sequencers, as one gel can assay two loci. In this case the PCR reamplification is performed with either the forward or reverse IRD labeled M13 primer (whichever gives the clearest view of the SNPs to be mapped), and the corresponding opposite unlabeled specific primer. The 2-step PCR amplification protocol was necessary, as we found that co-amplification of the specific primers with the IRD labelled primers was not successful. While the PCR reaction produced the correct fragment (confirmed using agarose gel analysis), very faint or no fragments were identified after CJE digestion and fragment separation on the LiCor 4200. Presumably either the specific PCR primers were out-competing the M13 IRD labelled primers during co-amplification, or the IRD dye was cleaved from the PCR product during CJE digestion. It is also possible to use directly labelled specific PCR primers, but due to the increased cost this involved, we utilized the 2-step PCR protocol for all CJE mapping.
Using multiple heterozygous SNP sites to join parental maps Multiple SNP sites heterozygous in the same parent can be used as confirmation of each other, and thus increase the accuracy of genotyping. However, SNP sites that are heterozygous in alternate
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Figure 2. CJE digestion of a PCR product amplified from 10 unrelated individuals. Panel 1 depicts labelled forward PCR primer (700 channel); panel 2 depicts labelled reverse PCR primer (800 channel). SNPs A-E in panel 1 have complementary sized fragments in panel 2 (A’–E’). The sum of fragment sizes of each SNP and its complement are equal to the full-length product. Note: for clarity not all SNPs are marked.
parents are also useful to integrate parental maps. In outbred species such as trees, both parents are often heterozygous, or at least heterozygous in either parent, allowing detection of recombination in both male and female parents. This results in two genetic maps, one for each parent. These maps can be integrated only at points where loci are heterozygous in both parents (if only dominant markers are utilized, the integration of maps is not possible). In the CJE method, if two SNPs within the same PCR product are heterozygous in alternate parents, then they will be segregating independently in the mapping population, and this locus can be used to integrate the two parental genetic maps. (Note this is not the same situation as crossing two heterozygous parents; here, the SNPs are heterozygous at different sites, and each segregates as a normal backcross).
Informativeness of heterozygous SNP sites common to both parents Alternatively, parents may be heterozygous for the same SNP site. While in an F2 mapping configuration alternative homozygotes cannot be distinguished using CJE digestion, there is still information about linkage. This is shown in Table 1, which classifies the four progeny classes, and each has probability of either 1/2 r(1 r) or r(1 r), where r is the recombination rate. The fact that their frequencies differ, and are functions of r, means that r can be estimated from such data. Also note the probabilities of offspring genotype depend upon the phase of parents; if progeny sizes are large enough and recombination rates sufficiently small, this phase can be inferred. Interestingly, with no linkage, each class is equally frequent.
266 Table 1. Probabilities of progeny banding types in an F2 cross (two double heterozygotes crossed with each other).
Both not heterozygous (AABB+AAbb+aaBB+aabb) Locus A heterozygous (AaBB+Aabb) Locus B heterozygous (AABb+aaBb) Both A and B heterozygous (AaBb)
Same phase
Different phase
1/2 r(1 r) r(1 r) r(1 r) 1/2 r(1 r)
r(1 r) 1/2 r(1 r) 1/2 r(1 r) r(1 r)
The expectations depend upon whether the parents share the same linkage phase. For example, parents have same phases when both are in coupling (AB/ab · AB/ab) or both are in repulsion (Ab/aB · Ab/aB). Other possible configurations correspond to the ‘different phase’ mating.
The amount of statistical information about linkage when CJE digestion is used to genotype SNPs in an F2 configuration, as compared to the case where alternative homozygotes can be distinguished, is found by computing Fisher information (found by taking expected second derivatives of the likelihood function). We assume known linkage phase. For the CJE method, the Fisher information (per observation) is ! 1 2rð1 rÞ rð1 rÞ ; ð1Þ 2ð1 2rÞ2
while the second locus would have distinguishable homozygotes. The information about recombination in this case would be in between that depicted in Figure 3 and the fully informative case (relative information of unity). However, existing mapping programs have not incorporated any case wherein alternative homozygotes cannot be distinguished, and we suggest that software developers consider this.
while when all nine genotypes are distinguishable (but with couple double heterozygotes indistinguishable from repulsion double heterozygotes), the Fisher information is 1 2rð1 rÞ rð1 rÞ ð2Þ 2ð1 3rÞð1 rÞ
For mapping in our Genome BC spruce project, we have utilized two mapping populations that share one parent, a highly heterozygous artificial white – Sitka spruce hybrid. Use of EST databases also allows the design of primers for loci that have the largest chance of success: ‘conserved orthologous set’ (COS) markers (Fulton et al. 2002). These markers are identified by self-BLASTING an EST database, and identify ESTs that lack hits to related ESTs. Ideally, an EST database from a second related species can also identify a subset of these markers that are slowly evolving (conserved) as evidenced by low e-values of BLAST hits. Of 105 COS primer pairs designed from spruce EST sequences, 60 amplified single PCR products that were the right size for mapping using CJE digestion. The remaining 45 primer pairs did not amplify any product, amplified multiple fragments, or amplified PCR products too large for CJE digestion. Of the 60 single fragments, 53 (88%) could be mapped in one of the two mapping populations, with only 7 not detecting polymorphism between any of the three parents. Furthermore, 36 (60%) of the PCR products could be mapped in both populations. Twenty-two (37%) COS markers had alternative heterozygous SNP loci in both parents of either mapping population, allowing these markers to be scored as co-dominant markers,
The relative information of the CJE method is the ratio of Equations (1) and (2), or ! 2 ð1 2rÞ ð3Þ ð1 3rÞð1 rÞ This equation is plotted in Figure 3. For low recombination rates, the loss of information by using the CJE method is quite low, less than 10% for recombination rates of less than 0.10, and ca. 25% for recombination rates of 0.20. Given that these are the values of r that are most informative for high density linkage maps, the CJE method seems practical to implement for F2 mapping populations. An intermediate case occurs when one PCR product is heterozygous for the same SNP site, while a linked PCR product (e.g., gene) is heterozygous for different SNP sites. In this case, the first locus would have indistinguishable homozygotes,
Results
267
Figure 3. Detail of a CJE genotyping gel showing the parents (P1 and P2) and 33 members of a mapping population. SNPs A, B, C and D are heterozygous in parent 1 and can be compared to each other to confirm the genotyping. Note that SNPs A and D are in opposite phase to SNPs B and C. SNP E is heterozygous in parent 2 and segregates independently to SNPs A–D, therefore this locus can be scored as a co-dominant marker.
therefore enabling these markers to be used to integrate the two parental maps. All loci mapped were in a backcross configuration. As mentioned previously, the F2 configuration can be informative; however we found that in most cases where F2 configuration SNPs were present there were also backcross configuration SNPs found on the same PCR product. In the seven loci where no polymorphism was detected between the parents, four had SNP loci in the F2 configuration, with only three loci not having any heterozygous loci at all. If we included the use of F2 configuration SNPs in our current mapping project, then 95% of single PCR products approximately 500 – 1500 bp in size could be placed on the genetic linkage map of spruce. As the remaining 5% of PCR products did not contain heterozygous SNP sites in any of the parents they are not informative for linkage mapping regardless of genotyping technique. The majority of markers that we have mapped using CJE digestion have not been previously mapped, and so the map positions could not be compared to other spruce genetic maps. However, we have identified novel orthologous markers
between white spruce and loblolly pine, that have been mapped in both species. Additionally, we have mapped in white spruce markers that have been identified as orthologous between loblolly pine and Douglas fir, and genetically mapped in both species (Krutovsky et al. 2004). In the cases where two or more of these orthologous markers are found on a white spruce linkage group, they are also found on the same linkage group and in the same order in the loblolly pine genetic map (Rungis et al, unpublished).
An indirect test of the robustness of the CJE mapping method Since we had two mapping populations (each with 88 progeny) and also potential segregation on either the male or female size of each mapping population, up to four estimates of recombination can be obtained for a given pair of loci. The concordance of these estimates can be used as an indirect test of the robustness of CJE digestion for estimates of recombination.
268 We used a likelihood-ratio test to detect any heterogeneity of recombination estimates as follows. Between two loci, suppose there are n progeny populations (n = 2, 3 or 4) and in population i there are Xi recombinant progeny and Yi nonrecombinant progeny; the estimate of recombination for population i is then rˆ i = Xi /(Xi + Yi). The likelihood of the data for all populations under the hypothesis of separate recombination n Q i ^ rates is L1 ¼ ri ÞYi while that under the rX i ð1 ^ i¼1
hypothesis of a common recombination rate P P is Xi, Y = Yi L0 = rˆX (1 rˆ )Y where X = and rˆ = X/(X + Y). The test statistic is 2ln (Lo /L1) which is distributed as chi-square with n 1 degrees of freedom. We performed this test on pairs of loci whose combined estimate of r were statistically significant at a LOD value (based 10) of 10 or greater, e.g., linkage was 10 times more likely than lack of linkage. Among 84 marker loci, 186 pairs showed significant linkage. 140 of these had n = 2 and 8 (5.7%) were significant at the 5% level, 29 had n = 3 and none were significant, and 17 had n = 4 and again none were significant. Thus the linkage estimates are homogenous indicating robustness of the CJE mapping method.
mapped. While both configurations are informative for mapping, both will result in a 1:1 segregation ratio in the mapping population, as CJE digestion only identifies heterozygous SNP sites. Furthermore, the digestion conditions are such that each DNA strand is only partially cut, allowing multiple SNPs on a single PCR product to be identified (Figure 4). Therefore, if more than one heterozygous SNP occurs in a PCR product from one parent, then the two SNPs can be used as confirmation of each other, as they are completely linked. While multiple SNPs in a PCR product (heterozygous in the same parent) provide no extra information about linkage, they can be compared to each other to increase the accuracy of genotyping. CEL I has been shown to detect 100% of polymorphisms, including deletions, insertions and missense alterations, regardless of sequence identity or context (Oleykowski et al. 1998). This polymorphism detection technique is robust and repeatable; in an ecoTILLING study in Arabidopsis, all polymorphisms detected by CEL I digestion were verified as true polymorphisms by sequencing (Comai et al. 2004). Some additional polymorphisms were detected by sequencing, but these were present in the terminal 80 nucleotides of each PCR product, which migrate in a noisy area of the gel, and so were not detected by CEL I digestion (Greene et al. 2003; Comai et al. 2004).
Discussion The CJE method of mapping seems ideal for placing candidate genes and other genes of interest onto genetic maps. Using this genotyping technique, any PCR product can be mapped if it amplifies a single locus from the genome (providing an informative SNP is present), in contrast to other SNP polymorphism detection methods such as cleaved amplified polymorphic sequence (CAPS), SSCP or DGGE, which do not detect all SNP polymorphisms, and often require optimization to maximize polymorphism detection. Also, compared to the TILLING and ecoTILLING protocol (Till et al. 2003; Comai et al. 2004), which assays for mutations and natural variation in populations, genetic mapping by CJE digestion does not require a second ‘reference’ sequence in the re-annealing mixture. One drawback to genetic mapping using CJE digestion is that the parents of the mapping population must be genotyped to determine the configuration (backcross or F2) of the SNP being
Application and efficiency of genetic mapping using CJE digestion As mentioned previously, 88% of single locus PCR products could be mapped in at least one population, while 60% could be mapped in both populations. Furthermore, 37% of the PCR products contained at least 2 SNP sites heterozygous in alternate parents, thus allowing these markers to be used as co-dominant markers to integrate the two parental maps. These results are for spruce, but this technique is applicable to any species. However, the mapping efficiency will depend on the polymorphism present within and between the parents used for the mapping cross and ease of developing single-locus PCR primers will depend on the characteristics of the particular genome in question. The success rates of detecting polymorphism from EST-based markers in spruce using other
269
Figure 4. Efficiency of the CJE method for estimating linkage in an F2 population, relative to the case when all 8 possible twolocus genotypes are distinguished.
methods, such as agarose gel electrophoresis or SSCP, has been somewhat lower. In a single pedigree of Picea abies, 46% of successfully ESTbased amplified fragments were able to be mapped using length polymorphism, PCR-RFLP or SSCP (Achere´ et al. 2004). Using only length polymorphism or SSCP only 28% of successfully ESTbased amplified fragments could be mapped in a single pedigree of P. glauca (Gosselin et al. 2002). The use of DGGE increased the rate of polymorphism detection, with up to 74% of successfully EST-based amplified fragments polymorphic in a panel of 10 pedigree parents for each of P. mariana and P. glauca (Pelgas et al. 2004). The CJE technique, which efficiently detects practically any SNP in a PCR amplified region, is also useful for transferring linkage maps across pedigrees, as if a particular nucleotide site is not polymorphic between a particular set of parents, then it may be possible to map the fragment using a different polymorphic site within the PCR product. Another application this technique is suited to is the creation of comparative maps between widely diverged species. Cross-hybridizing RFLP probes have been used for this purpose, as well as SSRs, but these are not highly transferable between widely diverged species. Furthermore, SSR primer pairs that do amplify across species tend to be less polymorphic in species other than their target species and produce null alleles in greater frequency; an example are ‘EST-SSRs’ or SSRs identified from ESTs, which are ca. 20% less polymorphic than SSRs identified from genomic DNA (Liewlaksaneeyanawin et al. 2004). As
mentioned above, COS markers are ideal for comparative genetic mapping of diverged species, and our current efforts are focusing on the loblolly pine–spruce comparison. In summary, the advantages of this technique are speed, simplicity and cost. It combines the precision of genotyping a particular SNP with the power of detecting multiple SNP sites per PCR product, which effectively transforms these into co-dominant markers. A particular nucleotide substitution does not need to be identified, and in fact, a priori knowledge of the presence of a SNP is not required, as the entire PCR product is interrogated for the presence of SNPs. No optimization is required, and the cost per genotype is similar to that of SSR assays (approximately 10c/genotype). CJE digestion has previously been shown to be accurate and sensitive (Oleykowski et al. 1998; Greene et al. 2003; Comai et al. 2004), and our results have confirmed this, both by confirmation of SNPs in the parental screens by labeling both ends of the PCR product, as well as by repeating the CJE digestion independently (in the parental screen and the mapping population genotyping). The use of CJE digestion allows for the rapid and efficient genetic mapping of PCR products, which enables candidate genes to be easily placed on linkage maps, as well as facilitates the comparative mapping of species by mapping previously developed orthologous markers or novel markers derived from bioinformatic analysis of EST databases.
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