D.

pseudoobscura Inversion Breakpoint Sequence Evolution

Haley E. Randolph1, Megan E. Lee1, Geovanny C Montoya1, Atousa Jahanshashi1,

Shannon Dugan2, Diahhulz Zhu2, Stephen Richards2, and Stephen W. Schaeffer1, (1)
Department of Biology, The Pennsylvania State University, University Park, PA 16801;
(2) Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX
Ancestral SC to TL Breakpoint

Mapping of Reads that Flank

the SC to TL Breakpoints

Abstract
The chromosomal arrangements located on the third chromosome of D.
pseudoobscura provide an exceptional system in which to study chromosomal
arrangement evolution and the mechanisms that generate chromosomal
inversions. All of the arrangements exist because of a series of overlapping
paracentric inversions located in the noncoding regions of the third
chromosome. As a continuation of previous research, the sequences of two
pairs of inversion breakpoints, Santa Cruz to Chiricahua and Santa Cruz to Tree
Line, were assembled and analyzed. Previously, the Hypothetical to Standard,
Standard to Arrowhead, and Standard to Pikes Peak arrangements were
determined and analyzed and demonstrated that the inversion breakpoints
occurred in noncoding sequences. Three kilobase large-insert paired-end
libraries were utilized to map the breakpoints; genes that flanked the
breakpoints were utilized as the beginning of the sequence assemblies. Once
the breakpoint sequences were constructed, the ancestral and derived
arrangements were used to determine the location of the chromosomal
breaks. In each of the arrangements, areas of heavily-repeated DNA
sequences are found, further suggesting that repeats play a role in the
mechanism of chromosomal inversion creation. Similar repeats are found in
the arrangements previously studied.

Proximal

81
63
64
65
66
67
68 79 76 75 7479 68
69
70
71 72 73 74 79 78 77 76 80
ABCDE ABC ABCD ABCD ABCDE AB CD A CBA C B CD ABCDE ABCD ABC ABC ABC AB A CBA BA CB ABC ABCD

Tree Line (TL)

SC

Santa Cruz (SC)

63
64
65
66
67
68 79 76 75 74 73 72 71
70
69
68 79 78 77 76 80
81
ABCDE ABC ABCD ABCD ABCDE AB CD A CBA CBA CBA CBA CBA DCBA EDCBA DC BA CBA BA CB ABC ABCD

Distal

79B

Reads that span the SC to TL inversion

breakpoints will map at distances greater than 3 kb
on the TL reference strain

Mapping of SC to TL Breakpoints

79A

17,154,179..17,153,168

17,150,663..17,150,163

Current contig locations of ancestral dSCTL

breakpoint

Common Repeats Among Inversion BPs

Proximal (ST ancestral)

Models of Chromosomal
Rearrangement in Drosophila

Distal (ST ancestral)

300

200

200

100

100
1

Proximal (AR derived)

ABC D
Ancestral Arrangement

8kb

GA17716

GA24334

Ancestral Arrangement

2kb

GA19155

GA17559

Distal (AR derived)

300

B C D

200

200

100

100
1

IGV plot of Tree Line reads at the distal breakpoint mapped to

the Arrowhead reference strain (reads marked in red flank the breakpoint).

3
5
Derived Arrangement
5

3
5

6kb

GA17559

Distal (ST ancestral)

120
80

D C B A

D C B A

5
3

40

ST

TL

PP
CH

HY

63
64
65
66
67
68 79 76 75 74 73 72 71
70
69
68 79 78 77 76 80
81
ABCDE ABC ABCD ABCD ABCDE AB CD A CBA CBA CBA CBA CBA DCBA EDCBA DC BA CBA BA CB ABC ABCD

SC
SC

SC

TL

The TL arrangement is derived from the SC

ancestral arrangement
The SC to TL breakpoints were mapped using
reads from a 3 kb insert library sequenced on an
Illumina HiSeq2000 machine

GA13487

9kb

GA22082

GA20777

Distal (PP derived)

40
2

6kb

GA22082

GA13475

5kb

GA20777

Similar repeats are found within proximal and

distal BPs and between independent BPs

Assembly of Breakpoint Regions

The breakpoints were assembled using reads from next
generation sequences of a Santa Cruz and Tree Line strain
Paired-end reads were identified with a BLAST search of
the sequence library starting using a query sequence from a
gene flanking the breakpoint region
The acquired reads were assembled using SeqMan Pro
from the Lasergene suite of programs available from
DNAstar
Stringent assembly parameters (> 99.01% match) that
only allowed reads that differed by a single mismatch from
contigs to be entered into the assembly

Analysis of Breakpoint Regions

The Lasergene program MegAlign was used to compare
breakpoint sequences and identify repetitive sequences
using the dot plot alignment option

Conclusions
Inversion breakpoints in D. pseudoobscura occur
in intergenic regions and do not always lead to
duplication of gene regions
Short repeat sequences are associated with
inversion breakpoints
Common repeats are found within and between
independent inversion breakpoints suggesting that
they play a role in the mutation process
It is possible that these repeats are replicated as
in the staggered cut model

Literature Cited

Preparation and Sequencing of Large

Insert Library for Illumina HiSeq2000
Proximal

NEI, M., 1987 Molecular Evolutionary Genetics. Columbia University

Press, New York.

Derived SC to TL Breakpoint

RANZ, J. M., D. MAURIN, Y. S. CHAN, M. V. GROTTHUSS, L. W.

HILLIER et al., 2007 Principles of genome evolution in the Drosophila
melanogaster species group Public Library of Science Biology 5:
1366-1381.

Breakpoint Region
1000 bp

Randomly Shear Genomic DNA

3 kb DNA fragments and Add Biotin Junction Adaptor

320
280
240
200
160
120
80
40

AR

81
63
64
65
66
67
68 79 76 75 7479 68
69
70
71 72 73 74 79 78 77 76 80
ABCDE ABC ABCD ABCD ABCDE AB CD A CBA C B CD ABCDE ABCD ABC ABC ABC AB A CBA BA CB ABC ABCD

Proximal (PP derived)

Phylogeny Chromosomal Arrangements in

D. pseudoobscura
ST

GA13475

ST

GA13487

500 bp

2000 bp

Ligate Ends of the 3 kb Fragments

160

Staggered-Cut Single-stranded
Break Model (Ranz et al. 2007)

SC

GA17716

GA19155

40

Repeat-mediated Model
(Richards et al. 2005)

5kb

Proximal (ST ancestral)

Derived Arrangement

Genomic DNA

GA24334

Pairing and
Exchange

Santa Cruz (SC)

10,627,111..10,627,437

10,625,908..10,627,095

TL

IGV plot of Tree Line reads at the proximal breakpoint mapped to

the Arrowhead reference strain (reads marked in red flank the breakpoint).

Tree Line (TL)

74B

74C

4000 bp

6000 bp

GA19582

6785 bp

GA14679

Distal

RICHARDS, S. Y. LIU, B. R. BETTENCOURT, P. HRADECKY, S.

LETOVSKY et al., 2005 Comparative genome sequencing of
Drosophila pseudoobscura: Chromosomal, gene and cis-element
evolution. Genome Research 15: 1-18.

1000 bp

Randomly Shear and Isolate 200-500 bp Fragments

Using the Biotin Tag

500 bp

Acknowledgements
2000 bp

GA13539
Genomic DNA

Breakpoint Region
Reads Mapped to Genome

4000 bp

14000 bp

15498 bp

GA24132

Repeat maps of the breakpoint regions. The histogram shows how often bases in the
region match other sequences in the genome. The colored bars indicate repeat blocks
between proximal and distal breakpoint regions.

This work was supported by a grant from the NIH

(Grant Number R01 GM098478) and a PSU
Discovery Grant.