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Solgenomics.wur.nl

Guideline for selecting seed BACs
The BAC stabs (or rearrayed BAC clones) are made directly from frozen glycerol stocks. Therefore, streaking the stabs and picking a single colony are strongly suggestedfor confirming marker—BAC associations and selecting seed BACs. However, if theBAC stab is made from single BAC clone/colony, it will be clearly stated in the shippingdocumentations.
Confirming the hybridization of genetic markers with BAC clones:
1. Verifying marker-BAC association by sequencing BAC clone using a customizedprimer Select two candidate seed BACs: If possible, two seed BACs are suggested for eachmarker based on the estimated clone size. The size of the selected BAC should be > 100Kb, if possible. If BACs with large inserts are not available, BACs with smaller insertsize of 60 to 100 Kb or unknown insert size from plate 1-260 could be selected. (note:Plates 1 - 260 of the HindIII library are from a ligation yielding larger insert clones). Verifying by direct sequence BAC using a customized primer: A customized primer isdesigned close to, but not overlapping, the overgo probe in the marker sequences (Fig. 1).
A distance of approximately two to three hundred bases between the overgo probe andthe customized primer is ideal to provide sufficient sequence for alignment confirmation.
Because most of the SGN markers are developed from EST sequences, SGN tool“Intron_Finder” should be used to predict splice sites for facilitating the primer design.
Then, the BAC can be sequenced using the customized primer. One end (SP6 or T7 forthe HindIII BAC library) of the BAC clone should be sequenced to confirm the quality ofthe BAC DNA. If the BAC end sequence is good and the sequence from customizedprimer fails, an alternative customized primer should be designed and used forsequencing. High quality BAC sequence obtained from the customized primer should bealigned with marker sequence. If sequences align perfectly or nearly perfectly, then theBAC clone is considered verified.
Figure 1. designing the customized primer based on the marker and the overgosequences. The splice sites are predicted using the SGN tool “Introns_Finder”(http://www.sgn.cornell.edu/cgi-bin/tools/intron_detection/find_introns.pl).
Intron_Finder predicts the splice sites based on the sequence alignment oftomato and Arabidopsis homologs with a changeable criterion of blast e value.
2. Rehybridizing BAC clones using overgo probes developed from the genetic markers.
DNA from BAC clones anchored to one genetic marker should be prepared from the overnight culture with 12.5 mg/ul chloramphenicol. After BAC DNA is completelydigested with HindIII (overnight or >5 hrs), DNA fragments are electrophoresed andtransferred onto a Hybond+ filter. Then Southern hybridization with the overgo probedesigned for the genetic marker is conducted. Positive BACs are the plausible candidateseed BACs. The sizes of HindIII digested fragments used for FPC are currently availableupon request and will be available on sgn.cornell.edu in the near future.
The Southern hybridization can also be performed on filters containing NotI digested DNA (good for evaluating the insert size on CHEF gel) or BAC colony stampsfrom the overnight culture.
3. PCR amplification of genetic markers from the BAC clones PCR primers can be designed from the sequence of a genetic marker (Note: For some markers, primers are already available in SGN database). Results from PCRamplifications using BAC DNA as template can be used to validate the BAC-markerassociation. Please be aware that most of the genetic markers are developed from tomatoESTs and PCR amplification might be problematic due to introns, which could bepredicted using the SGN tool “Intron_Finder”.
Verifying the location of BACs on chromosome
1. Fluorescence In-situ Hybridization (FISH) 2. Mapping in tomato IL lines (CAPS mapping in tomato IL lines) Search SGN for known information: If a candidate seed BAC has a confirmed marker-BAC association. The original genetic anchor marker information should be searchedfrom SGN database for any known polymorphyism between M82 (S. lycopersicum) andS. pennellii. If known information is available, the original CAPS information could beused for IL mapping.
Identifying sequence or digestion polymorphism: If there is no polymorphisminformation available in SGN database, the BAC sequence (BAC ends or sequence fromthe customized primer) can be compared with tomato ESTs or other sequenced genomesto identify conserved coding sequences. PCR primers should be designed fromconserved coding regions. DNA fragments from S. pennellii and M82 (two parents of theIL populatoins) can be amplified and sequenced to search sequence and enzyme digestionpolymorphism.
IL mapping: DNA of the chromosome ILs is amplified and the products are digested witha selected enzyme. If the BAC maps to the chromosome, we will see the S. pennelliipolymorphism on one or more of the ILs. If the sequence maps to a differentchromosome, the S. pennellii polymorphism will not be found on the ILs and then theentire set of lines will have to be amplified in order to map the BAC.
(Note: Dr. Dani Zamir will supply the genomic DNA of S. pennellii, M82 and ILs uponrequest. zamir@agri.huji.ac.il) Selecting seed BACs:
1) large insert size (>100kb, if possible, or with unknown insert size)2) reconfirm by sequencing, overgo hybridization or PCR amplification3) BAC physical location are tested using FISH or mapping in IL lines3) in a valid FPC BAC contig (optional).
Selecting the first extension BACs:
1) minimum overlap (5 to 10 kb) with seed BACs anchored to nearby genetic markers. Overlap should not be too extensive to avoid redundant sequencing.
2) confirmed by FISH or IL mapping on the same chromosome as the seed BAC (note: BAC end sequences are available on SGN, which should be used to verify the size of overlapping regions and pick the extension tilling path BACs.)

Source: http://solgenomics.wur.nl/static_content/solanaceae-project/docs/Guideline_v2.pdf

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