New Marker Generation: Approximately 8,000 new molecular markers

The expanding genome sequence coverage, and new S1-D2 EST sequencing, has offered an opportunity for significant expansion of molecular markers. We have generated five new sets of markers from these data: SSRs, InDels, STS (3-primer), CAPS and DCAPS. Since SSR markers are based only on C. reinhardtii (CR) sequence (no S1-D2 genomic sequence is available), we tested 100 selected at random, to see how often a useful length polymorphism would occur. The result was 62%. Only 10 primer pairs were tested for the other marker types, since they were based on comparative sequence analysis between CR and S1-D2; 73% worked as expected. Except for the 140 primer sets tested, researchers are expected to order their own primers; in general we aimed for one primer set per 100 kb, however in most cases saturation is much higher. Within the Stern laboratory, April Melvin, with participation from Jocelyn Handley and Linda Rymarquis, were responsible for the testing of primers. Qi Sun1, a Senior Research Associate at the Cornell Theory Center, developed the spreadsheets after discussing specifications with us. Below we summarize the results, and then give more details for each of the marker sets. Summary of Results Marker Type SSR InDel STS CAPS dCAPS ALL MARKERS Number Tested 100 10 10 10 10 140 Success Rate 62% 80% 60% 80% 70% N/A Predicted number of useful markers in spreadsheet 696 160 645 740 6,534 8,775

SSR Markers
Methods The CR genome (v2.0) contains many simple sequence repeats (SSRs) as shown by the repeat masker track on the genome browser. These repeats are highly polymorphic in other species and serve as a rich source of diversity for marker generation. In order to determine if SSRs are polymorphic in Chlamydomonas, Qi Sun developed a program to identify SSRs and design primers to regions surrounding these repeats. Primers were generated with lengths of 22-28 nucleotides and melting temperatures between 55 and 70C. Each primer pair was constrained to a 5C maximum difference in melting temperature and a predicted CR product size of 100-300 bp. To reduce the occurrence of multiple products within a PCR reaction, primer pairs were BLASTed against the CR genome. The following scoring system was developed to discard primer sets likely to yield undesired products.
1

To whom primer generation correspondence should be addressed; email: qisun@tc.cornell.edu.

Locations with an E value <0.2 received a score of 1. Locations with 0.2<E value<10 and homology extending to the 3 end of the primer received a score of 1. Homologous regions with 0.2<E value<1 not meeting the previous criteria were each given a score of 0.25. Locations with 1<E value<10 and homology extending to the second most 3 nucleotide were given a score of 0.25. Totals for each primer were calculated and primers scoring 3 or above were discarded. If multiple primer pairs were generated for the same SSR, the one with the lowest overall score was kept. The resulting primer pairs were organized into a spreadsheet including location on the scaffold, primer sequences, and predicted CR product size. 100 primer sets were tested to identify polymorphisms within SSR regions between CR and S1-D2 (CC-2290). PCR reactions consisted of 3.1 L 80% glycerol, 0.25 L formamide, 7.0 L Go Taq Buffer (Promega), 0.5 L 10 mM dNTPs, 0.3 L Go Taq or Taq polymerase (Promega), 1.0 L of each primer (10 mM stock), and 25 ng of template DNA, in a final volume of 30 L. DNA was prepared using a protocol modified from Rochaix, Meths. Enzymol. 65:7852. The PCR program was the same as that used by Rymarquis et al.3, with 35 cycles instead of 40. Annealing temperature for each reaction was approximately 3C below the melting temperature indicated by the primer manufacturer (IDT). Ten L of each PCR product was electrophoresed in a 1% agarose TBE gel. If no polymorphism was evident, the sample was also examined in a 3% agarose TBE gel. Results Out of the 100 primer sets analyzed, 62 yielded an identifiable polymorphism between CR and S1-D2. Of the remaining primer sets, 4 had no evident polymorphism, 18 did not yield an S1-D2 product, 1 did not yield a product in CR, 1 had an incorrect product amplified, and 15 yielded multiple bands in either CR, S1-D2, or both strains. To determine if the markers with multiple bands were due to a lack of stringency in the BLAST criteria, all 100 primers sets were manually BLASTed against the CR genome. There appears to be no correlation between success rate and the total number or length of homologous regions, indicating that the BLAST criteria are sufficiently stringent. The number of homologous regions per primer pair ranged from 3 to 31, with an average of approximately 10. Markers exhibiting multiple bands had 4 to 20 homologous regions, averaging 9 per pair. The length of homologous regions for each primer were also considered and ranged from 14 to 23 nucleotides, with 14 nucleotides being most common.

InDel Markers
Methods
2 3

Method available upon request. Rymarquis, L., Handley, J., Thomas, M., and Stern, D.B. (2005) Beyond complementation. Map-Based Cloning in Chlamydomonas reinhardtii. Plant Physiology 137, 557-566.

Insertion/Deletion (InDel) markers were developed by Qi Sun, using CR genome v2.0, the combined CR EST data (projects 1024, 1030, 1031, 1112, 1115, 832, 833, 874, 894 and 963), the S1-D2 BLAST database, and recently generated S1-D2 EST data contributed by JGI. The InDel regions considered for marker generation were insertions in S1-D2 EST sequences 20-250 bp in length, when compared to the CR ESTs and genome. Primers were constructed and BLASTed against the CR genome using the same criteria as for SSRs. The spreadsheet consists of 200 primer sets and includes scaffold location, primer sequences, and predicted product size for CR and S1-D2. Results Ten primer sets were tested using the PCR reaction conditions outlined for SSRs, with annealing temperatures of 53-56C. Eight primer sets were successful and yielded the predicted size shift, 1 lacked a S1-D2 product, and 1 showed no polymorphism. The release of new S1-D2 ESTs has made InDel marker generation a rapid and efficient method for mapping. PCR reactions are straightforward with two primers and have a higher success rate than SSRs because the sequence from both parents is known.

STS Markers
Methods Sequence tagged site (STS) markers were generated using CR and S1-D2 EST data, as well as the CR genome to locate intron-exon boundaries. Polymorphic regions were identified between strains, and primers were generated so that one primer was unique for each of CR and S1-D2, one primer site was conserved in both strains, and neither primer set spanned an intron. Using the three primers in a single reaction generates products differing in size for CR and S1-D2, as previously reported4. Primers were BLASTed against the CR genome using the same criteria as used for SSRs. The spreadsheet consists of 1,075 STS primer sets and provides scaffold location, primer sequences, and predicted product sizes for CR and S1-D2. Results Ten primer sets were tested using the same conditions used for SSRs, with annealing temperatures ranging from 54-58C. Six markers were successful, yielding polymorphism between CR and S1-D2. One of the six exhibited an S1-D2 product larger than predicted, which may be due to an intron not found in CR. Two markers did not show a polymorphism, and two exhibited multiple products. With the 60% success rate, approximately 645 STS markers in the spreadsheet will be effective tools for mapping.

CAPS markers
Methods
4

Kathir, P., LaVoie, M., Brazelton, W.J., Haas, N.A., Lefebvre, P.A., and Silflow, C.D. (2003). Molecular map of the Chlamydomonas reinhardtii nuclear genome. Euk. Cell 2, 362-379.

Cleavable amplified polymorphic sequence (CAPS) markers were produced using CR and S1-D2 EST data. Primers were developed to amplify regions containing restriction sites in either CR or S1-D2, or at different locations in both strains. The resulting spreadsheet contains 926 possible CAPS markers with restriction sites for commonlyused enzymes including XhoI, PstI, BamHI, HindIII, EcoRV, DraI, EcoRI, SalI, and XbaI. PCR conditions were the same as those outlined for SSRs, with annealing temperatures of 55-58C. Digestions consisted of 5 L PCR product in a final volume of 10 L, and were analyzed in agarose gels. Results Ten CAPS primer sets were tested. Eight markers exhibited clear polymorphism between CR and S1-D2. One marker showed no polymorphism and one did not yield an S1-D2 product. With an 80% success rate, it is expected that 740 CAPS markers will perform as predicted.

dCAPS markers
Methods Derived cleavable amplified polymorphic sequence (dCAPS5) markers were also generated using CR and S1-D2 EST data. Single nucleotide polymophisms between the two genomes were identified and primer sets generated. For a given primer set, one primer contains a single base mutation that will convert the SNP into a restriction site in one of the strains. The mutation was constrained to bases 4-2 from the 3 end of the primer. The 3 end nucleotide of the primer was never altered, to allow for proper amplification. Since the mutation creates a restriction site in the product of only one strain, a 20-30 bp size difference is evident after the digestion is analyzed in a 3% agarose gel. Qi Sun generated 9,335 dCAPS primer pairs with restriction sites for the same enzymes listed for CAPS markers. Scaffold location, primer sequences, necessary enzymes and undigested product sizes are listed within the spreadsheet. PCR and Digestion conditions were the same as those used for CAPS. Results Ten markers were tested and seven were successful. One primer pair exhibited no polymorphism, one yielded no S1-D2 product, and one produced multiple bands in CR and S1-D2. With this success rate, it is estimated that 6,534 dCAPS markers will be useful for mapping.

Neff, M.M., Neff, J.D., Chory, J., and Pepper, A.E. (1998). dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J. 14, 387-392.