1. Plant Genomic Structure
Phillip SanMiguel
Director/Purdue Agricultural Genomics Center

2. DNA Sequencing: Machines and Methods

3. “Next Generation” Sequencing Platforms at the Purdue Genomics Core Facility
Roche “454” GS-FLX
Long read (~400 bp) instrument
Replacing “Sanger” sequencers
(for most high-throughput assays)
Applied Biosystems SOLiD
Short read (50 bp) instrument
Mainly for re-sequencing (SNP discovery)
Also expression analysis

4. Sequencer Niches Sanger 454 SOLiD
Smaller sample sets (eg, check a construct)
Longer reads (~800 bases)
Most accurate base calls
454
most de novo sequencing projects
SOLiD
resequencing, especially for SNP discovery
RNA sequence – microarray-like

5. Raw Capacity First Gen Sequencers Second Gen Sequencers
Sanger
Roughly 1 million $1200 per million nt
Second Gen Sequencers
454
Roughly 400 million $25 per million nt
SOLiD
Roughly 6 billion $0.50 per million nt
Third Gen Sequencers ???

6. “Library” Construction
No currently existing DNA sequencing instrument can “read” the order of bases in a DNA strand for long stretches
~1000 bases is the maximum read length (Sanger sequencers)
Two limitations:
“clone” length
read length

7. Length Limitations “Clone” length
“Cloning” is classically defined as storing a fragment of DNA from one species in a bacterial (E. coli) “host”
Also effectively amplifies that fragment
Next gen sequencing methods use PCR “amplicons” to accomplish
Both bacterial clone and amplicons have length limitations

8. Bacterial (1st gen) Clones
~300 kb for single copy vectors (BACs)
~50 kb for phage mediated (fosmids, cosmids, phage lambda)
~20 kb for high copy number vectors (pUC, pBluescript, pGEM, etc.)

9. Amplicon (2nd gen) Length Limits
Ultimately limited by PCR amplification length limits
Standard PCR limited to ~2 kb
But amplicons are created, millions at a time under conditions of limited reactants (microreactors)
~1 kb limit for 454
~0.5 kb limit for SOLiD

10. Read Length Sanger read lengths max out at 1 kb
Have heard of >1500 base reads with the ancient Licor (Sanger) sequencer
454 read lengths will go to ~ bases within 6 months
SOLiD read lengths 50 bases
Illumina read length max ~100 bases
Future: Pacific Bioscience. 10 kb?

11. “Paired ends” and “Mate pairs”
Can read both ends of a fragment of known length
Currently “paired end” denotes a normal fragment library amplicon – read both ends
span limited to amplicon size
Currently “mate pair” denotes a specialized library construct
span not limited to amplicon size

12. Plant Genome Structure

13. What is a Genome? All the DNA (chromosomes) in a cell
Exceptions?
Each cell has a complete copy

14. Useful approximate conversion of base numbers to mass
1 billion (109) bp ~= 1 pg of DNA
1 trillion (1012) bp ~= 1 ng of DNA
1 quadrillion (1015) bp ~= 1 ug of DNA

15. The C-value Paradox Arabidopsis 120 Mb Rice 400 Mb Sorghum 750 Mb
Maize Mb

16. Small Genome Size Angiosperms
Genome Size (Gbp)
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Cardamine amara
"Large Bitter-cress"
.046
Aesculus hippocastanum
"Horse Chestnut"
.104
Rosa wichuraiana
.104
Retroelements are transposable elements that transpose via an RNA intermediate.
Here 4 types are shown. The Retroviruses to my knowledge have only been shown to exist in animals. The next two lines are called viral-like retroelements or retrotransposons. They are related to retroviruses and share a numer of features with them.
They are flanked by long direct duplications “Long Terminal Repeats” LTRs anywhere from 30 bp to 4.5 kb. I don’t want to dwell too much on the internal structure of retroelements -- but they ususally have a long ORF or a few ORFs with lots of identifyable features. Lines do not have LTRs.
For my purposes LTRs are critical features of a retrotransposon...
.124
Epilobium palustre
Thlaspi alpestre
"Penny Cress"
.124
Arabidopsis thaliana
"Thale Cress"
.145

17. Smallest Genome Size Cereals
Genome Size (Gbp)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Oropetium thomaeum
0.21
Chloris gayana
“Callide Rhodesgrass”
0.29
Agropyron smithii
“Western Wheatgrass”
0.33
Brachypodium sylvaticum
"False Brome"
Retroelements are transposable elements that transpose via an RNA intermediate.
Here 4 types are shown. The Retroviruses to my knowledge have only been shown to exist in animals. The next two lines are called viral-like retroelements or retrotransposons. They are related to retroviruses and share a numer of features with them.
They are flanked by long direct duplications “Long Terminal Repeats” LTRs anywhere from 30 bp to 4.5 kb. I don’t want to dwell too much on the internal structure of retroelements -- but they ususally have a long ORF or a few ORFs with lots of identifyable features. Lines do not have LTRs.
For my purposes LTRs are critical features of a retrotransposon...
0.39
Hygroryza aristata
0.41
Oryza sativa "Rice"
0.41

18. Brachypodium sylvaticum
Cereal Genome Sizes
Genome Size (Gbp)
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Oropetium thomaeum
Chloris gayana
Agropyron smithii
Brachypodium sylvaticum
Hygroryza aristata
Oryza sativa
Retroelements are transposable elements that transpose via an RNA intermediate.
Here 4 types are shown. The Retroviruses to my knowledge have only been shown to exist in animals. The next two lines are called viral-like retroelements or retrotransposons. They are related to retroviruses and share a numer of features with them.
They are flanked by long direct duplications “Long Terminal Repeats” LTRs anywhere from 30 bp to 4.5 kb. I don’t want to dwell too much on the internal structure of retroelements -- but they ususally have a long ORF or a few ORFs with lots of identifyable features. Lines do not have LTRs.
For my purposes LTRs are critical features of a retrotransposon...
Sorghum bicolor
Zea mays
Triticum urartu
Lygeum spartum
"Esparto grass"
Triticum aestivum

19. Angiosperm genome sizes, linear scale
Genome Size (Gbp)
0.0
20.0
40.0
60.0
80.0
100.0
Oropetium thomaeum
Chloris gayana
Agropyron smithii
Brachypodium sylvaticum
Hygroryza aristata
Oryza sativa
Sorghum bicolor
Zea mays
Triticum urartu
Lygeum spartum
Triticum aestivum
Cardamine amara
Retroelements are transposable elements that transpose via an RNA intermediate.
Here 4 types are shown. The Retroviruses to my knowledge have only been shown to exist in animals. The next two lines are called viral-like retroelements or retrotransposons. They are related to retroviruses and share a numer of features with them.
They are flanked by long direct duplications “Long Terminal Repeats” LTRs anywhere from 30 bp to 4.5 kb. I don’t want to dwell too much on the internal structure of retroelements -- but they ususally have a long ORF or a few ORFs with lots of identifyable features. Lines do not have LTRs.
For my purposes LTRs are critical features of a retrotransposon...
Aesculus hippocastanum
Rosa wichuraiana
Epilobium palustre
Thlaspi alpestre
Arabidopsis thaliana
Fritillaria davisii
Trillium rhombifolium
Fritillaria assyriaca

20. Angiosperm genome sizes, log scale
Genome Size (Gbp)
0.0
0.1
1.0
10.0
100.0
Oropetium thomaeum
Chloris gayana
Agropyron smithii
Brachypodium sylvaticum
Hygroryza aristata
Oryza sativa
Sorghum bicolor
Zea mays
Triticum urartu
Lygeum spartum
Triticum aestivum
Retroelements are transposable elements that transpose via an RNA intermediate.
Here 4 types are shown. The Retroviruses to my knowledge have only been shown to exist in animals. The next two lines are called viral-like retroelements or retrotransposons. They are related to retroviruses and share a numer of features with them.
They are flanked by long direct duplications “Long Terminal Repeats” LTRs anywhere from 30 bp to 4.5 kb. I don’t want to dwell too much on the internal structure of retroelements -- but they ususally have a long ORF or a few ORFs with lots of identifyable features. Lines do not have LTRs.
For my purposes LTRs are critical features of a retrotransposon...
Cardamine amara
Aesculus hippocastanum
Rosa wichuraiana
Epilobium palustre
Thlaspi alpestre
Arabidopsis thaliana
Fritillaria davisii
Trillium rhombifolium
Fritillaria assyriaca

21. The Elements of Genomes That Are Shaped by Evolution
To a first approximation, there are only two components of genomes:
“Genes”
Defined here as “functional genes” – those that directly contribute to cellular structure and function
Transposable Elements
Can catalyze their own duplication via “transposition”
“Selfish DNA”, cellular “purpose” ambiguous

22. What is a Gene?
Y1-Q60
In a study of the maize genome is it appropriate to start with a gene.
Y1 was cloned by Brent Buckner and I sequenced it.
Y1-Q60 is a non-mutant allele -- a “typical” maize gene. 6 exons, introns,
transcription start sites, poly-adenylations sites -- and transposable elements.
A number of Miniature Inverted-repeat Transposable Elements (MITEs) elements are associated with Y1 and many other, if not most other maize genes.
An Ins2 and a Stowaway elements are just 5’ of the gene. Keep in mind this is a non-mutant allele of Y1. Further, in another non-mutant Y1 line --
B73 -- a tourist is inserted in the 6th intron, in the non-coding region but before the poly-adenylation sites of the Q60 lines. In B73 the tourist provides the poly-adenylation site.
The checkerboard element is the Mu3 element used by Brent to tag Y1 and subsequently clone it.
Finally, back to theQ60 allele, a couple of kb upstream of Y1 is another transposable element, a retroelement.

23. Transposable Element Interlude

24. Why Consider TEs Differently From Genes?
Although their fate is tied to the fate of the organism they “inhabit”, TEs have their own agenda
Maintenance of the sequence integrity of genes from generation to generation is critical to survival of a species
Deleting a TE is rarely detrimental to an organism

25. Why Not Ignore TEs?
TEs frequently compose the majority of a eukaryotic genome
TEs contribute regulatory regions to genes by transposing upstream of a gene
TEs serve as focal points for deletion events that can delete genes as well
TEs can carry genes!

26. Types of Transposable Elements (TEs)
Type I aka “retroelements”
Transpose via an RNA intermediate
Tend to dominate larger genomes
(More detail in next slide)
Type II
DNA transposable elements
Thousands of different types
Usually “cut and paste” but can be “copy and paste”

27. Retroelements Retrovirus Ty3/Gypsy-like Ty1/Copia-like LINE
PBS
PPT
Retrovirus
LTR
gag prot RT RNase H int env
LTR
PBS
PPT
Ty3/Gypsy-like
LTR
gag prot RT RNase H int
LTR
PBS
PPT
Retroelements are transposable elements that transpose via an RNA intermediate.
Here 4 types are shown. The Retroviruses to my knowledge have only been shown to exist in animals. The next two lines are called viral-like retroelements or retrotransposons. They are related to retroviruses and share a numer of features with them.
They are flanked by long direct duplications “Long Terminal Repeats” LTRs anywhere from 30 bp to 4.5 kb. I don’t want to dwell too much on the internal structure of retroelements -- but they ususally have a long ORF or a few ORFs with lots of identifyable features. Lines do not have LTRs.
For my purposes LTRs are critical features of a retrotransposon...
Ty1/Copia-like
LTR
gag prot int RT RNase H
LTR
LINE
gag RT RNase H
(A) n

28. Long Terminal Repeat PBS 5' LTR
PPT
LTR
gag prot int RT RNase H
LTR
PBS
5' LTR
Here is the LTR of a retrotransposon I’ll introduce to you later. Here I’m just using it as an example of a typical retrotransposon.
Retrotransposons, once you have sequenced them, have obvious ends. This is because they have Long terminal repeats. An LTR, by itself, doesn’t look like much. Sure, it usually begins with a “TG” and ends with an “AC” but that is not much to go on -- unless you hav ethe other LTR to compare it with. Upon integration of the retrotransposon, the LTRs should be identical. If you compare one with another it is obvious where the element begins and ends. Further, immediately flanking the element are small target site duplications of host DNA -- generally 5 bp -- caused by the staggered cutting mechanism of the retrotransposon’s integrase and subsequent DNA repair.
Also the internal end of the LTR should either be next to a Primer Binding Site or a Polypurine Tract. The PBS is usually homologous to the 3’ end of a transfer RNA -- in this case tRNA Methionine. The polypurine tract is less identifiable -- just a stretch of “G’s” and “A’s”.
Generally, if you are focused on a lambda clone sized insert of genomic maize DNA containing a gene you may not be overwhelmed by the presensce of retrotransposons. Yeah, you’ll find part of one or two some distance from the gene, but these are large elements, not like MITEs. But genes only make up a small part of the maize genome. To study the maize genome you need to characterize a bigger clone.
5 bp dup. of
PBS
host DNA
5' end of LTR
3' end of LTR
Opie2
-LTR
GGACC
TGAAAGGGAAA
AGGTGCTCTCA AT
TGGTATCGGA...
1254 bp

29. LTRs Can Be Used as Molecular “Clocks”
The 2 LTRs of a retrotransposon are initially identical (upon insertion)
Subsequent mutations will tend to cause LTRs to diverge
Hence, the higher the divergence, the older the element

30. Insertion Times Millions of Years Ago Estimated Substitutions/kb 10 110 1 20 2 30 3 40
adh1-S and 4
adh1-F diverge 50 60 5 70 6
10 kb 80
adh1 -F
u22

31. Forces that Shape Genomes
Genomes are linear so there are only 2 possible ways to change genome size:
Insertion
Duplication – copy number increase
Deletion
Copy number decrease
But either type of change can happen at any scale – from a single base to the entire genome

32. Types of changes Translocations Whole genome duplication
Polyploidy
“Tandem Segmental Duplication”
Vastly different scales
Transposition
Catalyzed by TE encoded protein
Simple Sequence Repeats
Polymerase Slipping

33. Gene Duplications – Substrates for Evolution
Single genes may have crucial roles
Their mutation would have detrimental effects upon the organism
After duplication, one copy of a gene becomes expendable
Frequently the extra copy is deleted
In cases where it is retained, mutation may change the function of one copy

34. Putting Together the Pieces
The complexity of a genome is generated via moderately simple mechanisms
Duplications and transpositions occur
Extra gene copies and TEs may mutate under neutral or even positive selection pressures.
The result:
Genic areas are fairly stable, whereas upstream and downstream regions rapidly evolve.

35. The Picture? Complex, but Driven by Understandable Mechanisms