Locating markers from the genetic to the physical rice map

1
Locating markers from the genetic to the physical
rice map
2
Review Linkage map of molecular markers (Genetic
maps)

• Shows order map distance of various molecular
  markers along a linkage group (chromosome)
• Generated by linkage analysis

SSR marker
RFLP marker
3
A physical map of the rice genome that is aligned
with genetic maps is available
Genetic map Cornell
Physical map
4
Terms and facts about the physical map

• Chromosome assembly of contigs
• Contig assembly of clones
• Clone the only physical entity (BAC or PAC
  clone)
• A veeerrry small chromosome piece sequenced
  100 kb DNA
• Assigned a GenBank accession ID
• Gap introduced by
• Recombination
• No data (assembly unfinished or data non-existent
  yet)
• Anchor marker molecular marker that is
• physically located on a clone (sequence is there)
  
• has an assigned cM location
• 1 cM 247kb (from 420Mb japonica genome / 1700
  cM total)

5
Molecular markers from genetic map can be located
into physical map
Genetic map Cornell
Clone AL772426
Anchor marker C460
6
Locating a marker in the physical map enables you
to..

• Determine gene(s) in silico that may be
  responsible for the phenotypic effect of a marker
• Gene structures in the region of clone where
  marker sequence can be found
• Identify more markers that can be used for fine
  mapping
• Use existing markers
• Design STS primers new markers

7
Where are these map databases???

• Gramene
• http//www.gramene.org/
• TIGR rice genome map
• http//www.tigr.org/tdb/e2k1/osa1/BACmapping/descr
  iption.shtml
• Arizona Genomics Institute rice physical map
• http//www.genome.arizona.edu/fpc/rice/
• RGP (Japan)
• http//rgp.dna.affrc.go.jp/

8
Sample problem 1

• You mapped a gene for salt tolerance (SALTY) 5 cM
  away from marker RZ649 in chromosome 5. What SSR
  marker(s) can you use near this region?
• Use Gramene comparative maps resourceCornell
  SSR map

9
Gramene output

• Further exercise
• Which clones contain these SSR markers?
• How far apart (in cM) are these clones?

10
Sample problem 2

• You have a clone of a putative salt-tolerance
  gene, sequence known (SALTY)
• Candidate gene approach
• You want to map it by RFLP/SSR combination
• Strategy
• Locate the sequence in the rice genome in silico
  for better targetting (BLAST is a good tool)
• For sequences gt 50 nt, 50 identity
  hybridization signal
• Locate SSR/RFLP markers flanking the location
• Gramene or other databases mentioned
• Additional Tips
• Also use markers anchored to clones
• Use standard-spaced markers for good genome
  coverage

11
Step 1. Locating the clone

• Find similar sequences in the rice genome (IRGSP)
  using BLAST
• Predict the genes in the clone in the region
  where there is significant BLAST hit using
  FGENESH
• Ensures that you are hitting a functional gene
• Identify the gene
• Search the Protein Family database for homologous
  proteins with the predicted gene using HMMPFAM
• Ensures you are working with the target gene of
  interest

12
Gene as a data text file

• FASTA Format the most common format
• Loosely formatted text file containing a
  descriptor line the sequence data
• Saved as a text-only file
• Best to use Notepad or a text-only editor
• Most sequence database centers offer this option

13
Sample FASTA file

• gtSALTY gene Oryza sativa putative salt tolerance
  gene mRNA,complete cds
• TTCTCTCTCTCTCTCTTCTTCTTCTTCTTCTTCTCCATATCTCCTACTCC
  TCGTGAAGATCGATCGACCATCGGCAATTT
• CATTCGGTAATAGTTAAGCTAAGATCAAATCAAGATTGGCGAAACGATGG
  AGATGGTGCTGCAGAGGACGAGCCACCACC
• CGGTGCCCGGGGAGCAGCAGGAGGCGGCGGCGGAGCTGTCGTCGGCTGAG
  CTCCGGCGAGGGCCGTGGACCGTCGACGAG
• GACCTCACCCTCATCAATTACATCTCTGATCACGGCGAGGGCCGCTGGAA
  CGCACTCGCACGCGCCGCCGGTCTGAAGAG
• GACTGGGAAGAGCTGCCGGCTCCGGTGGCTGAACTATCTCCGGCCGGATG
  TGAAGCGCGGCAACTTCACCGCAGAGGAGC
• AGCTGCTCATCCTCGACCTCCACTCCCGATGGGGCAACCGATGGTCCAAG
  ATAGCACAACATTTGCCTGGGAGGACCGAC
• AACGAGATCAAGAACTACTGGAGGACCAGAGTGCAAAAGCATGCCAAGCA
  ACTCAATTGTGATGTCAACAGCAAGAGGTT

14
Database search using Basic Local Alignment
Search Tool (BLAST)

• Most popular sequence alignment tool available
• Similarity/Homology Alignment
• BLAST hist significance is quantified by various
  parameters
• Alignment for the Maximal-scoring Segment Pairs
  are reported
• 6 Different BLAST programs from NCBI
• BLASTN, BLASTP, BLASTX, TBLASTN, TBLASTX
• Usually we use BLASTN

15
BLAST Score its Statistical Significance

• One alignment has a score , S, associated
• local random alignments are given a probability
  density function named extreme value distribution
  
• When you relate an observed alignment score (S)
  to the EVD, you can calculate statistical
  significance known as E value
• E value is the number of alignments with scores
  S that would be expected by chance alone
• Lower E value, higher MSP match

16
Where to BLAST

• Web-based one to tens of sequences
• NCBI
• TIGR http//tigrblast.tigr.org/euk-blast/index.cg
  i?projectosa1 - my favorite
• IRRI Local computer, via command line
• Palay Alphaserver
• Good for heavy-duty searches

17
Gene Prediction

• FGENESH predicts multiple genes in genomic DNA
  sequences (Solovyev 2001)
• Used by the rice genome sequence authors (BGI,
  TMRI)
• Available in web server http//www.softberry.com/b
  erry.phtml?topicgfindprgFGENESH and command
  line version (Palay)

18
FGENESH command line

• fgenesh /usr/local/fgenesh/Monocot seqfilename
  gt outfilename

19
Identify the gene Search the established protein
database for homology

• PFAM database HMM search
• Web based thru the TIGR site
• http//tigrblast.tigr.org/web-hmm/

20
Locate the surrounding SSR markers in the region

• BLAST reports the accession ID where you have
  significant hit(s)
• Get more information on this clone from Gramene
  or AGI