1. genes, genomes, and markers
DNA sequencing:
genes, genomes, and markers

2. Steps in Genetic Analysis
Knowing how many genes determine a phenotype (Mendelian and/or QTL analysis), and where the genes are located (linkage mapping) is a first step in understanding the genetic basis of a phenotype
A second step is determining the sequence of the gene (or genes)

3. Steps in Genetic Analysis
Subsequent steps involve….
Understanding gene regulation
Understanding the context of the gene in the sequence of the whole genome
Analysis of post-transcriptional events, understanding how the genes fit into metabolic pathways, how these pathways interact with the environment

4. Genome sequence of a diploid plant (2n = 2x = 14)
5,300,000,000 base pairs
165% of human genome
Enough characters for 11,000 large novels
60,000,000 base pairs of expressed sequence
~ 1% of total sequence, like humans
125 large novels

5. Molecular tools for determining DNA (or RNA) sequence
Genomic DNA (or RNA) extraction
(RNA cDNA)
Manipulate DNA with restriction enzymes to reduce complexity and/or facilitate further manipulation
Manage and/or maintain DNA in vectors and/or libraries
Selecting DNA targets via amplification and/or hybridization
Determining nucleotide sequence of the targeted DNA

6. Extracting DNA (orRNA)
Genomic DNA (or RNA):   
Leaf segments or target tissues
Key considerations are
Concentration
Purity
Fragment size

7. RNA-seq: mRNA to cDNA
Reverse transcriptase

8. Restriction Enzymes
Restriction enzymes make cuts at defined recognition sites in DNA
A defense system for bacteria, where they attack and degrade the DNA of attacking bacteriophages
The restriction enzymes are named for the organism from which they were isolated  
Harnessed for the task of systematically breaking up DNA into fragments of tractable size and for various polymorphism detection assays
Each enzyme recognizes a particular DNA sequence and cuts in a specified fashion at the sequence

9. Restriction Enzymes
Recognition sites and fragment size: a four-base cutter ~ 256 bp (44); more frequently than a six-base cutter, which in turn will cut more often than one with an eight base-cutter
Methylation sensitive restriction enzymes
Target the epigenome

10. Restriction Enzymes
Palindrome recognition sites – the same sequence is specified when each strand of the double helix is read in the opposite direction.
Sit on a potato pan, Otis
Cigar? Toss it in a can, it is so tragic
UFO. tofu
Golf? No sir, prefer prison flog
Flee to me remote elf
Gnu dung
Lager, Sir, is regal
Tuna nut
CRISPR: Clustered regularly interspaced palindromic repeats

11. Vectors
Propagate and maintain DNA fragments generated by the restriction digestion
Efficiency and simplicity of inserting and retrieving the inserted DNA fragments
Key feature of the cloning vector: size of the DNA insert
Plasmid ~ 1 kb
BAC ~ 200 kb

12. Libraries
Repositories of DNA fragments cloned in their vectors or attached to platform-specific oligonucleotide adapters
Classified in terms of
cloning vector: e.g. plasmid, BAC 
in terms of cloned DNA fragment source: e.g. genomic, cDNA
In terms of intended use: e.g. next generation sequencing (NGS)

13. Genomic DNA libraries
Total genomic DNA digested and the fragments cloned into an appropriate vector or system
Representative sample all the genomic DNA present in the organism, including both coding and non-coding sequences
Enrichment strategies: target specific types of sequences
unique sequences  
Methylated sequences

14. cDNA libraries
Generated from mRNA transcripts, using reverse transcriptase
The cDNA library represents only the genes that are expressed in the tissue and/or developmental stage that was sampled  

15. DNA Amplification: Polymerase Chain Reaction (PCR)
K.B Mullis, 1983
in vitro amplification of ANY DNA sequence

16. Synthetic DNA: oligonucleotides
Primers, adapters, and more …~$0.010 per bp...
< ~ 100 bases

17. DNA Amplification: Polymerase Chain Reaction (PCR)
Design of two single stranded oligonucleotide primers complementary to motifs on the template DNA.

18. DNA Amplification - PCR
A Polymerase extends the 3’ end of the primer sequence using the DNA strand as a template.

19. PCR Principles The PCR reaction consists of: Buffer
DNA polymerase (thermostable)
Deoxyribonucleotide triphosphates (dNTPs)
Two primers (oligonucleotides)
Template DNA

20. PCR Principles
Each cycle generates exponential numbers of DNA fragments that are identical copies of the original DNA strand between the two binding sites.

21. PCR Principles
The choice of what DNA will be amplified by the polymerase is determined by the primers
The DNA between the primers is amplified by the polymerase: in subsequent reactions the original template, plus the newly amplified fragments, serve as templates
Steps in the reaction include denaturing the target DNA to make it single-stranded, addition of the single stranded oligonucleotides, hybridization of the primers to the template, and primer extension 

22. PCR Applications
Amplify a target sequence from a pool of DNA (your favorite gene, forensics, fossil DNA)
Start the process of genome sequencing
Generate abundant markers for linkage map construction molecular markers

23. DNA Hybridization
Single strand nucleic acids find and pair with other single strand nucleic acids with a complementary sequence
An application of this affinity is to label one single strand and then to use this probe to find complementary sequences in a population of single stranded nucleic acids
For example, if you have a cloned gene – either a cDNA or a genomic clone - you could use this as a probe to look for a homologous sequence in another DNA sample 

24. DNA Hybridization
The principle of hybridization can be applied to pairing events involving DNA: DNA; DNA: RNA; and protein: antibody
Southern blot
Northern blot
Western blot

25. DNA sequencing
Advances in technology have removed the technical obstacles to determining the nucleotide sequence of a gene, a chromosome region, or a whole genome.

26. (classic but still relevant)
Sanger DNA Sequencing
(classic but still relevant)
Start with a defined fragment of DNA
Based on this template, generate a population of molecules differing in size by one base of known composition
Fractionate the population molecules based on size
The base at the truncated end of each of the fractionated molecules is determined and used to establish the nucleotide sequence

27. Sanger Sequencing - ddNTPs
A dideoxy nucleotide lacks a 3' OH and once incorporated, it will terminate strand synthesis. L-1. No free 3' OH

28. Sanger Sequencing Buffer DNA polymerase dNTPs Labeled primer
Target DNA
ddGTP
ddATP
deoxinucleotyde (dNTP)
dideoxinucleotyde (ddNTP)
ddCTP
ddTTP

29. Next Generation Sequencing - Illumina

30. Sequencing - PAC Bio

31. Sequencing considerations
Read length
Accuracy
Speed
Cost
Assembly

32. Sequencing – up and coming (?)

33. Genome sizes and whole genome sequencing
Plant
Genome size
# Genes
Arabidposis thaliana
135 Mb
27,000
Fragaria vesca
240 Mb
35,000
Theobroma cacao
415 Mb
29,000
Zea mays
2,300 Mb
40,000
Pinus taeda
23,200Mb
50,000
Paris japonica
148,852Mb ??
Credit: Karl Kristensen, Denmark

34. Sequencing a plant genome

35. Sequencing a plant genome
Fragaria vesca
Herbaceous, perennial
2n=2x=14
240 Mb
Reference species for Rosaceae
Genetic resources
Credit: commons.Wikimedia.org
Fragaria x ananassa: 2n=8x=56. Domesticated 250 years ago

36. Sequencing a plant genome
Short reads
No physical reference
De novo assembly
Open source

37. Sequencing a plant genome
Roche 454, Illumina
X39 coverage (number of reads including a given nucleotide)
Contigs (overlapping reads) assembled into scaffolds (contigs + gaps)
~ 3,200 scaffolds N50 of 1.3 Mb (weighted average length)
Over 95% (209.8 Mb) of total sequence is represented in 272 scaffolds

38. Anchoring the genome sequence to the genetic map
Sequencing a plant genome
Anchoring the genome sequence to the genetic map
94% of scaffolds anchored to the diploid Fragaria reference linkage map using 390 genetic markers
Pseudochromosomes ~ linkage groups

39. Sequencing a plant genome
Synteny
Homologs
Orthologs
Paralogs
Credit: Biology stackexchange.com

40. Sequencing a plant genome
The small genome size (240 Mb)
Absence of large genome duplications
Limited numbers of transposable elements, compared to other angiosperms

41. Sequencing a plant genome the transcriptome
Fruits and roots – different types of genes

42. Sequencing a plant genome
Gene prediction
34,809 nuclear genes
flavor, nutritional value, and flowering time
1,616 transcription factors
RNA genes
569 tRNA, 177 rRNA, 111 spliceosomal RNAs, 168 small nuclear RNAs,
76 micro RNA and 24 other RNAs
Chloroplast genome
155,691 bp encodes 78 proteins, 30 tRNAs and 4 rRNA genes
Evidence of DNA transfer from plastid genome to the nuclear genome

43. DNA (molecular) markers Linkage mapping, quantitative trait locus (QTL) mapping, anchoring genome sequences

44. Why use markers rather than whole genome sequences?
A way of addressing plant genetics and breeding challenges:
The large number of genes per genome
Huge genome sizes
Often a subset of the total genome is of interest

45. Applications of Markers
Establish evolutionary relations: homoeology and synteny

46. Applications of Markers
Are trait associations due to linkage or pleiotropy?
Identify markers that can be used in marker assisted selection
Locate genes for qualitative and quantitative traits
A starting point for map-based cloning strategies

47. Markers are based on polymorphisms
Amplified fragment length polymorphism
Restriction fragment length polymorphism
Single nucleotide polymorphism
The polymorphisms become the alleles at marker loci
The marker locus is not necessarily a gene: the polymorphism may be in the dark matter, in a UTR, in an intron, or in an exon
Non-coding regions may be more polymorphic
Molecular markers are abundant

48. Marker polymorphisms are based on
mutations
Silent
*** CTG GGA GAT TAT GGC TTT AAG***
*** CTG GGA GAT TAT GGC TTC AAG*** alignment
Leu Gly Asp Tyr Gly Phe Lys
Leu Gly Asp Tyr Gly Phe Lys translation
Missense
*** CTG GGA GAT TAT GGC TAT AAG*** alignment
Leu Gly Asp Tyr Gly Tyr Lys translation
Nonsense
*** CTG GGA GAT TAG GGC TTT AAG*** alignment
Leu Gly Asp Tyr Gly Phe Lys
Leu Gly Asp STOP translation

49. Markers
Polymorphisms can be visualized at the metabolome, proteome, or transcriptome level but for a number of reasons (both technical and biological) DNA-level polymorphisms are currently the most targeted
Regardless of whether it is a “perfect” or a “linked” DNA marker, there are two key considerations that need to be addressed in order for the researcher/user to visualize the underlying genetic polymorphism

50. DNA Markers
Finding and understanding the genetic basis of the DNA-level polymorphism, which may be as small as a single nucleotide polymorphism (SNP) or as large as an insertion/deletion (INDEL) of thousands of nucleotides
Detecting the polymorphism via a specific assay or "platform". The same DNA polymorphism may be amenable to different detection assays

51. Marker examples: Simple Sequence Repeats (SSRs)
Simple sequence repeats (SSRs) (aka microsatellites) are tandemly repeated mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs
SSR length polymorphisms are caused by differences in the number of repeats
Assayed by PCR amplification using pairs of oligonucleotide primers specific to unique sequences flanking the SSR
Multiple platforms

52. Simple Sequence Repeats (SSRs)
Marker examples
Simple Sequence Repeats (SSRs)
Simple sequence repeats in hazelnut:
Note the differences in repeat length AND the consistent flanking sequences
Credit: mind42.com

53. Marker examples Simple Sequence Repeats (SSRs)
Highly polymorphic
Highly abundant and randomly dispersed
Co-dominant
Locus-specific
Amenable to high throughput assays

54. SSR Concept
Individual 1 (AC)x9
Individual 2 (AC)x11
51 bp
55 bp

55. Marker examples: Single Nucleotide Polymorphisms (SNPs)
DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered
Alleles
Single
Nucleotide
Polymorphisms
(SNPs)
…..ATGCTCTTACTGCTAGCGC……
…..ATGCTCTTCCTGCTAGCGC……
…..ATGCTCTTACTGCAAGCGC……
Consensus…..ATGCTCTTNCTGCNAGCGC……

56. Marker examples: SNPs Highly abundant (~ 1 every 200 bp)
Locus-specific
Co-dominant and bi-allelic
Basis for high-throughput and massively parallel genotyping technologies
Connectivity to reference genome sequences

57. SNP Detection Strategies
Locus specific systems
Many samples with few markers
Markers for key target characters
Example: KASP
Genome wide systems
Fewer samples with many markers
Germplasm characterization
Genotyping panels for Genome Wide Association Studies Example: Illumina

58. SNPs on KASP and Illumina 9K

59. available for every plant !!!!!
Abundant markers are
available for every plant !!!!!