1.
Cotton Marker Database (CMD) for Genetic And Genome Research Anna Blenda, Pengfei Xuan, David Camak, Feng Luo, Don Jones (presenting author) ICGI-2010, Canberra, Australia

2.
CMD Objectives
Collect and integrate all public cotton molecular markers (SSRs and SNPs) as a cotton community resource.
Accelerate utilization of molecular markers in cotton breeding.
Provide data retrieval and search tools.
Provide stand-alone data mining tools.
Facilitate collaboration domestically and internationally
The CMD database resourse is available for deposit and data mining of the cotton genomes sequencing data

3. CMD New Features 1.Primer Redundancy 2. Traits
CMD New Features
1.Primer Redundancy
2. Traits
3. Updated Cmap Viewer with QTLs
4. New System Platform
- powerful computing
5. Future/Work in Progress   

4. View section: 1. Primer Redundancy SSR Projects SNP Projects
1. Primer Redundancy
View section:
     SSR Projects
SNP Projects
SSRs    Markers
Homology    SSR Primers
Primer Redundancy    Panel    Publications    Maps

5. Importance of the Primer Redundancy Check:
Importance of the Primer Redundancy Check:
Initial step in the analysis of the CMD cotton SSRs collection redundancy.
Avoidance of generating marker redundancy.
Financial component is critical (spending money for non-redundant SSR markers only).
Direct effect on the efficiency of the molecular breeding research.

6. Primer Redundancy Summary Page:
Primer Redundancy Summary Page:
- 18,002 primer sequences analyzed;
2,570 (14.2%) redundant primer sequences;
Types of primer sequence match:
forward-forward;
reverse-reverse;
forward-reverse;
reverse-forward.

7. Threshold value for primer sequence match: 81%
Threshold value for primer sequence match: 81%
The threshold value (81% or higher for sequence match):
chosen based on the threshold value analyses (from 70% to 100% match);
- below 81% match primer redundancy increases dramatically

8. List of Redundant Sequences
List of Redundant Sequences

9. Primer Redundancy Individual Pages
Primer Redundancy Individual Pages

10. Redundant primer Info from View/Search SSR pages
Redundant primer Info from View/Search SSR pages

11.
Downloads Page
CMD SSR Primer Redundancy results available from the Downloads page:
-excel format

12. Search by Primer Redundancy
Search by Primer Redundancy

13. Example of published traits and QTLs associated with traits
2. Traits in Cotton Linked to the Genetically Mapped SSR
David Camak, undergraduate student (Erskine College)
Example of published traits and QTLs associated with traits

14. QTL Start & Stop Positions Publication Reference
Spreadsheet with Annotated Trait Data
Trait Symbol
Marker Interval for QTL
Trait Name
QTL/gene Name
R2 Value
Trait-linked SSR
QTL/gene?
Cross
QTL Start & Stop Positions
SSR Genetic Position
Publication Reference
Linkage Group
QTL Span
Marker Type
This is what David Camak did – he annotated into the excel spreadsheet the information from current pubs regarding ag.important traits in cotton and mapped cotton SSRs linked with those traits.
Trait Description

15. Results
Twenty-nine agriculturally important traits were analyzed overall
Total number of SSR markers associated with those traits was 142
The total number of crosses/genetic maps analyzed was 15
Initial results of David Camak’s undergraduate research project. The annotation of traits is being continued.

16. Agriculturally Important Traits Annotated
Boll Size
Boll Weight
Bolls per Plant
Color Components
Yellowness
Fiber Span Length (2.5%, 50%)
Fiber Elongation
Fiber Fineness
Fiber Maturity
Fiber Perimeter
Fiber Strength
Fiber Micronaire
Lint Index
Lint Percentage
Lint Yield
Number of Seed/Boll
Reflectance
Seed Cotton Yield
Seed Index
Seed Weight
Spiny Bollworm Resistance
Short Fiber Index
Wall Thickness
Weight Fitness
Uniformity Index
Genic Male Sterility

17.
Number of Cotton SSRs Associated/Linked with the Analyzed Agriculturally Important Cotton Traits

18. Agriculturally Important Traits 1
Agriculturally Important Traits
Number of SSRs Associated with Each Trait
Fiber Elongation 15
Fiber Length
Yellowness 14
Fiber Strength 13
Fiber Strength (kNm/kg) 12
Fiber Reflectance
Micronaire
Boll Size (g) 11
Lint Percentage 10
Short Fiber Index
Lint Cotton Yield (kg/ha) 9
Maturity
2.5% Fiber Span Length (mm) 8
Fiber Maturity
Seed Cotton Yield (kg/ha)
Seed Index (g)
Fiber Elongation Percentage 7

19. Agriculturally Important Traits 2
Agriculturally Important Traits
Number of Unique SSRs Associated with Each Trait
Wall Thickness 7
50% Fiber Span Length (mm) 6
Boll Weight
Genic Male Sterility
Micronaire Reading 5
Weight Fitness
Bolls per Plant 4
Fiber Perimeter
Fiber Strength (cN/tex) 2
Spiny Bollworm Resistance
Fiber Length (mm) 1
Fiber Length Uniformity
*These numbers are continually updated as molecular research and breeding uncover more trait-linked SSRs

20. View Traits
Go to CMD
Click on Traits

21. Search SSRs Listed by Traits Traits by Published Symbol
Results
Search SSRs Listed by Traits
Traits by Published Symbol
Click on any Trait

22. SSR Linked with Selected Trait Choose Trait Based on SSR
List of SSRs
Choose Trait Based on SSR
Click

23. Trait Data From Spreadsheet
Trait Information 1
QTL and Marker Information
Positions for Genetic Mapping
All Relevant Data from Spreadsheet
Click on 1 or 2 2

24. 1 Specific Molecular Marker Source Page
Forward and Reverse Primer Sequences
Molecular Marker (SSR)
Other Useful Information Related to Specific SSR

25. Search feature available on any page, including the homepage2
Trait Search Page
Simple search for agriculturally important traits in cotton
Search feature available on any page, including the homepage

26. 3. Updated CMap Viewer with QTLs
26 cotton genetic maps are available to view and compare in CMap Viewer;
QTL information was added
5) Updates in CMAp (26 maps, QTL info)
Consensus map:BC1-RIL: ("Guazuncho2" (G. hirsutum) x "VH8-4602" (G. barbadense)) 2009
Reference map: Comprehensive Reference Map (CRM) CottonDB 2010
BC1: (G. hirsutum "Emian22" x G. barbadense "Pima3-79") x "Emian22" 2008
BC1: Hai-7124 x Junmian
BC1: [(G. hirsutum "TM-1" x G. barbadense "Hai7124") x "TM-1"] 2007
BC1: [(G. hirsutum "TM-1" x G. barbadense "Hai7124") x "TM-1"] 2006
BC1: (("Guazuncho2" (G. hirsutum) x "VH8-4602" (G. barbadense)) x "Guazuncho2") 2004
BC2: (("Guazuncho2" (G. hirsutum) x "VH8-4602" (G. barbadense)) x "Guazuncho2") 2005
DH: Vgs x (TM-1 x Hai-7124) 2005
F2: Deltapine x Giza
F2: Deltapine-61 x Texas
F2: G. arboreum ("Jianglingzhongmian" x "Zhejiangxiaoshanlushu") 2008
F2: Xinluzao-1 x Hai
F2: G. hirsutum "CRI36" x G. barbadense "Hai7124" 2007
F2: G. hirsutum "Handan208" x G. barbadense "Pima90" 2007
F2: G. hirsutum "Handan208" x G. barbadense "Pima90" 2005
F2: Hai-7124 x Junmian
F2: G. hirsutum race "Palmeri" x G. barbadense Acc. "K101" 2007
F2: G. hirsutum race "Palmeri" x G. barbadense Acc. "K101" 2004
F2: TM-1 x WT
F2: Yumian-1 x T
F2: Acala-44 x Pima S
RIL: 7235 x TM
RIL: Zhongmiansuo-12 x
RIL: "TM-1" (G. hirsutum (AD1)) x "3-79" (G. barbadense (AD2)) 2006
RIL: "TM-1" (G. hirsutum (AD1)) x "3-79" (G. barbadense (AD2)) 2005
4WC: (Simian-3 x Sumian-12) x (Zhong-4133 x 8891) 2008

27. 4. New Virtualization /HPC System Platform
Palmetto
HPC
- CMD was moved to
virtual machines for
high-performance computing (HPC);
jobs submitted by users transfer to
Clemson Palmetto HPC;
- very powerful computing
resource ( more than computing notes);
daily remote backup.
ssh
Event Channel
Virtual MMU
Virtual CPU
Control IF
Hardware (SMP, MMU, physical memory, Ethernet, SCSI/IDE)
GuestOS
(CentOS 5.4)
Device
Manager &
Control s/w
VM0
CMD web page
Cmap Cgi-bin
CMBioTools
VM1(cmdweb)
Front-End
Device Drivers
(CentOS GuestOS
5.4)
MySQL
PostgreSQL
VM2(databases)
(CentOS 5.4))
Unmodified
User
Software
VM3(gmod)
Safe HW IF
Xen Virtual Machine Monitor
Back-End
SMP
Warriors HPC Cluster
- Live and development website host one virtual machine based on virtualization technology. (ready to cloud computing)
Computing job submit by user will transfer to Clemson Palmetto HPC. Very powerful computing resource ( more than 5000 computing notes)
Daily remote snapshot backup.
Remote Daily Backup
snapshot

28.
Future
- 100 SSRs from Siva Kumpatla (Dow Agro): a collaborative project with Texas A&M , SSRs mapped on TM-1 x 3-79 map;
- 200 SSRs from Ramesh Kantety;
Updating of the mapped SSR data is in progress
More SNP data is coming;
Annotation of traits/genes that are mapped and linked to SSRs/SNPs is in progress

29. 3 pipelines were designed (Pengfei Xuan):
Future (cont.)
3 pipelines were designed (Pengfei Xuan):
1. Eukaryotic Automated Structural Annotation Pipeline 2. Transposable elements denovo 3. Transposable elements annotation

30. 1). Eukaryotic Automated Structural Annotation Pipeline
(work in progress)
EST based refinement (PASA)
Finalize best annotation
Phase 3
Genome Sequence
Gene Finders
EST Database
(PASA)
Database
Comparisons
Consensus prediction
Manually build gene
models (200 genes)
Gene Finder
Use gene models as
Training set
Repeat Masker
Preliminary gene finding
Phase 1
Phase 2
Manual check
- Aimed to identify a vast majority
of genes;
raw sequences are run through
a series of programs and scripts (“pipeline”) in an automated way;
generates a basic working
gene set as a starting point for further work.
The pipeline was designed by Pengfei for CUGI initially, but we are planning to implement it into CMD. It will be very handy when genome sequences are available.

31. 2). Transposable Elements Computational Identification
(work in progress)
This pipeline is searching the genome sequences for TEs and creates a library file of TEs for a genome of interest
this pipeline starts by comparing the genome with itself using BLASTER. Then it cluster matches with GROUPER, RECON and PILER, clustering programs specific for interspersed repeats. For each cluster, it builds a multiple alignment from which a consensus sequence is derived. Finally these consensus are classified according to TE features and redundancy is removed. At the end we obtain a library of classified, non-redundant consensus sequences.

32. 3). Transposable Elements Annotation
(work in progress)
This pipeline mines a genome using a library
of TEs from TEdenovo
pipeline.
Identified TEs are filtered and annotated.
TEannot: this pipeline mines a genome with a library of TE sequences, for instance the one produced by the TEdenovo pipeline, using BLASTER, RepeatMasker and CENSOR. An empirical statistical filter is applied to discard false-positive matches. Short simple repeats (SSRs) are annotated along the way with TRF, RepeatMasker and MREPS. Then the pipeline chains, with MATCHER via dynamic programming, TE fragments belonging to the same, disrupted copy. A "long join" procedure is subsequently applied to connect distant fragments. Finally annotations are exported into GFF3 and gameXML files.

33. CMD TEAM Anna Blenda, PI Feng Luo,collaborator Pengfei Xuan
CMD TEAM
Anna Blenda, PI
Research Assistant
Professor,
Genetics and Biochemistry
Clemson University
Feng Luo,collaborator
Assistant Professor,
School of Computing
Clemson University
Pengfei Xuan
M.S. student
Computer Science
Clemson University
David Camak
former member,
currently
M.S. student
Biology SELU

34. Acknowedgements Cotton Incorporated
Acknowedgements Cotton Incorporated

35.
Thank you!