The document discusses genotyping by sequencing (GBS), a molecular technique that enhances breeding cycles by allowing efficient DNA polymorphism discovery and marker assaying with reduced costs and sample handling. It details the process and requirements for GBS, including DNA extraction using specific enzymes and the design of adapters for sequencing. Additionally, GBS aids in significant SNP discovery and studies related to genetic diversity, genomic selection, and phylogenetics without needing prior genome knowledge.

2. Sequence based genotyping
• Molecular techniques essential for lessening
the breeding cycle
• Genotyping
– Find DNA polymorphism
– Assaying markers across a full set of material
• Involves marker discovery ,assay design and
genotyping
• Sequence based genotyping-marker discovery
and genotyping together

3. Why to go for GBS ???
• Genotyping by sequencing : enzyme based
complexity reduction and multiplexing
approach
• Low cost
• Reduced sample handling
• Fewer PCR and purification steps
• No reference genome limit
• Efficient barcoding for multiplexing

4. RAD markers – An initial form of GBS
• Sequenced “ restriction site associated genomic
DNA” for SNP discovery and genotyping
• Restriction site used to reduce complexity of
genome
• Adapters with barcodes are used for cost
effectiveness
• Mapped induced mutations in Neurospora crassa

5. • Modified RAD- less complex and cost effective –
“GBS”
• In maize and Barley
• Works well with complex genomes and small
genomes (organellar, microbial and chloroplast)
Genotyping by sequencing

6. • Materials
– DNA samples
• From mapping populations like RILs or DH
• Extract using standard CTAB method
– Restriction enzymes
• that leave 2 to 3 bp overhangs
• do not cut frequently in the major repetitive fraction of
the genome
• ApeKI for maize
GBS – Requirements

7. ApeKI enzyme
• type II restriction endonuclease
• recognizes a degenerate 5 bp sequence
(GCWGC, where W is A or T)
• creates a 5’ overhang (3 bp)
• has relatively few recognition sites in the
major classes of maize retrotransposons
• Partially methylation sensitive
GBS – Requirements
5’ G C W G C 3’
3’ C G W C G 5’

8. –Adapters
• Two different types of adapters
– Barcode adapter
– Common adapter
• Adapters were designed so that the ApeKI
recognition site did not occur in any adapter
sequence and was not regenerated after
ligation to genomic DNA
GBS – Requirements

9. Adapters
Barcode adapter
–‘‘barcode’’ adapter terminates with a 4 to 8
bp barcode on the 3’ end of its top strand
–3 bp overhang on the 5’ end of its bottom
strand complementary to the ‘‘sticky’’ end
generated by ApeKI (CWG)
–The second, or ‘‘common’’, adapter
–Contain only an ApeKI-compatible sticky
end
GBS – Requirements

10. –Primers
• with primers for sequencing and flow cell
oligos
–Sequencers
• Next generation sequencers like Illumina
–Sequence filtering and alignment
softwares
• BLAST( Basic Local Alignment Search Tool)
GBS – Requirements

11. GBS – An overview
• Steps
–Preparation of DNA samples and select
adapters
–Preparation of Libraries for Next-Generation
Sequencing
–Filtering Raw Sequence Data
–DNA sequence alignments
–Mapping

12. GBS – An overview

13. GBS – An overview

14. • The reminder sequences except barcodes and
primers are trimmed out
• The rest of the sequences are aligned
according to reference genome data using
BLAST
• Mapping of SNPs is done using softwares like
TASSEL
GBS – An overview

15. Advances in GBS
Poland et al (2012)

16. How GBS is useful???
• A large amount SNPs are discovered
• Identification of minor alleles or QTLs
• There is no requirement for a prior knowledge
of the species genomes
• SNP discovery and genotyping are completed
together

17. How GBS is useful???
• Rapid and low-cost tool to study
– Genotype of breeding populations
– GWAS- genome wide association studies
– genomic diversity study
– genetic linkage analysis
– molecular marker discovery
– genomic selection (GS)
– Phylogenetics
– Germplasm characterization