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Published online 2 December 2005
Published in Crop Sci 46:12-21 (2006)
© 2005 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Single Nucleotide Polymorphisms and Insertion–Deletions for Genetic Markers and Anchoring the Maize Fingerprint Contig Physical Map

I. Vroh Bia,*, M. D. McMullenb, H. Sanchez-Villedac, S. Schroederc, J. Gardinerd, M. Polaccob, C. Soderlunde, R. Wingf, Z. Fangc and E. H. Coe, Jr.b

a Inst. for Genomic Diversity, Cornell Univ., Ithaca, NY 14853
b Agronomy Dep., Plant Sciences Unit, Univ. of Missouri, Columbia, MO, and USDA-ARS Plant Genetics Research Unit, Columbia, MO 65211
c Agronomy Dep., Plant Sciences Unit, Univ. of Missouri, Columbia, MO 65211
d BIO5 Inst., Univ. of Arizona, Tucson, AZ 85721
e Arizona Genomics Computational Lab., Univ. of Arizona, Tucson, AZ 85721
f Arizona Genomics Inst., Univ. of Arizona, Tucson, AZ 85721

* Corresponding author (biv2{at}cornell.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Single nucleotide polymorphisms (SNPs) and insertion–deletions (InDels) are becoming important genetic markers for major crop species. In this study we demonstrate their utility for locating fingerprint contigs (FPCs) to the genetic map. To derive SNP and InDel markers, we amplified genomic regions corresponding to 3000 unigenes across 12 maize (Zea mays L.) lines, of which 194 unigenes (6.4%) showed size polymorphism InDels between B73 and Mo17 on agarose gels. The analysis of these InDels in 83 diverse inbred lines showed that InDels are often multiallelic markers in maize. Single nucleotide polymorphism discovery conducted on 592 unigenes revealed that 44% of the unigenes contained B73/Mo17 SNPs, while 8% showed no sequence variation among the 12 inbred lines. On average, SNPs and InDels occurred every 73 and 309 bp, respectively. Multiple SNPs within unigenes led to a SNP haplotype genetic diversity of 0.61 among inbreds. The unigenes were previously assigned to maize FPCs by overgo hybridization. From this set of unigenes, 311 (133 SNP and 178 InDel) loci were mapped on the intermated B73 x Mo17 (IBM) high-resolution mapping population. These markers provided unambiguous anchoring of 129 FPCs and orientation for 30 contigs. The FPC anchored map of maize will be useful for map-based cloning, for genome sequencing efforts in maize, and for comparative genomics in grasses. The amplification primers for all mapped InDel and SNP loci, the diversity information for SNPs and InDels, and the corresponding overgoes to anchor bacterial artificial chromosome (BAC) contigs are provided as genetic resources.

Abbreviations: BAC, bacterial artificial chromosome • cM, centimorgan • FPC, fingerprint contig • InDel, insertion–deletion polymorphism • MMP, Maize Mapping Project • PCR, polymerase chain reaction • PIC, polymorphic information content • RFLP, restriction fragment length polymorphism • SNP, single nucleotide polymorphism • SSR, simple sequence repeat


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAIZE, one of the most important crops in the world, is a diploid species with an estimated haploid genome size of 2500 Mb, and a high level of sequence complexity due to the abundance of multiple families of repetitive elements (SanMiguel et al., 1996; Myers et al., 2001). Knowledge of the maize genome will allow the improvement of desired traits by refining gene isolation and molecular breeding strategies. Molecular markers provide a means to estimate diversity, to assess genetic relationships, and to map genes underlying important agronomic traits in crop species. The utility of DNA markers for determining relationships and genetic similarity in maize was reported for DNA markers such as restriction fragment length polymorphism (RFLP) and for simple sequence repeat (SSR) (Taramino and Tingey, 1996; Smith et al., 1997; Senior et al., 1998; Bernardo et al., 2000, Romero-Severson et al., 2001).

Sequence variants of SNPs and/or InDels are the markers of choice for genotyping and mapping because of their abundance and amenability to high-throughput screening. Furthermore, SNPs and InDels can contribute directly to a phenotype (Thornsberry et al., 2001) or they can associate with a phenotype as a result of linkage disequilibrium (Daly et al., 2001). Despite the extensive use of SNPs to study human genetic disorders, there have been fewer extensive surveys of SNPs in plants (Tenaillon et al., 2001). Previous studies in maize indicated, however, that SNPs and InDels occur at a relatively high frequency in maize genes (Rafalski, 2002; Bhattramakki et al., 2002; Batley et al., 2003a), and in flanking regions of maize microsatellites (Matsuoka et al., 2002; Mogg et al., 2002; Batley et al., 2003b). Single nucleotide polymorphism discovery projects routinely identify InDel polymorphisms that can be used as diagnostic or mapping tools by analyzing size polymorphisms of polymerase chain reaction (PCR) products on agarose gel, as well as SNPs that can be mapped by a number of assay systems (Kwok 2000).

Integrated genetic and physical genome maps are essential for map-based cloning, comparative genomics, and as templates for genome sequencing efforts. A primary goal of the Maize Mapping Project (MMP) is to develop an integrated genetic and physical map of maize to enable the cloning of maize genes controlling phenotypes and crop productivity. Positioning genes to chromosomal location is important for establishing correlation with phenotype, thus facilitating our understanding of biochemical pathways and biological mechanisms controlling agronomically important traits (Davis et al., 1999; Matthews et al., 2001). The MMP has assembled a high-resolution genetic map for the intermated B73 x Mo17 (IBM) population (Lee et al., 2002) consisting of {approx}1000 RFLP, and {approx}1000 SSR markers (MaizeGDB, www.maizegdb.org, verified 4 Aug. 2005). Many of the RFLPs and SSRs were generated from EST sequences (Davis et al., 1999; Sharopova et al., 2002), and therefore, directly mark the position of candidate genes for phenotypes and traits. The parents of the IBM population, B73 and Mo17, represent the two major heterotic groups of U.S. maize germplasm, namely Stiff Stalk (B73) and non-Stiff Stalk (Mo17). The IBM population (Lee et al., 2002) includes 302 recombinant inbred lines that underwent four generations of random mating at the F2 stage. The combination of a large number of lines and the map expansion due to the random mating generations result in a genetic map resource with approximately 17 times the resolving power of the prior maize map standard (Coe et al., 2002).

To develop the resources for physical mapping, three BAC librairies representing approximately 27-fold genome coverage were constructed from the inbred line B73. HindIII fingerprinting of all three libraries and contig assembly using FPC software (Soderlund et al., 2000) are being performed at the University of Arizona (Cone et al., 2002; www.genome.arizona.edu/fpc/maize, verified 4 Aug. 2005). The IBM markers have been hybridized to the fingerprinted BACs and the BAC-marker associations have been entered into FPC. This anchors and orders contigs based on the genetic information, hence, providing an integrated genetic and physical map.

Recently, >10 600 maize unigenes were used as the sequence source for overgo probes that were hybridized to high-density BAC filters (Gardiner et al., 2004). These probes identified BACs corresponding to >9300 unigenes. The mapping strategy adopted by the MMP included, in silico correspondence, BAC pooling to anchor mapped markers, and the use of SNPs/InDels as a complementary tool to anchor unmapped unigenes has been outlined previously (Cone et al., 2002). Although a number of the FPCs containing overgo hybridizations were located on the genetic map by sequence similarity of the unigene sequences to RFLP or SSR loci on the IBM map, or by the BAC pooling strategy (Yim et al., 2002), the majority of the unigenes www.agron.missouri.edu/files_dl/MMP/Cornsensus/) are currently without a genetic map position. To speed integration of the maize genetic and physical maps we have undertaken a mapping approach to place unigenes that contain B73/Mo17 polymorphism on the IBM genetic map through SNPs or InDels, thereby anchoring the corresponding contigs.

The goals of the present research are (i) to analyze the utility of InDels identified within the MMP and address how these InDels can serve as general genetic markers for the maize research community; (ii) to conduct physical mapping of unigenes using SNPs and InDels to anchor BAC contigs to the genetic map; and (iii) to disseminate the maize SNP and InDel resources to the scientific community.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
The genomic DNA from 12 inbred lines was used for SNP and InDel discovery. Among the 12 lines, B73 and Mo17 are the parents of the IBM population (Lee et al., 2002). Four of the lines, Tx303, CO159, T218, and GT119, are the parents of two additional SSR mapping populations (Sharopova et al., 2002). The lines NC7A, Mp708, and Tx501 were chosen to further broaden the germplasm examined. The line W22R-scm2 was included because of its extensive use by many maize geneticists. Illinois High Oil (IHO) and Illinois Low Oil (ILO) were added as part of a collaboration project on application to agronomic trait analysis. The SNPs and InDels that were polymorphic between B73 and Mo17 were genotyped in 286 individuals of the IBM population (Lee et al., 2002, Sharopova et al., 2002). Eighty-three maize inbred lines that represent much of the diversity in maize (Remington et al., 2001) were used to assess InDel diversity (Supplemental Table 1). The origin and the composition of the 83 lines are described at http://www.maizegenetics.net/germplasm/lines.htm (verified 4 Aug. 2005).


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  		Supplemental Table 1. Origin of the 83 diverse inbreds of maize assayed for 173 insertion–deletion polymorphism markers.

 
Amplification of Genomic DNA and Sequence Analysis
The unigene set www.agron.missouri.edu/files_dl/MMP/Cornsensus/, verified 4 Aug. 2005) was generated at DuPont using publicly available maize ESTs to seed their proprietary EST collection (Cone et al., 2002, Gardiner et al., 2004). The 3'-untranslated regions (3'-UTR) of unigenes were targeted to design PCR primers that amplify about 300 to 500 bp, using Primer3 (Whitehead Institute, Cambridge, MA). All PCR reactions were performed using a PTC-225 thermocycler from MJ Research (Watertown, MA). The following touchdown PCR program was used to amplify all the unigenes in the same conditions: 10 cycles of 1 min at 94°C, 1 min at 65°C with –1°C increment per cycle, and 90 sec at 72°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 90 sec at 72°C. The PCR amplification of unigenes was confirmed on 2% SFR agarose gels (Amresco, Solon, OH) stained by ethidium bromide.

Large InDels, visible on a 2% SFR agarose gel (Amresco, Solon, OH) between B73 and Mo17, were further analyzed to assess the polymorphic information content (PIC) of each InDel in the 83 inbred lines listed in Supplemental Table 1. The PIC values were calculated using the formula PIC = 1 – {Sigma}i=1 to n x fi2, where fi is the frequency of the ith allele, and n is the number of alleles (Smith et al., 1997).

Loci polymorphic due to InDels detectable on agarose gels between B73 and Mo17 during primary screening were PCR-amplified in 286 individuals of the IBM population, and genotypes were visually determined directly from agarose gels. Primers that did not reveal InDels between B73 and Mo17 and gave consistent, single product amplification in all 12 lines were advanced to the sequencing phase for SNP and short InDel discovery. Nucleotide diversity parameter {pi} (Tajima 1983) and haplotype diversity were analyzed using the DnaSP program (Rozas et al., 2003).

Identification of Sequence Variants and Data Management
A single PCR fragment per unigene was sequenced on both strands. Only SNPs that occurred in at least two of the 12 inbred lines were considered for mapping to minimize sequencing errors and PCR artifacts. Sequence variants were highlighted as described in Fig. 1, and SNP interrogation primers were typically designed with a 50–61°C annealing temperature using the Oligo Analyzer (v. 2.0, IDT, San Jose, CA). Oligonucleo tide primers were synthesized by MWG Biotech, Inc. (High Point, NC).



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  		Fig. 1. Pipeline for polymerase chain reaction amplification of unigenes, sequencing, and data processing for genotyping and mapping single nucleotide polymorphisms and insertion–deletion loci.

 
Bioinformatics routines developed to process data in the SNP project required integration of existing programs with custom PERL and Visual Basic scripts for sequence alignment and quality evaluation, automated detection of sequence variants and their position in the sequence, and generation of mapping files (Fig. 1). The SNP discovery and mapping pipeline was integrated into the Laboratory Information Management System (LIMS) of the MMP (Sanchez-Villeda et al., 2003).

Multiplex Single Nucleotide Polymorphism Assays
The approach for determination of SNP genotypes has seven steps: (i) separate PCR amplification of each unigene, (ii) pooling of up to six different unigene PCR products for simultaneous detection of sequence variants, (iii) exonuclease and phosphatase digestion, (iv) multiplex SNP primer extension reaction, (v) second phosphatase digestion, (vi) resolving labeled interrogation primers on an ABI PRISM 3700 Sequencer, and (vii) analysis of data with GeneScan and Genotyper software (Applied Biosystems, Forster City, CA). Sequence variants detected by sequence alignment were first validated in the parental lines B73 and Mo17 before determining genotypes in the IBM population following the manufacturer's protocol for the SNaPshot Kit (Applied Biosystems, Forster City, CA). The multiplex SNP extension consisted of a 0.5-µL aliquot of the enzyme-treated mix per individual SNP and the extension products were resolved on an ABI PRISM 3700 Sequencer (Applied Biosystems, Forster City, CA).

Linkage Mapping
Genotypes for loci exhibiting SNPs and/or InDels between B73 and Mo17 were determined for 286 individuals of the IBM population. Linkage mapping was conducted using Mapmaker/EXP version 3.0b (Lander et al., 1987) on a UNIX platform. The SNP and the InDel loci were integrated into a framework IBM map consisting of 247 RFLP and SSR loci. Markers were assigned to chromosomes with a minimum LOD score of 15 using the assign command. The SNP and InDel loci were ordered on the chromosomes with the build (LOD 3) and place (LOD 2) commands. Map distances were calculated using the Haldane mapping function.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Optimization of Single Nucleotide Polymorphism Reactions
At the beginning of the genotyping process, it was necessary to optimize reaction conditions and determine the number of SNPs per multiplex reaction. The DNA of 286 individuals of the mapping population was arrayed into three 96-well plates. After PCR amplification, plates of different unigenes of a given multiplex group were bulked into final plates for enzyme treatment before use in SNP reactions. In a six-plex reaction, this results in avoiding enzyme treatment for 15 plates per multiplex reaction. For the number of SNPs per multiplex reactions, we tested three- to eight-plex. Under our conditions, we found that the maximum number of amplicons per multiplex reaction to achieve a good resolution of SNP peaks on an ABI 3700 capillary sequencer was six. In this case, the maximum volume of multiplex reaction analyzed on the ABI 3700 was 3 µL to keep the background noise at the minimum level. To assess the accuracy of the genotyping and mapping process, the genotype determination process was repeated at three different SNPs, in two different multiplex reactions for three unigenes; AY110160 on chromosome 1, AY110063 on chromosome 5, and AY109644 on chromosome 7. For each unigene, both SNPs mapped to the same genetic location.

Maize SNPs and InDels as Genetic Markers
Genomic regions corresponding to 3000 unigenes were screened by PCR amplification across 12 maize lines. Of these unigenes, 194 (6.4%) showed size polymorphism on agarose gels between B73 and Mo17 due to InDels, herein called large InDels, as opposed to short-length InDels that were present in sequences but did not result in visually obvious size polymorphism on 2% SFR (Amresco, Solon, OH) agarose gels. Accounting for large and short InDels resulted to an average of one InDel every 309 bp. Since the first objective of this research was to anchor the maize FPCs, unigenes showing large InDels between B73 and Mo17 were not sequenced. Rather, they were genetically mapped by agarose gel screening of 286 individuals of the IBM population.

We conducted SNP discovery on the first set of 592 unigenes sequenced in the project. The analysis showed that 260 unigenes (44%) contained B73/Mo17 SNPs, while 8% of the unigenes did not show any polymorphism among the 12 inbred lines. On average, 5.5 sequence variants occurred per unigene, with SNPs and InDels occurring every 73 bp and 309 bp, respectively. A previous study in maize found a frequency of one SNP every 70 bp, and one InDel every 160 bp (Rafalski et al., 2001). To assess the utility of SNPs as genetic markers, more detailed analysis was conducted on 470 unigenes having aligned sequence longer than 200 nucleotides across the 12 lines. The genetic diversity ({pi}, the average pairwise difference) assessed in all the 470 unigene alignments varied from 0 to 0.016, with an average of 0.009. Individual SNP are almost always biallelic, leading to a theoretical maximum genetic diversity level of a single SNP marker of 0.5. However, when combinations of SNPs present in the 470 unigenes were used to derive allele haplotypes the genetic diversity range from 0 to 1, with an average of 0.61. This range of SNP haplotype diversity compares favorably with the genetic diversity of RFLP and SSR markers, and demonstrates that SNPs can be highly informative across maize germplasm when analyzed and used at the haplotype level. Unigenes with three haplotypes were the most frequent, with every class from 1 to 12 represented (Fig. 2).



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  		Fig. 2. Distribution of single nucleotide polymorphism haplotype class among 470 maize unigenes analyzed for sequence diversity.

 
To assess the discriminatory power of InDels as molecular markers, 173 of the 194 large InDels identified between B73 and Mo17 were PCR-amplified in 83 diverse inbred lines (Supplemental Table 1). Figure 3 shows an example of PCR amplification in unigene AY104252 in a set of these inbred lines. The 173 markers detected a total of 539 alleles among the 83 inbred lines. The number of alleles per unigene varied from 2 to 6 with the three-allele class (39.8%) as the largest (Table 1). These results showed that InDels are often multiallelic markers in maize. The PIC values for these InDel markers for the 173 unigenes ranged from 0.04 to 0.76, with an average value of 0.47 (Fig. 4). For assessment of SSRs in the same germplasm, one SSR was chosen per chromosome and tested against the 83 inbred lines. The number of SSR alleles varied from 2 to 5 and PIC values ranged from 0.22 to 0.73, with a mean of 0.54, only slightly higher than that of the large InDel markers. Information on allele number, PIC values, overgo identification, and sequence of PCR primers are given in Supplemental Table 2 for each InDel marker.



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  		Fig. 3. Profile of a B73/Mo17 insertion–deletion marker in 2% SFR (Amresco, Solon, OH) agarose gel for unigene AY104252 showing three alleles in diverse inbreds of maize.

 

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  		Table 1. Distribution of allele class for 173 InDel markers tested in 83 diverse inbreds of maize.

 


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  		Fig. 4. Distribution of polymorphic information content values for173 insertion–deletion markers tested in 83 diverse inbreds of maize.

 

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  		Supplemental Table 2. Allele number, polymorphic information content (PIC) values, and sequence of polymerase chain reaction primers for 173 B73/Mo17 insertion-deletion polymorphism markers derived from 173 maize unigenes, and displaying size polymorphism on 2% SFR agarose gel among 83 diverse inbreds of maize. Accession numbers were sorted by ascending order. Overgo names used to view contigs and hybridized bacterial artificial chromosomes in WebFPC http://www.genome.arizona.edu/fpc/WebAGCoL/maize/WebFPC/, verified 2 Aug. 2005).

 
Genotyping of SNPs and InDels in the IBM Population
Using one SNP interrogation primer per unigene, we tested 260 unigenes on B73 and Mo17, the parental lines of our IBM mapping population, to validate the SNPs. The B73 and Mo17 genotypes for 246 (95%) SNPs were in concordance with the SNPs predicted by the sequencing data. Among the validated SNPs, 133 were used to determine genotypes in 286 individuals of the IBM population by multiplex primer extension assays. The genotypes of 178 unigenes displaying large InDels between B73 and Mo17 were scored also on the IBM population by direct size polymorphism on 2% SFR (Amresco, Solon, OH) agarose gels. Therefore, a total of 311 unigene loci (represented by 178 InDel and 133 SNP markers) were derived for placement on the IBM genetic map via the LIMS (Sanchez-Villeda et al., 2003) by automated data processing and mapping.

Anchoring FPCs on the IBM Genetic Map
The 311 maize unigenes genotyped in this study were mapped relative to a framework of 247 genetic markers evenly distributed across the 10 maize chromosomes, thus totaling 558 markers (Fig. 5). The genetic map spans 5630 centimorgans (cM), with the number of mapped unigenes ranging from 21 on chromosome 9 to 54 on chromosome 1.





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  		Fig. 5. Integrated genetic and physical map of the intermated B73 x Mo17 (IBM) population based on single nucleotide polymorphisms (SNPs) and insertion–deletion (InDel) markers with the fingerprint contigs of June 2004. The types of markers (SNP or InDel) used to map the unigenes are given in Supplemental Table 3
Supplemental Table 3. Unigenes mapped by single nucleotide polymorphisms (SNPs) or insertion- deletion polymorphisms (InDels) to anchor bacterial artificial chromosome (BAC) contigs on maize chromosomes and their corresponding polymerase chain reaction and SNP interrogation primers. Unigenes are listed in map position order from short to long arm of maize chromosomes. Overgo names can be used to view contigs and hybridized BACs in WebFPC http://www.genome.arizona.edu/fpc/WebAGCoL/maize/WebFPC/, verified 2 Aug. 2005).

Chrs
 Accession
 Overgo
 Type
 Forward primer
 Reverse primer
 SNP interrogation primer
Chr1 AY110314 CL24410_–1 SNP TCATCCTTTTGCAACCACTACTCA GCATAGATTGGGTTACCAGTTGGA ATCACCATGAAGCAAGCA
AY110401 CL809_1 SNP TGCACCTGCTCCACAAGTACAG CGTAACGTGTCATTATGTTCTCCG GTCAATGTTGACGGCGGT
AY108650 PCO072650 InDel AGTCGCAGAGCCACAGGTTC AACATGGAACAGAGGCAGACGTA –
AY103844 PCO132874 InDel catactgaagggaacggacgac tgcaactgagttcagaacagtaagc –
AY107207 PCO128140 InDel agatcatccacagaggagaccatc aagcagatgcgcatatcaatgag –
AY109929 CL4401_1 SNP TCAAACTGTCTGAAGATCCCAATG TTGACTGCTGAACCACGATTAGAA AAAAAATCGGCGTCTCTACT
AY110052 CL33106_1 SNP TTGTTTACGAGGATGTTGAGCTTG GAACAACAACTGCGAGATGGC AAAAAATATCGCCGTGCCCGA
AY110028 CL302_1 SNP GAAAACAATCAGGGAAGAGGAAGG GTTCACAGCACCAGCATTTGTTAC AAAATCAGATCGCATTGAACACACT
AY106592 CL14065_1 InDel attttgatgggaaacagtccttga ccaaatcctaaagacactgccatc –
AY106736 PCO116807 InDel GACAAGCAGGATGAGAGGAAGAAG GCACAGTCTTAAATTGCACACCAC –
AY110640 CL833_1 SNP ACAAATTCTACAGACACGGGAGGA AATTGCGCTTCGAATAGACTGAAC AAAAAATAGAGTTAGACACTCAGAC
AY110632 CL5778_1 SNP ATCAAGACCAAGGTGCTCGTCT GCACACCTTCTGAATCCTTCAAGT CCAAGGTCCACTTCACCGC
AY110393 CL5132_1 SNP ATATTCATCGTCGTCGTCAAGCTG GACCCTCCAAGATCGGACCTC CTACAAGTTGCAAACCATGGAAAT
AW400087 si707051B04 InDel AAGACCAGAGAGAGCCTGCAGTTA ACATATAGTTGAATCCGCAGCTCA –
AY108090 CL62610_1 InDel agctggttcctactttggctcttt ttacctactgagcgaatggtagcc –
AY110330 CL946_1 InDel GCAACGACGACTTACCAAGAGTTC CTCATGCGACAGTAGGTAGGAAGC –
AY110241 CL8900_1 SNP AACATCACCAGGTTCAGCTTCTTC TTCGGAAATATCCGCACTAAAAGA AAAAGGTTGACTCTCTTTTACATCC
AY112354 CL34571_2 InDel TTTCCCCACCAATACAAACATCTC ATCATCAGCGTCCTGAAAATGTGT –
AY106088 PCO099462 InDel cggagaccggtagaggaaattaag tttttgcgagacataaagtggtagtg –
AY109646 CL1599_-1 SNP GGGGACGACACTGATACTACTGCT TTAACAACAACGGTTGGACACTTG AAAAGTGGTGACCACGACGACAG
AY111680 CL11621_2 InDel TACCACATTCAGGTATTCGGGTTC TAAGCCAAAGATCTTGAGGGGAAG –
AY109678 CL18174_1 SNP CGGACCAGACAATATTCCAAGTTC CTTTCCCAAATAATTCGTGGTCAA AAAAAAAAATTCAGGTACATGACGGAGACATC
AY110396 CL18635_1 SNP AACCATACAGTACAGGAGGGTCCA CAAAGGTTGGATATCCCAAGTGAA CAAAATGCTTCGAAGCAAGCC
AI855190 si603009F08 SNP AGATGTCCAGGTACTTGAGCCG GAGCCTGTGGCTGGAGAAGAT AAAAAACCTCACCGAGCTGCC
AY112092 CL14065_1 InDel GTGAATAGCTTCAGTTCTGCGTGA GTCTCCTCAGCTTGACTCTGGC –
AY109499 CL2801_1 SNP GTTCTTTCCGTCGATGTATCCCT GCTATGCAGAGCCTCCTCGT AAAAAAAAAAATAGCGGTTGATTGCAGATTG
AY110566 CL45690_1 SNP ATCAGGTTGATTTCACAGCAGTTG CAGCTCAATCATCGAATTCTTGC AAAAAAAAGATGGCTGCTCTCCAGGTA
AY110296 CL11152_1 InDel GCGTGCTGAGACTTACCTGGTATT AACGCAGCATAATCTGGAAATTCA –
AY104360 PCO094248 InDel GATGGAATAGCTGGCATCCATAAG ACAGCTGAGGTAAGCACAAAGTGA –
AY111153 CL12437_1 InDel CCCTTTCGGTAGAATCAAGGATCT AGCCAAGGCAGAAAGTCCAATATC –
AY107847 PCO116807 InDel gagaagacccactactcagggacc aatacacagatcgagggagcagag –
AY111834 CL67476_1 InDel CATTACACACGTAGGCATCAAACC TGTGAGATGACGAGGATGAGACA –
AY110356 CL22447_1 SNP GCAGTGGCCATTCTCGTAGTATTT AGTCTAGGAAACACTTGGGAAGCA GTCTTGACGAACCACCACT
AY110191 CL21485_1 InDel GTAGAGCGTGTAGTCCGTCACCTT AATCCTCCTCCTCCAGCCAC –
AY110159 CL11942_1 SNP AGAAAGAGGGGAGCATATAAACCG TCACGTGATCTGAAGTACCCATACA TTCTGCATCACTGGGGAGAC
AY110313 CL31053_1 InDel TGTGAACATCTTGAGCTTCTTTGC CGACATTGGATACACTGAGGAAGA –
AY110349 CL11942_1 SNP AGAAAGAGGGGAGCATATAAACCG TCACGTGATCTGAAGTACCCATACA AAAAAATTTTCGTATTGAATTGGTTGCAAG
AY109506 CL7587_1 InDel GCTCTACTCCAACCTCAAGGACAC AAACATAGTCATCGGCAGGACAGT –
AY110452 CL23944_1 SNP GTCGAGGATTACTCTCCCTCCTGT CATCGCAACAACAGGATCATAGG CACCGGATGCCAAGCTGTT
AI665421 si605011A07 InDel GTGAAGGTCTTCCACCACGTATTC GATTCTTCGGGCTGATAGTACCCT –
AY110019 CL471_1 SNP GTATGGGAACTGGCAATAATGTGG GAAACTGGGGCTAATAGCAAGCTC AAAAAAAAAAAAAAAGAATCAAGGGTTCTAAGTACAAAAC
AY108625 PCO087393 InDel CAAACTGTTGATCTGGGTGTTGAG TGGCAGTTGACACTACAGAAAATCA –
AY111936 CL22280_1 InDel GTCTCTGCATCGGCATCATAATC TTGGTTTACCGAATCTCACCTCAT –
BE639426 si946033A04 SNP AGGTACTCGTCGGAAGATTCTTTG CTCCCTCCTGACCACCTCACT ATTTATCTGATCATATGAGGTATTGCCATTT
AY108136 PCO095183 InDel ACTCCTGACATTCCTGAGATGGAG GCAAATGTTTGCCCTAAGTGTGTC –
AY109834 CL7866_1 SNP TACAGTTTGCAGGAGCAAGACAAG ATTTCAGGTCCTTCATTCCATTTG AAAAACAAATTCCGCACAGCTGAATCAATA
AY108778 PCO075654 InDel ACTGGGCCACTATGAGATTGTAGG ACATTTTGCACGCAAATAGATCCT –
AY110426 CL31361_-2 SNP GAGATGCCGTAGACAGTAACAGCC AACTACATCAACGGCAACGTGTC AGGACAGCTGAACCGTATGATGATTGA
AY110479 CL16056_1 SNP GGGCTAGACTGCTGAAGGTAACAA ATTCCAAGTATCAGTGCCGAACTC CAAAATCAAGAAGAATCTAATCATCAGCTAA
AY106606 PCO135617 InDel agacgatagcattggatcctcttg gcatccctgcttaaagagtccata –
AY110160 CL7400_1 SNP ACACCTCTTTGACGAACCTCTGTC CAAGGACCTCATCCATGGTTTC AAAAAAAAAAAATGGAACTTAATGAGGTGCTTTA
AY111767 CL62610_1 InDel gatcaacgtcaaggatacgtgctc ttagctagcgaccgtggtactcat –
AY110983 CL778_1 InDel GCTAAGGCAGCACAAATGAATCTT ACCTTAGACCGTCTTCGGCG –
AY109916 CL11745_1 SNP GTTATCAAGAGGCTGAAAGTGGGA GTGTCTGATTCTGTGGTGGCTATG GAGCATACACCACCTTTAG
Chr2 AY108849 PCO091677 InDel AATCCGCTTTCCTAGCTCTAATCG GAACAGCCTTATGTTCTTCTCGGA –
AY109692 CL301_1 SNP ACAGAAGTATGCCATGGGCTAGAA GCTCACTGGAACGTCTTTCTTTCA TGGTCCTCAAAAGTCCATTCATACTTG
AY109603 CL18778_1 SNP AGCCTGTCCTTCGTTACTGACATC CATCTTGTGGGTTTGGTGCAG AAAAAAAAAAATAATGCTCCTCCACATCCTGC
BE640649 si945021E10 SNP CGGTATGTCTCATTTCCTATTGCC AGCAACAGGCTCTGGTGTTATCTC AAAAAAAACCGGTGAGGTTCACTAGTCATTA
AY109516 CL5703_1 InDel TGTCGTTTACGGTTACTGGTCCTT ATTCCATGCATTCATCCTGACAC –
AY112075 CL52019_1 InDel AATGCTGACTTGTAGAGGGTGGAG GCTTGGTATGAGGAGGCAGAAAAT –
AY112131 CL12768_1 InDel TCGTCCTCCGCTTGTTACTATCTC CAGCTTTACGCAACTACAGAAGCA –
AY106040 PCO125510 InDel AGCGACACTTTCTCCAGTTACCAC GTAACCTCCTTTGATGTAGTCGGC –
AI745852 si605074C02 InDel aagtcctcaggaatggtcagtgtc tttgggagctgctaggattatcaa –
AI920398 si603020H12 SNP CTGCTTTCATCCATCACAATTCAC CTTCAAGGAGATAAGGGAGCGACT AAAAAAAAAAAAAAAGATCACGTTAAGAGAAACAAA
AY104214 PCO099155 InDel CTGTAATACAATGCGTGCGCTAAG GCCAGCAAGATCATAATCTCCATC –
AI691686 si606023F08 InDel AGTCCCGACATCATTGTGACTTTT GAGGCTGGAGTATTCTTTGGCTTT –
AY110266 CL17243_1 SNP CCCTCTTGAAACTAATACTGCCGA GTCAAAGAGGTGATCACAGTGGTG TTTGCAAGGTAGAAATTACATAATC
AY106712 PCO129934 InDel CCTTCGTGCTCAGGTACTACGTCT GCGTTACAAATATGACATTACAAAACCA –
AY103590 PCO098412 InDel ggtcgatttgcttctgagttgttt tgttctatgtgtgttttcctgtgga –
AY107218 PCO140184 InDel actacgaatccagcgacaagaaag acctcatttatcaggctgaaagca –
AY111434 CL58207_1 InDel aacaagatcagatcggcgaagtag cttccccataaatgcaatccataa –
AY112466 CL10221_1 InDel TTGAAGTTCTTGTAGAAGTCCGCC GGCATATGGAGTACAAGGTTGAGC –
AY110485 CL1980_2 SNP CAAGATTGAGACTGAGCTGACCAA AAGCCTAATAGGGTGAGTTGGAGC TGCACCACAAACCCCTTTTCAG
AY104386 PCO096835 InDel cgagttcgacaccagctacatct gacgagcgtagtgcaagatgatta –
AY109687 CL45878_1 InDel TATAGCATCATTGCCATCACGTTC GAGAGAAGAGAAGCACCGCAAC –
AW681281 si707094H09 SNP CAACCATACAAGTTTCCTCTCGCT ATGACGAATTTGCAGATCATTGTG AAAAAAATGCAGTATTTATTTCGTAT
AY110336 CL54827_1 SNP TGACAAGTTTATCTGACGGAGGCT GGAATTATTCAAATTGCGCAGAG AATTCATCCATCGGGCTCTCAGAAA
AY105915 PCO131593 InDel GTGCATCAAGTTCTCTCATCCCTT TCCACTCTAAACATCTCTCTGAGGC –
AY109981 CL18929_1 SNP CTTGCTGCATCTCTCACATCAAGT TCTCCTTTATGCAGTTCACCAACA CAGAGTACTACTTCTCTAGGTATTC
AY108804 PCO063114 InDel GGTGTGACAAATTTCAGCAGTACG TTTTTATCAGCAAACAAGCAGCAG –
AY110410 CL7336_1 SNP GGTCTGTAGTGACACGCTGAAGAA TGTTATTATACGCAACATGCGGTC ACGCGCCTCTTCTTCGGT
AY109722 CL2887_1 InDel TGCAAAAGAATTGGTTGTAAATGG AGGCAGTATTCAAAGGCATTTCTG –
AY109917 CL13558_2 SNP ACTGGGAGTGTGCTAAATGAGGTT GGCGATGTTGGAGAAACACTATTC AAAAAACCATGCTCTTCCTGCGACTA
AY109583 CL18551_1 SNP TCAAAGTCTGATTCACGAGAGCAC CCACCTTACAAATCTCTTGCTGCT TGGATCTTTGTTTGCAAGATGGGC
AI668346 si605031A07 SNP CAGCCCTTATTTCATTTCCATCAG TGATGGTAGATTTGCCAAAATTCA AGGTGGCCTCTTATCATC
AY109645 CL4356_2 InDel AGCATCATCTTTGAGGTTACCAGC CTGCGTTTCTTCTCCTGTTCATC –
AY107622 PCO102097 InDel atttcatttttcttccccctcttg atggatcaaacggatctttaagca –
AY109575 CL1708_1 InDel CTACATCAAGGACGACAGCAAGAC TTGATGGACATACTAACCAACCCC –
AY109592 CL34788_1 InDel AACACTTGCCAATGACCAATCTTT TCTAACCTTGTCAGCCTCAAATCC –
AY110467 CL42453_1 SNP GATCTTCCAATCTGGCTGTTCATT AAGTTCTACGTCATTGGTGGGAAA AAAAACCCCAGCCATGAACTGATGTATA
AY111236 CL31103_1 InDel TGCTTGTTTTGTTGCTGATCCTAA CTTCTCAAGGCCGAGCAGAC –
AY109586 CL860_-1 SNP TAAAACTTTCGTGGGGTTACCCTT CATTCAATCGTTGTTTGCATTCTC CAAATCTCGATGACAGAAACAAACG
Chr3 AY109549 CL1501_1 SNP AACTCAACGTCCTCTTCATCAACC CCATACAGTCCTGCAATTCTTGG TCCCTGTTTGTTAGTAATGCTGTTTT
AY107363 PCO107756 InDel AAGAGATGACGCCGTGAGAACTAC GAGGATGTGTTTTGCTGGATTCTT –
AY109870 CL2099_1 SNP AGGAACTGATCCAGAAAGCTGCTA AAGCATATTACACCGTATCGGAGG AAAAGCAGCAGGTGGCGGA
AY110403 CL5194_1 SNP CGCTGATGCCTGTTAATAGTGAAG ATGCAACCGGAAATATGTACTTGA CCTTCCGTCCACTGCACAT
AY108004 PCO068796 InDel CGTTCTAAAATATCCGGTCTGCAT CTAGATCAGATCATGTTTGCACCC –
AY106948 PCO141323 InDel ccaggatgcttccttcagtacagt tccatatatacacatgggcaactga –
AY110151 CL4217_1 SNP AGGCATGTAGGAATTGACCGAATA TTACATCGTAAACAACCAGACCGA AAACAGCACCAACAAAGATCATCAAA
AY110297 CL25043_1 SNP CCACCGTACATAGAAACAGTGTCG CTGTTATCTGCCATAAAAGGAGCG CTCGTACAGAGGACTCAGCTC
AI714716 si606017F01 SNP ATCCTAATGCTCACCACCGTAGAC AAAACAAAGCTGGAGGCGACTT ATTATTCATGTTGGTGAGAATTAGTGGTAG
AY110352 CL12989_2 SNP CATATCACGCACACTACCACAACA TTCTGGAACTACCGAAGAAGATCG AAAAAAAAGATGAAGCTCAAGAAACAATACATG
AY112215 CL7356_1 InDel CAGGCAAGCTGCTAGAACTTTAGG TCCAGTCGTCTCAACTAGTGTTGC –
AY111507 CL7343_1 InDel TGGTTGTTGGTAGCACTAACTGGA CAGAGGGAGTATAAACGTTAGCTTCAA –
AY111541 CL56108_1 InDel TGCTTCCAATTCCAACCAGTAGAT CGGCACGAGTGTAAATCTGGTAAT –
AW179494 si618046E03 InDel ACCTCATCTACCTTGCTCATCAGG TAGCCCTAAAGCAATGCATCAGTA –
AY106230 PCO119904 InDel CACATGGTACAAGGAGAACAGGTG AAGTTCTCGCAGTGGTCTTGTTTC –
AY111296 CL62634_1 InDel TGAACACCTTCTCTTCGAGGTACG GCTGTAGGAATTCGGCACGAG –
AI770873 si606059F03 InDel GGTGGGTTGGGTTTGACTACTATG GAAGAAGAACCGCAGTACAAGGAC –
BE639846 si946038G12 SNP CTCCTACATCCTCGCTACGGC AGAATCACCAACCTCTCAGCACTT GTCTTACTGCACTGATGGG
AY105347 PCO089398 InDel gtggccaactatatccagaagacg acagattaaagccaaacaaaccga –
AY110055 CL10277_1 SNP TTTGGAACCCTGCATTATACACCT GAAACAGCAGTGCAATACCACAAC CCAAGTAGGATACACCTGTCG
AY110812 CL13054_1 InDel acactcaaatccctccaatccata ttgctgcgtcttgatgatctgtat –
AY111125 CL6928_1 InDel GGTCAACGTCGGGAAGAACAC CGGGTGGCTTTTGAATGTAAATCT –
AI770795 si606058D10 SNP TGCCTGGGCTTATAATACCATCAT GGAACTTCCCGGTGCTACTAGATT AAAAAAAAAAAAAAAAAAAAAGGTCAAGTTGTTAGCTTTG
AI920617 si618016E09 InDel TTCTTCCACCTCTACTAGGCACCA TGTTCAAAGAATGAACATGATGCG –
AI745823 si605077F08 InDel GTCGAATTCTATGGGAACTCGTCA CTGCTGCTTCGTCCCTGATG –
AY107303 PCO142509 InDel TCAAGGATTACTCCAGCTACGTGA GCACGAGCTAGTCTCGAGTTTTT –
AY104511 PCO117256 InDel CTCCCTTCTCAAACATCAGGAAGA TCGTCGACTCAAACTTTATTCCAA –
AY105849 PCO098078 InDel GAAGGGTTCATTTGATGCTGAAAA TATTACAGGTGTCCCTCGGTTGTG –
AY109934 CL64932_1 SNP CGTACAACTTCACCATCAGCATGT CAGGTCCCGTTCTCGAAGTG AAAAAAACCATCAACGACCTGGAGC
BE639338 si946021A07 InDel GGAACTAGCCATCTAGTTTGCGTT GAAATGAACATATTAGGCGAAGCG –
AY111254 CL29988_–2 InDel GGAACTAGCCATCTAGTTTGCGTT GAAATGAACATATTAGGCGAAGCG –
AY110567 CL7839_1 InDel GAAGTGTGTGATGTGTCAGACGTG TAGAAGACACATGAAGCAGCCAAC –
Chr4 AY109715 CL333_1 SNP GTTATCAGCGACTGATGAATGTGG CAAAGTTTTATGATTTGAAACGGGA AAAATATGTGGGGCTAAGTCCGGT
AY107692 PCO146629 InDel caaagttcattgaatcggtgaaaa ctgaaacttatttgctcacctatgatct –
AY110398 CL736_1 SNP CGATGGTTGTTGCTTTCAGTGTAG ATTTGTAAACATGGTTGGCGTTTC TGTATCACTCATGTTAGTTTGTTAAAAA
AY110253 CL871_1 SNP TACTGGATCCTCAAGAACTCCTGG AAAGGTCATGTTCTTTGCATTCGT AAAAAAAAAAAAAAAAAAAACACAAATCTTCAGATAAAGC
AY110573 CL1596_1 SNP AGATGCTCAACAGAATGTCCATGA ATAGCGTGTTTTACATGAGGACCC TGCTCACACCCATGATGTTACC
AY110290 CL17864_1 SNP ACGATTGCTCAGCAGCTCTAATCT GGGATCAGTGTACGGGTCAAATAC GAATAGAAAGTTAGTCAACAACTTAAAAAA
AY110872 CL65845_1 InDel actcaattaggacaagtgcagcct acgctcatactggcatacctcact –
AY110562 CL26590_1 SNP GCTACTATTGACACGAACGAGGGT AGATGTACAAGCTTGGCAAGAAGC AAAAAAAGAGACGAGTACGGCCAC
AY110355 CL11612_1 SNP GGAGGCAAGTTGATGGTTCTTATG TCGCTGTTCTCTGAACAAGCAAT TGTTCAACTGGTGGAGCCACA
AY110310 CL4235_1 SNP GGCTGTCCACGGTCAAGAAC CAACCCAATAATGCTTCATCCTGT ACTGCAGGACGGCAAGATCAT
AY108440 PCO119336 InDel gatcatctccccattgttcttgtc catattctatggaacactgcacgg –
AY109534 CL50733_1 SNP GCCTGCAGATGGCTACTGAACTAT TTGTGGATGGAAAGCATGTAATGA GGGCTTCCTCGCCTGATGAATTT
AY110185 CL32627_1 SNP TTTCAGGATAATGGCCTTGTAGGA GGCGTTCAGAATATAAGCCAGATG GGCATTCTTGGTATTCTGAAAAAA
AY104784 CL32627_1 InDel gagaagaagactaacggcacgaaa gtgcgtctggtacacaacacaag –
AY112127 CL11712_1 InDel ATCAAGCACCTGAAATGCTACACA GTTTTAAGCAGTGGGTTCATGTCC –
AY110631 CL19053_1 SNP CCTGACAAGGTCCTCTGGTAGCTC TCATGAACTTCCTCGAAGAGAACG AAAAAAAAATACCTGCGCTCAGAAACAAG
AY108096 PCO136722 InDel CGAGGAGAAGAAGCCTTACATTGA TCATTCATGTAGCAACGATAACGG –
AY105971 PCO076386 InDel AACCTCACCTTGTGGACTTCTGAC CCATACCACATTTGATTATTGCCA –
AY110989 CL5869_1 InDel GGTTTGCATTTCAGTATCGTAGCC AGGAATGACGTATGGATTCTCTCG –
AY109980 CL10245_1 SNP GAGAAATATGGCCTGGTAGTGGTG CAGTCCTTTCCAAACAGTCAGAGC GTTGATGAAAAACAACTCTTAGATAA
AY110949 CL12681_1 InDel TGACTGTTGCTGAGCCTGTTACC GTTTCACTGACTCGAGGGTTCG –
AY110170 CL65658_1 SNP CATCTCAACTAGCCGTGAAGGAAG GGTCGCCTCCACCTCAGACT AAAAAAAAAAAATCTCACAGGTACCTATCCTT
AY106822 PCO104784 InDel gaagacccagatgatctcactgct gtacaaatcgattgacagtggcag –
AY109933 CL27530_1 SNP AAGGACCAAAACCGTTCTCTTCTC GAAGGAGAGGTGGGTACAACAAGA TACTGACCTAGAAAGGTGGCTCCT
AY109730 CL2227_ 3 InDel gttggaagtccggagactggat gcaaacataggtcccaaatggtaa
AY110064 CL27003_1 SNP TAGACGTTATAGGAGCCCCTCCAG CGTAGGAAACCAACCCTTTCTTG AAATGCGTAGCTCATTCTTGTGG
AY110231 CL852_1 InDel GAATAAGTCGTACTCCGGCACAGT CCATCTAGATCTCGTGCTACCGAT –
AY108514 PCO088312 InDel CTCTGTTCTTGTTTCCAACGGACT AGGAGCTTGGCTCTCTTGAAACTC –
BE997321 si947017G02 InDel AGCCTCAATCTCAGAAGAAGAGCA CCGATCTATCAATGTGTTTGTGGA –
AY106414 PCO109372 InDel tgtgacacaccagcaagtcctaat tatgaggtcacatctgctatggct –
AY109668 CL6375_1 InDel GATGTTTGCTGGTTTGTCTCTCCTT TTAACAGCTTTTGCGACGGATAA –
AY109859 CL6375_1 InDel ACGCTAAAGACAAGCAACCAAAAG CCATGTTCAAGTGTTCAACTGGTC –
AY109611 CL37807_1 SNP CTACCGCCATACTCCTGTACGTCT TGACAAGGCAAAGCACAAATTTTA GGACAGTACAGATGTTCATCAGGA
Chr5 AI676903 si605046E08 SNP CGCTTGTCAGTCTAATACGCCTCT GTCCAAGGCCTGATGTCAATAATC AAAAAAACATGGATCTGCAGGATTC
AY110625 CL1125_2 SNP AAAGGTTGAGTTGCTTGGTGAGAC CCCACTAATATCAACTCGGCAAAG AACCATTACACCACTCCCATGTTCC
AY109758 CL1976_1 SNP CACATCTACTTCTGCGGTTTGAAG ACTGCCAAAAGCAACTGCAC TCGATTCAATTTCAATCATGTGTTTGTCTCC
AY111819 CL21419_1 InDel gcagtagaacccgatcagaaagaa atatgtcaactctccccttatgcg –
AY106472 PCO062666 InDel ccacacataaaacacagacccaga ttttgacacacaaagcagcaagat –
AY109733 CL5075_1 SNP AGGGTTTCTGTTTAGGCAGTTTCC ACGTGAAACGATGAAATTGAACCT GGATGAGACTTAATTCTGGTTTGACTC
AY106398 PCO135705 InDel GCTTTCCAGAACATAAATTGGTGG TGGTAACAGTCGTCAAGTACCACAC –
AY111142 CL9565_1 InDel AGTAGATCATGCATCCATCGTCAG TCAAGGAAGGTGAAACCATTAGGA –
AY109606 CL4591_1 InDel ACAACAGACTCTGACAGCTTGCAC GATTCATTCTTCCGGGTTGTACTG –
AY109995 CL9999_1 SNP CTTTCCTTCGTAGCCTGTGCTAAA CCAGATGACCGTGTTGAGTATGAG AAAAAAAAAAAAAAAAGCATATCACATTTAGGCCC
AY104079 PCO134626 InDel AGGCAGTACAGAAGAGGAGCAAGA AGGGAGTACATCGCAAGTAACAGC –
BE639933 si946044B06 SNP GTACAGACATACGAGGGGCACAG CACGAGGTTAGTTGGTTCTGTGTG TGACAACCTTCTGCACAT
AY110906 CL14503_1 InDel GCAACGTCACTTCAACAGATTGTC CAAACAATGAGGCAGAAGTATGGA –
AY105029 PCO082331 InDel ATCAAGGCCTAGGTCAGAACTGTG TTTCTGGGACGGAGTACATACCAT –
AY105906 PCO103687 InDel accctgtctacttcttgacgaacg agacagacagacagacaccaggaa –
AY109532 CL1742_1 SNP GGACTATTTGGTGGATTTAGCAGG CGAAAACTCTATGTTTAATTGACACCT AAAAAAAAGCTGCGTTGTCCTCTCTATATAT
AY109682 CL2492_–1 SNP GCTGTTCATGTACCAGAGCTTTCA AGATGGATGTACAGAGCTCAGGCT AATGGATCCGGACGGGGCTC
AY106487 PCO060271 InDel GCTTTCCAGAACATAAATTGGTGG TGGTAACAGTCGTCAAGTACCACAC –
AY108049 PCO078116 InDel CCTGATCGAGCTCAGGAGGAT AGTATTCTGGCAAATTCCCCATCT –
AY112581 CL11475_1 InDel GCAGAGAGCATAGGTGATCGTTTT CTGCAGTGAGTTTAACAAGACCGA –
AY105069 PCO099796 InDel TGGGTATGTTACAATTCCTGCAAA AGGATGAAGAAGCTGTTATGGCTG –
AY110825 CL16923_1 InDel gccactgttgctttcatacttgtg aaccagtggcaaatatccgataaa –
AY110063 CL863_1 SNP TTTCAGGTATTGTGTTGTCGTGCT ATAACATCCGAGTCAGAGGAGCAG AAAAAAAAAAAAAATGGATGCATCGAGTGAGTC
AY109938 CL19962_1 SNP GACACACAAGTTGCGATCCATTAG CGATCAAGCTTTTGCCATTCTCTA AAAGGGAACTCTTGCTCA
AY105176 PCO111982 InDel agcacactgatgcactaatgaagg aacatcagaaaccacctaccagga –
AY110369 CL10716_1 SNP CAGATGTAAGCACAGGTGATCTGG GGTAGAAGGGTCTTGGAGTGGAGT AAAATCTTCAGTACTATGGTTAGCATTGGTA
X64446 siX64446 InDel CTCAAGGTGTGGTACGTGGTCAG GGCCATGGCACCAGCTAGTAT –
AY110413 CL8857_1 SNP ATCAACACAGTCCTTGTGGAAACA ATATGCTGTTGCCTTCCCTGATTA ACGCAGTACGTCTATCTTGAATTTTTAGTT
AY110182 CL16596_1 SNP TGGTCGAACCTTCTTGGCTTACTA GGGTCTTGGCCTTTTAGAATTCAC AAAAGAAGTTTGCAGAACAGGC
AY105910 PCO106969 InDel TACAGAGGTTACCCACGAGGATTT CGGAGTAAGACTCAAAAGCAACAG –
AW065811 si614062B02b InDel TGGTCTCGATGTTAACCGTACTGA GGCAAATAAGTATTCCCCTCCATC –
Chr6 AY106921 PCO069699 InDel AGTTCCTCAACGAAGAATCCACTG AACAGACGGTCGCTTAATCTTCTG
AY110100 CL1035_1 SNP ACTAGCAGACTGAACTCTTCGTGGA ATCAAATCAAAGGAAGCCCACAG GTGTGTTTTAAAGCTTGATGCTATATAATA
AY110213 CL1225_1 SNP GATCACGTGGTCATTGACAAGAAG GTAACTTCGGTCAACCTCTACCCC AAAAAAAAAAAAAAACCTCATACATGGTGG
AY104775 PCO061308 InDel GCATTTCCTGGTGGTGAAGTTATC AAGCTCTTGAACCGCATTAAGGAT –
AY111964 CL9771_1 InDel ATGGTACATGCATCACCTGAGAAA CCCCTTCTCTGTTCATGGTTAATG –
AY105303 PCO075489 InDel ctcctcatctgggtcaccatct caagcatgagataaactggatgga –
AI737983 si606044D05 InDel ATCAGATGTTACCGGTTTTGATGC CATGTCTAAAGGTTGGAGGGAGAC –
AY104742 PCO152525 InDel ggcatgatctgaagaagtttttgg agacgtccgaacatctgtcatgta –
AY107240 PCO146525 InDel GGGAACTGGATCATTCTAGTCGTG CTTCTCCTGCTCCTACTCCTGCT –
AI665560 si605012H03 SNP GAGCACTAGGATGGACGATTGC GTTCTTCAGGAATTATCAAGGGGC AATTTAATATATACTTTGGCAAGAACTGAGA
AY110542 CL4202_1 SNP ATGTACATGGTGTCGCAGAACG TCTATCTATCGCCATCGAGCAAT AAAAAAATGGTGCGGGCCGAC
AY110435 CL57831_1 SNP GGTGCACCTTCTTGACGAGG ACGGCACAAAGATAATGGGCTAC AATTCAACAGCACAGTCGTTGG
AY108243 PCO134814 InDel gagatggcaagaatgatagcctgt aaagagcaaaaacagagagggagg –
AY110260 CL26716_1 SNP GCTTCCTGGTAAGGTTTGCTGATA GAGAATTGGCTTTTGGAACATTTG AAAAAAAAATCAACCGTCCCATTTCATTGTTAA
AY109873 CL6571_1 InDel GCTTATGGAGATGTTATGCCAAGG GAAGGGAATATTAACACGGCTGCT –
AY110873 CL10452_1 InDel TCAATCGATGGTCAGAGTAGGTGA ATTTAACAAGGTGAGGCTGTTGGA –
AY110050 CL392_1 SNP CTTCCTGACAGGTACATCGACCAT GTAACTGTTGTTCCGGCGACAC AAAAAAAGATCTGGGCCTATAGAGATG
AI737346 si606039C09 InDel ACAGAATAGCTGTGATAGGCACCC GCAATTCGATACCAAGATCATTCC –
AY112405 CL10251_1 InDel GATCAGCTCACGTGCAATAACATC ACCTTTGTGCTATAGTGGGAGGTG –
AY104923 PCO144363 InDel TGTTTTGCTTCATGACATGTGTGT GCTTCACAGACCAAATTCAATGTTT –
AY105728 PCO142478 InDel AAAGGCCTTGAGAAGACAGGAGAT GTACATCAGTTGCATAGGGTCCGT –
AY110400 CL15423_2 SNP ATGAAGAGCTGGATTCAAGATTGG CGAGTTGAGTTCAGTTCCTAAGGG CAATGAATGTGTTGGCTGTTGGGT
AY104289 PCO114336 InDel CTGTTGGAAATAAACCTGCCATTC TCGCCTAACCTAGACCAACATCAT –
AY109797 CL10211_1 SNP CTGGTAAATTGGTAGGGCAACTGA CCTCCCACGACCTCTTCGTC AAATGCCAAAGAAGTGGGGCAATGT
AY109996 CL2349_1 InDel ACTCCAGTTTAGGCACTGATTCCA CAGTAGAGAATTGCGCACACCAG –
AY106902 PCO068526 InDel TGCTATGACAAATGAATGAGCGTT GACATGTTTTGCGCTTGAAGAAAT –
Chr7 BE056994 si945036H05 InDel aggactacaaggataagctggcg ctatctccttggagaccctggagt –
AY104465 PCO087457 InDel CTCCCTCAACACACTTCTCAGGAT TATGAGAATGGCAGTCGAAAAACC –
AY108063 PCO143084 InDel CATAGCGCAGTTGATGTGTGATGT GATTTTTACACCAAGCGACAGGAA –
AW308691 si618080H02 SNP GCAGTTGTTAGCAAGAGGGAACTC AATGAAACTGTCAAAGGAGGATGC TGTCTTCTTCGCGACTTCACC
AY109536 CL5684_1 SNP CTCTAAGACGAGCTGCTGAAAAGG TGAAAAGAGAAAGGAGCTGACCAA AAAAAAAAATGTCTATGCACCCACAA
AY105589 PCO140163 InDel CTCAGCATTGTAGGCTCGTCAATA AGACAATGTCAACTTGGTTTCTCG –
AY110473 CL10705_–2 SNP ATAGGGTCTCACTCTCATGGCAAG CGAAGTTCATATCTTCGGGAATTG CCTTCTCCGTCTGCCAGC
AY110576 CL21448_1 SNP TGCTCCAGACTGACAACTCAATTC TACATTACAAACATGGCAGCTGGT AATCCTTATATTTGGGACTAAACTTGT
AY112356 CL4745_2 InDel taccccatacattttcctcgctaa ttcacaaaggctctgttgagattg –
AY109809 CL29132_1 SNP TTTAGAACTTCTGGTTTTGCTCCG GTGATTCTGAGGAAGATAGCCAGC CAACTGATGCTGAGATACCCTGAACAA
AY105254 PCO115023 InDel agtttgcaaatctgctcaagatcc ttgagttcggagtcaggggtaac –
AY109968 CL376_1 SNP ACAGCACTCTTCGCCTACAATTTC TCATGCTACCAAAGAAAAGCTGAA AAAAACAGTTGTTCAGTTTGTCCATACA
AY110374 CL34066_1 InDel AACACTGTCCGTTCCAGATCATTT GGTTGCTATAGACAAGCACTGCAC –
AY106517 PCO071075 InDel cagcatgttcagtccaagtgctac ccccttcaattacattacatgactgc –
AI987507 si614054G01 InDel AACCACTTGAATTAACACCACGAAA GAGATGCGAGAAGGGAAGTCAATA –
AY107797 PCO101826 InDel AAGTGCTTGATTTCGTTACTGCCT TTTTTGAGACCTTGGAATGTTTGTT –
AY109644 CL12102_–1 SNP GTAGGCGGTCTTCTGACTTTCGTA ACAAGGGAGAGAGCTGCCATC AAAAAAAATCGCGAAGGGGACTCC
AY108039 PCO102751 InDel ggtgttactggactacggttgagg ttaattcacacagtgcatggacaa –
AY110023 CL41684_1 SNP TGACCTGAGCTTTTAGGGTGACTC CACAATTACTATCGACTCTCACGGTC AAAAAAAAAAAAAAAAAAAATATGGTTTCATCCACCTTGA
AY110439 CL194_1 InDel CAGGCGCTCCATATATAAACTTCG GCCATGAAATCAGTCTTGAGGTGT –
AW267377 si829001F06 InDel GGGATCTACGTACCCCAAGCTTTA CGCTGTCTCCTAAAGAAAACTACACAG –
AY104976 PCO136133 InDel gagagaacggtgttctttgacgac cgacagggatgagctatacaaacc –
AY103848 PCO061754 InDel atctgccttatcctcaccttcctc caagcaaccaacccagtactacaa –
AY109703 CL4455_1 SNP CTACACTAGGGGTGCAGGTGAGAT TCCAATCTCCAAGAGTCTTTTATTCAA GTTATTCTCAGGCACCCTTCTTGCC
Chr8 AY111865 CL16874_1 InDel TCCTCAGGATGGTCAGGTACAAG ACCGACACTTGGTTGATGAGAGAT –
AY109699 CL7702_2 InDel CAAGGTTTCTTATACCATGCGAGG CAGCCAATCTGATCGAAAAGTTCT –
AY111073 CL39692_1 InDel ttccaagagacattacacgcagaa ctcatccatcataactgtccaccc –
AY111071 CL51477_1 InDel aagcccacctgatgatacatgact tattagggctgatggagctattgg –
AY106269 PCO138212 InDel CCAATTACACCACCGAGATCTACC AACCGCTTAAACAGCACGATTAGT –
AW244963 si687017E04 SNP
AY110450 CL27631_2 SNP ATGGGTATGTACAGGTACAGGGCT GCATACTGTTTGAGTCCAGCTGTT AAAATGACACATGCAGGTTCAATGCTA
AY103821 PCO084551 InDel GGAGTTGGGTTTTGCCTCCTATAC GATAAGCATGCAACAACTTCCAGA –
AY109740 CL2942_1 SNP TTGATCAGGATTCGTGTCTATCCA GGACAGCACATAAACCTTGAAATATG TTTGTTGGTGACTTGTTTCTTATGA
AY105457 PCO149506 InDel AATGCAGGAAATACAAAGAAGGCA CCATTCAACTTGGAGAACATAAAAGG –
AY110032 CL34676_1 SNP CCTTCTTGATCTATTCCCTGCTGA AAACATACCCAATATACCTCCCGC AAAAAAAAAAATGGCTGTTGTAAAAGAGAGAA
AY109626 CL1397_1 SNP GATTACAGAGCACAAGTTCGGACA GTGACCATGAACAGCTTGTCGTT AAAAAATTTCAGTTTCTCACCTGAAGGAAAAGAAAG
AY108084 PCO147505 InDel TAAATGCCATGCTGAAGATTTCCT TCCAAATGTAAGCTAAGTGTGGTTCA –
AY110056 CL10675_1 SNP GTACTCTCCCTTGTTGTAGGGCAA TGGCGTGTCTACCTCTGAAATACA AAAGTTTTGAGCACCTGTATC
AY104017 PCO119482 InDel GGAGATTGCCCACATGTACAAGAC GAACATTAACCAAAGAGCACCGAC –
AY104566 PCO130617 InDel GATTCTGACAGTTTCCGTCAACCT GGACAACCCGCATCAAACATA –
AY109883 CL42920_1 SNP TCTCTTCTTGGTTTCAAGGTCCAC TCCATGCTCCAGTGCTACGATA AAAAAACTTTCTTATCGGTGCCCC
AY107140 PCO079694 InDel ggaaggaaaacttctccccctagt ttccctgctgtaaaatatctggttg –
AY111629 CL9311_1 InDel ctagtattcgaggaactgagcgga cgttgtcagaagaaatgcagattg –
AY110569 CL10335_2 SNP TTTAGCAAGCGAACATGTCTGAAG GTGGTGTGCGTATCAAGAAGCAT TTAAGAATGTACTGTAGTTAAAACTGGAGAA
AY110539 CL13530_1 InDel CAGCCAAATAACTGATCTGCAATG ATTCTTTGGTAGCAGCAGGTCTTG –
AY109593 CL7515_1 SNP GCCAAGAATGATACCAAAACGAAG CTGTCAATGCAGGCTTATGATGTC AAAAAAAGAGTATGAGGATTATCATGTGGGCAT
AY110053 CL2103_2 SNP ATCCTCCTTGGGTTTACTCTCTCG CAAGCTCACCAAGATGTCGGA AAAAAAAAAAAACAATCCCTTCAATCTGGCG
AY110127 CL4812_1 SNP GCAGTAACCTTTGTAGGCGACAGT TCAATAGCGAAGCATTCAGTTCAG AAAAGAATGTAATTATGCAATGTAACTGTATGC
AY103806 PCO111734 InDel AAGAGGTGAACATTGGTGCTAAGG TTTTTAAGCGCAACTCCAGATGAT –
AY109853 CL2096_2 InDel GTAGAGCAAGGCCTGTGAATGG TCAGAAGCAGTTCAATCACACACA –
Chr9 AY104252 PCO110637 InDel ATTGCATTAACCATTGATTCCTGG CAAACCACTCCAAACTACATGGGT –
AY109531 CL1198_1 SNP CCTGCCAAGAACTGGGAGAAC CACCGAACAGCAGGGATTATTTAC AAAAGCGCGAGATCAGGGG
AY109570 CL1864_2 InDel CTCCTTGATCATCTCGAAATAGGG GTGATCACTGATTTCTGGTCATGG –
AY107559 PCO061815 InDel TGTTGTAAGAAGCATCCTGTTCCC ATCGCGATGCACTGGTTCTG –
AY109816 CL0_–2 InDel GCCATATTTCTTTACGACGACGAC CTTTTAGTATGCGCCTAGCATTGG –
AI812156 si605086B11 InDel CCTTGGTACACTACCTGCTCCAGT CAGCATCTTGAGTGCTTACCCATT –
AY103770 PCO100327 InDel ATCCGTCTTACCCTTATCCCAAAA GCGCATTTCTAGTTTTTAGCAACG –
AW257883 si687063E06 InDel CTTGTGGTTGTTTCTCCTGATGTG CAAGCATATACAGCTTCTGCTCCC –
AY109764 CL5759_1 SNP GCTTCTACGTTCTCCCAGATGC TTATTACAATGCATAGCTCCCACG TCTTGGTTAGATTTTGCCTTCCCATTCTGTT
AY110217 CL9357_1 SNP CCCACCCGTTGTTTATAACTCTGT ACCAGGGCAAGATCAAATCTGTTA AAAAAAAAAATGGTTGAGGAAAGAAAGA
AY109792 CL22067_1 SNP GACTTGCACCTTTTCAGAACGAAC CCAGGGCAAGAATAACTACAACCA TCTTGTACATCTTTCACAACGGTG
AW215946 si687046G05 InDel gcctctgtaggatggatgtgatct tatgggcattgttaccccttgtag
AY109550 CL135_2 InDel CTTCGATGCAGTGTTCTCAGTTGT AGCATTCGGCACAATTTTGTACTT
AY110141 CL39723_1 SNP AACCAGAGAACTGCTGTTGATTCC AAGAGTCCAGGAACTTCAATGCAA ATGAAATGGCCATACATGGCAGAGTA
AY109819 CL3318_1 SNP GATGAAGCCTAAGCAAGAGGTGAA CTTCTTTGCAGCCTCGTTAAACAT AAATTTGGATTTACAGTTCATTGTTGTCCTTCT
AY109543 CL854_1 SNP ACATGAATCCAAGGTGGAACCTAA TTGTGTCTACTGTCAAATCCTGGC CTTGAAACTCTCCCACGCTCAC
AY110382 CL32455_1 InDel AAATTCATCACGCTGATTTGGTTT CAAGGCTGATAATATCTGGGCATC –
AY106323 PCO084517 InDel CGAGAACACAGGGAAATCTGAAGT TCCTGCAATAAACTTGTGTAATGTGTG –
AY106075 PCO127444 InDel ttcatcaaccagaggaagaggaac gcgtccggacaaactatgctatac –
AI901738 si618009H12 InDel GCATCCCATATTCCCATTGACTAA CCATGGAAGTGGGAGATGCTAC –
AW216329 si687049E06 SNP CTTGAACCATGCTAAGCTTCATCC ATCGGTTTTGCGAAGTACAAAAGA AATTAACCCTCAAGAGACTTCATATTGAA
Chr10 AY110060 CL35548_1 SNP TAGAAAATGGATGACCATCCCAAG TCAGAAGGCACAGGTCTATGAACA ACTACCTTCAGAACGAAAGC
AW330564 si707029B01 SNP CGCACTATGAGTCGGCTGAAC CCACTTGGTACTACACACGCATTC GTGTGTTTTTAGTTTATTATTGTGCTA
AW225120 si687024F01 InDel GGGAATGTATAGTTTCTGGGCTGA ACACCAATAAAGCGAGTCAAGGAA –
AY110360 CL2011_1 SNP TATTTTAAGTTCGGCCTTTCATGC AAGGATACAAGCGAAAGAATGCTG AAATTTCCTGGTTACAATTTATCTT
AI795367 si605011H02 SNP GCATGGATCATAATGTTCTCCACA GAGGGTGGATACTTGACTGCTGAT TCGTGAAGCTTCAACATGGCGAATA
AY109994 CL768_1 SNP GACGATCTGTCTGTTTCACGAAGA TATGACTTGCCACAAAAGCAATTC GAGGGCAAAGGCAGACAGA
AY107726 PCO062847 InDel ATGCAGATGCAGTGTGAGATGTTT GGATCATATGGGATTTCCTGTTCA –
AY105746 PCO099975 InDel TTCACACTGATTTTGAAAGGGGTT ACAAGCCATACAATTGTGCATGTT –
AY110411 CL242_1 SNP ACTCTACTCCTCGCGTCAATGTTC ACAACGAGATAATTCTGGAGCGAG AAAGAGAGATCGCGGACTGGG
AY110248 CL4753_–2 SNP TGTCCTGTTCTCCATTTGTTGTGT CATTGCTAAACACTGGGTCAGTTG AAAAAAAAAAAAAAATAGGGCTGGGGGCA
AY112073 CL6800_1 InDel AGATTGCTAGCTTTATTCCGGTCC GACCATCTGGAGGAGTACAGCAG –
AY111178 CL5772_1 InDel CAAGGATCGTCACCTTCAACTC CCCATAGTCCATACATACATGAAAGA –
AY110514 CL948_1 SNP AAGAATGCTGAACCAAGAGTTTCG CCCAAACTCCAAACTCAATTCAAA GCTTGTAACATTCCCTAATTAATGTAAATTTATG
AY109584 CL2436_1 SNP GAGGAGAACAAACTGCTCATAGGC TCCTTCCTACGGTATCTCAGAAAACT AAAAAAACACTAGCCAAACTGTCA
AY109920 CL36143_1 SNP ATCCAGTGATCCCCTAAAATCCTC CATACTTTTGGATGACAGTGGTGC AAAAACAAAAGGTTAAGGATTCCGAGCT
AY109876 CL1675_1 SNP GTGCAACGTTCTGCTTCCTTTACT TGGAATGGGAGACGTGTCTTTAATA AAAAAAAAAAAATAATGCCATCGTACAAGAATGGATG
AY106635 PCO086427 InDel CTAGCTAGGGCTGCCCGTGT GTGCAAGCCGGGAAACAATAC –
AY110365 CL38215_1 SNP GCGATGCCACTACAGCTACAACTA ACTCTATGGCAAGACCCATTCAAG AAAAAAACACTGCACCAACACCTG
AY109698 CL4149_1 InDel ACGAGCATCACGAGAAGAAGG AAACACACGTAAATTCACATTATTCAA –
AY108476 PCO126344 InDel CGGATTTCACTCTTAGGTTGAGGA CCACGTTCTCATCTGGGTTAAAAG –
AY110634 CL3121_1 SNP TACTACAACAGGCTAAGGGCGGTA CAGGACGTGCCCTCAAAGAC AAAGCACCTGCAGCTTCATGTT
AY106733 PCO107634 InDel ATACCTGCTGGGTTCTTAGAAGGG CAGCCTCGATGAATAGGAACAAGT –
AY107822 PCO087182 InDel CAGTTGCAATCTTAGGGTACGAGG CACAATCCAAAATGTTTTACATCCG –
AY110167 CL14540_1 InDel CGACATCTAGATCAATCCAAGCAA ACTCGCTTCGACTCCGACAG –
AY110016 CL16508_1 SNP GGCTCATCTGGAACGAATACAAAT CCAAATCCCTGAGCTCCAAAG ATGGCACCATATGCCGATG
AY109829
 CL35744_1
 SNP
 TGACATAGCTCGTCCAACTGTAGC
 ATGCTGCTATTGGTCTTCTTCTGG
 CCAATAGTATGCCTCACGATAATCATCAAA

. Bars are bacterial artificial chromosome (BAC) contigs detected by two or more adjacent markers. The BAC contigs associated to unigenes are noted (ctg#). Unigenes that detected several BAC contigs are underlined. Off-frame markers are in italic, and markers that are not listed in Supplemental Table 3 are the framework markers used to assign SNP and InDel markers to maize chromosomes.

 
The mapped unigenes were evaluated to determine which might serve to anchor contigs to the genetic map. The 311 unigenes mapped in this study provided unambiguous anchoring of 129 contigs by association with overgo probes used for BAC hybridization (Fig. 5). The unigenes that did not provide unambiguous contig assignment resulted from overgo hybridizations that either failed to identify a contig, or identified multiple contigs (underlined markers in Fig. 5). The multiple contigs identified by a single overgo may represent duplicate gene regions, or contigs that should be merged during manual editing. In 33 cases, two or more adjacent unigenes identified a single contig (Fig. 5), allowing 30 contigs to be oriented relative to the genetic map. The remaining three contigs correspond to off-frame markers, and therefore await the precise placement of theses unigenes for orientation. These results allowed a physical-to-genetic ratio to be calculated for 33 regions distributed throughout the 10 chromosomes, from one contig on chromosome 5 and chromosome 9, to seven contigs on chromosome 4. The genetic-to-physical distance varied from 23.1 to 1143 kb cM–1, where the physical distance is measured in the FPC metric CB (consensus band) units, which is approximately 4096 bases per CB. Thus, genetic-to-physical distance showed a variation of almost two orders of magnitude. It is worth noting that the genetic map of IBM is expanded nearly fourfold relative to a standard genetic map (Coe et al., 2002; Sharopova et al., 2002); therefore, these size estimates would be expected to be about four times greater on a standard genetic map.

The amplification primers for all mapped InDel and SNP loci and the interrogation primers for SNP loci are provided in Supplemental Table 3.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study SNP discovery was performed on 592 genes across 12 maize inbred lines. The results confirm those of earlier reports (Tenaillon et al., 2001; Rafalski et al., 2001; Batley et al., 2003a) that both SNP and InDel frequency in maize is very high (one SNP every 73 bp and one InDel every 309 bp). This level of polymorphism is greater than other crop species examined to date such as rice (Oryza sativa L.) (one SNP per 268 bp; Shen et al., 2004) and soybean [Glycine max (L.) Merr.] (one SNP per 328 bp; Zhu et al., 2003). Although only a single {approx}300 sequence alignment for each gene was obtained, 92% exhibited sequence polymorphism, indicating that an SNP strategy will permit genetic mapping of essentially any maize gene of interest. Although the maize genome contains many duplicate genes and gene families, these did not present a major problem in genotyping assays because the same primers were used for the amplification of genotyping fragments as used in the initial sequencing. Primer sets that amplified multiple genes were detected by the presence of multiple fragments in screening before sequencing or near identical paralogs were identified by the presence of heterozygous bases within inbred lines. Primer sets yielding either of these problems are excluded from use in SNP genotyping assays.

The integration of genetic and physical maps, which is done by locating the FPCs on the genetic map, is a complex task in eukaryotic genomes such as maize. Through the use of SNPs and InDels as complementary tools to BAC pooling and in silico anchoring in the MMP, we have placed 311 unigenes of maize onto the IBM genetic map, among which 293 unigenes (94%) are assigned by overgo hybridization to one or more contigs. Of these unigenes, 167 (54%) correspond with overgo probes that hybridized to fingerprinted BACs, providing unambiguous locations for the corresponding FPCs on the genetic map of maize. Contigs associated with the remaining 126 unigenes were directed toward the manual editing pipeline for anchoring. Only 18 unigenes (6%) failed to identify contigs, probably because of the failure of overgo hybridization. These results clearly demonstrate the power of SNP and InDel analysis to provide genetic markers for maize genetic and physical map integration. The study further shows that in addition to the traditional BAC-end sequencing, SNPs and InDels can be used to orient contigs or to confirm contig orientation on a transcript map.

Thirty-three contigs, anchored by two or more unigenes each, provided the relationship between physical and genetic distance at the anchored points for all 10 maize chromosomes. The present results show that the genetic to physical distance in maize varies more than an order of magnitude. That 311 markers identified 33 pairs of linked markers within the {approx}1600 contigs as of June 2004 suggests that much of the recombination in maize may occur in small physical regions. This agrees with results from studies on recombination rates within the bronze1 (Dooner, 1986), anthocyaninless1 (Brown and Sundaresan, 1991), and waxy1 (Okagaki and Weil, 1997) loci. Because the IBM population underwent successive rounds of intermating, differences in recombination rates between regions should be magnified as each region accumulated recombination events in each generation. Thus, a more nonuniform distribution of recombination is expected for the IBM map than for standard, one-generation maps (Sharopova et al., 2002). The anchoring and ordering of more contigs will give us a comprehensive comparison between the genetic and the physical map distances in maize.

The unambiguous anchoring of FPCs to the IBM genetic map is the main focus of our SNP project. We continue to sequence and map unigenes through SNPs and InDels with the objective of mapping 1000 unigenes to the IBM genetic map. The maize FPC map has now advanced to the state that we can focus our sequencing and SNP mapping efforts solely on unigenes that identify single contigs. This will allow all mapped loci to serve as contig anchor points. These data are being integrated into an iMap for public display www.maizemap.org, verified 4 Aug. 2005). In addition to the detailed information on the SNPs and InDels provided in Supplemental Tables 2 and 3, a SNP database identifier was included in the bioinformatic resources of the MMP http://www.maizemap.org/cgi-bin/SNPDataDisplay/SNPData4.cgi, verified 4 Aug. 2005). Such resources are significant aids to map-based cloning of genes with agronomic or biological significance, to marker-assisted selection, to genome sequencing, to understanding of genome structure and functions, and to comparative genomics in the grass family.


   ACKNOWLEDGMENTS
 
This research was supported by NSF grant DBI# 9872655. The authors would like to thank Bertrand Lemieux, University of Delaware, for DNA of the IHO and ILO lines. Names of products are necessary to report factually on available data: however, the parties neither guarantee nor warrant the standard of the product, nor imply approval of the product to the exclusion of others that may also be suitable. We thank Karen Cone and Georgia Davis for shared genome mapping and Linda Schultz and Ngozi Duru for technical assistance.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Research was performed at the University of Missouri, Columbia, and was funded by NSF Grant DBI# 9872655 to the University of Missouri.

Received for publication December 6, 2004.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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