1. DNA sequencing and genome architecture

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. 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 
The microarray concept:

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. Sanger DNA Sequencing - classic but not obsolete
The gold standard for accurate sequencing of short (~ 650 bp) DNA sequences

26. Next Generation Sequencing - Illumina
A tremendous amount of short read data in a short time,
at a good price

27. Sequencing - PAC Bio Long reads!

28. Sequencing - Nanopore
Potentially, cheap, fast, portable

29. Sequencing considerations
Read length
Accuracy
Speed
Cost
Assembly

30. 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

31. Sequencing a plant genome

32. 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

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

34. 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

35. 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

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

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

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

39. 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

40. Genome architecture and evolution
Key considerations:
Genes
Chromosomes
C value paradox
Gene regulation
Epigenetics
Transposable elements

41. Genome architecture and evolution
Plant
#genes (est)
2n = _x = _
Genome size
Arabidposis thaliana
27,000
2n = 2x = 10
135 Mb
Fragaria vesca
35,000
2n = 2x = 14
240 Mb
Theobroma cacao
29,000
2n = 2x = 20
415 Mb
Zea mays
40,000
2,300 Mb
Pinus taeda
50,000
2n = 2x =24
23,200Mb
Paris japonica ??
2n = 8x = 40
148,852Mb
S. Wessler on Transposable elements:
Review of retroviruses:
Review of cut and past transposition:

42. Genes Other RNAs? DNA ……..……..mRNA……….…….Protein Plant
Transcription
tRNA
rRNA
Translation
Plant
Estimated # genes
Arabidposis thaliana
27,000
Fragaria vesca
35,000
Theobroma cacao
29,000
Zea mays
40,000
Other RNAs?

43. Genes (classically..) DNA specifying a protein 200 – 2,000,000 nt (bp)
promoter
Coding region
Exon
Intron
Exon
Intron
Exon
Start
codon
Stop
codon
5’UTR
3’UTR
Basal
promoter +1
Termination
signal
ORF
mRNA
CDS

44. F. vesca: 35,000 genes/7 chromosomes = 5,000 genes/chromosome?
Plant
2n = _X = _
Arabidposis thaliana
2n = 2x = 10
Fragaria vesca
2n = 2x = 14
Theobroma cacao
2n = 2x = 20
Zea mays
F. vesca: 35,000 genes/7 chromosomes = 5,000 genes/chromosome?

45. Genes, chromosomes, and genomes
F. vesca: 2n = 2x = 14
genome = 240 Mb
average gene ~ 3kb
79,333 genes? 11,333 genes/chromosome?
35,000 genes….. ~ 5,000 genes/chromosome
What’s the rest of the genome???????

46. C-value paradox “Organisms of similar evolutionary complexity
differ vastly in DNA content”
Federoff, N Science. 338:
1 pg = 978 Mb

47. The C-value paradox
Fig. 1.The C-value paradox. The range of haploid genome sizes is shown in kilobases for the groups of organisms listed on the left. [Adapted from an image by Steven M. Carr, Memorial University of Newfoundland]
Fedoroff Science 2012;338:

48. C-value paradox 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 ??

49. C-value paradox Junk??????? Shining a Light on the
Genome’s ‘Dark Matter’

50. 40% of all human disease-related SNPs are OUTSIDE of genes
Gene regulation
Pennisi, E Science 330:1614.
40% of all human disease-related SNPs are OUTSIDE of genes
The dark matter is conserved and therefore must have a function
DNA sequences in the dark matter are involved in gene regulation
~80% of the genome is transcribed but “genes” account for ~2%
RNAs of all shapes and sizes:
RNAi
lncRNA

51. Epigenetics
Observe changes in phenotype without changes in genotype - due
to alternative regulation ( 0 – 100%) of the gene
Methylation expression
Acetylation expression

52. Epigenetics Facultative heterochromatin:
Holoch and Moazed Nature Genetics

53. Transposable elements
DNA sequences that can move to new sites in the genome
More than half the DNA in many eukaryotes
Two major classes:
Transposons: Move via a DNA cut-and-paste mechanism
Retrotransposons: Move via an RNA intermediate
Potentially disruptive – can eliminate gene function. Therefore, usually epigenetically silenced

54. Transposable elements
Federoff (2012) argues that TE’s, via altering gene regulation, account for the “evolvability” of the “massive and messy genomes” characteristic of higher plants
Create new genes
Modify genes
Program and re-program genes
Transposition events lead to genome expansion and help to explain the C value paradox

55. Transposable elements
Transposition events lead to genome expansion and help to explain the C value paradox:
TEs nested within TEs nested within TEs

56. Transposable elements
The arrangement of retrotransposons in the maize adh1-F region
Fig. 6.The arrangement of retrotransposons in the maize adh1-F region. The short lines represent retrotransposons, with the internal domains represented in orange and the LTRs in yellow. Younger insertions within older insertions are represented by the successive rows from the bottom to the top of the diagram. Small arrows show the direction of transcription of the genes shown under the long blue line that represents the sequence in the vicinity of the adh1 gene. [Adapted with permission from (102)]
N V Fedoroff Science 2012;338:
Published by AAAS

57. Transposable elements
Sequence adjacent to the bronze (bz) gene in different lines of maize
Fig. 7.The organization of the sequence adjacent to the bronze (bz) gene in eight different lines (haplotypes) of maize. The genes in this region are shown in the top diagram: bz, stc1, rpl35A, tac6058, hypro1, znf, tac7077, and uce2. The orientation of the gene is indicated by the direction of the green pentagon, pointing in the direction of transcription; exons are represented in dark green and introns in light green. Each haplotype is identified by its name and the size of the cloned NotI fragment. The same symbols are used for gene fragments carried by Helitrons (Hels), which are represented as bidirectional arrows below the line for each haplotype. Vacant sites for HelA and HelB are provided as reference points and marked by short vertical red bars. Dashed lines represent deletions. Retrotransposons are represented by yellow bars. DNA transposons and TAFTs (TA-flanked transposons), which are probably also DNA transposons, are represented by red triangles; small insertions are represented by light blue triangles. [Redrawn with permission from (113)]
Published by AAAS
N V Fedoroff Science 2012;338:

58. Transposable elements
85% of the maize genome consists of transposons
Transposition events are in real time: differences between maize inbreds
Transposons can move large blocks of intervening DNA
Transposases are the products of the most abundant genes on earth

59. Transposable elements
~ 24% of the cacao genome
~ 21% of the Fragaria genome
~68,000 TE-related sequences in cacao
“Gaucho” is a retrotransposon ~ 11Kb in length and present ~1,000 times
“The lack of highly abundant LTR transposons is likely to be the reason F. vesca has a relatively small-size genome”

60. Transposable elements
Reid and Ross. Mendel’s genes….An Ac/Ds-like 0.8 kb insertion: round vs. wrinkled peas
Transposon tagging – find genes by mutation due to TE
Holoch and Moazed. RNAi….Transposons held in check by epigenetic mechanisms

61. Genome architecture and evolution
Plant
#genes (est)
2n = _x = _
Genome size
Arabidposis thaliana
27,000
2n = 2x = 10
135 Mb
Fragaria vesca
35,000
2n = 2x = 14
240 Mb
Theobroma cacao
29,000
2n = 2x = 20
415 Mb
Zea mays
40,000
2,300 Mb
Pinus taeda
50,000
2n = 2x =24
23,200Mb
Paris japonica ??
2n = 8x = 40
148,852Mb
S. Wessler on Transposable elements:
Review of retroviruses:
Review of cut and past transposition: