Positioning Maize Knobs Relative to B73 Maize Sequence
Danielle N. Charley1&2, Leslie R. Nelson2&3 , Ethalinda Cannon2, Takeo Angel Kato Y4, and Carolyn J. Lawrence2&5
1Northern
Arizona University, Flagstaff, AZ USA; 2Iowa State University, Ames, IA USA; 3University of New Mexico, Albuquerque, NM USA;
4Colegio
Abstract:
de Postgraduados, Chapingo, MEXICO; 5USDA-ARS
CICGRU, Ames, IA USA
Graph 4
Table 1
In the past knobs have been used to characterize the diversity of
maize accessions and have been key in understanding genetic crossing
over in maize as well as for other problems. Over time, they have become
less widely utilized. During the assembly of the maize genome sequence,
some repeat rich regions were removed which included some of the knob
sequences. Because most knob sequence has been removed an objective of
this research is to find where the knobs might be located relative to
sequence. Regardless of where sequence has been removed, there remain
some copies of the repeats. Using the MaizeGDB website and the tools
offered within the site, I found regions where knob repeats are high,
indicating that the knob itself may reside in that region. This research
will aid researchers in using the maize genome assembly by alerting them
to regions of the assemblies where sections of the genome sequence may
be missing.
Background:
Maize has 10 chromosomes, on some of these chromosomes are
heterochromatic regions called knobs. A heterochromatic region is a
place in the genome where the DNA is tightly compacted and mainly
consists of repeats. These regions contain the 180-bp repeat and the
350-bp TR-1 repeat (Peacock et al., 1981 and Ananiev et al., 1998).
Studies have shown that there may be some association between knob
constitution and phenotypic characteristics including yield (Wellhausen
and Prywer 1954; Moll et al., 1972; and Chughtai and Steffensen 1987) .
Knobs also were used by Barbara McClintock as cytological markers in
her seminal discovery that links genetic crossing over with physical
crossovers observed in chromosomes (Creighton, H., and McClintock
1931). There has been little research done which associates knob
locations with the genomic sequence. TA Kato Y has been studying maize
knobs for many years. Figure 1 is an idiogram of the maize karyotype,
showing where he has observed knobs to exist (Kato unpublished). The
goal of the project was to mark positions on the genome where the knobs
may reside.
Data TA Kato Y collected for chromosome 1 from various maize lines as well as the conversion of micrometer measurements to
cytological map units for those data.
Figure 2
Graph 1
Conclusions:
Researchers understand the maize genome in many ways: cytologically,
genetically, and sequentially. Placing knob data onto the various maps
on all map types will help researchers to understand their placement
across all major paradigms of biological understanding. Because all
the work done here is inferential, the knob locations relative to the
genome sequence represent hypotheses for location and must be further
tested. This work serves as a framework for understanding where knobs
may lie relative to markers mapped to the maize genome sequence.
Materials and Methods:
There are three different maps types used in this project: a genetic
map, a sequence map, and a cytological map. All three of these have
their own units: centiMorgan (cM), base pair (bp), and centiMcClintock
(cMC). Each map type is collinear but each unit type is different. A cM
is a statistical measure of distance based on segregation of traits in
offspring of two inbred parents. A bp is a single nucleotide in a DNA
sequence. A cMC is the percentage distance of a locus from the
centromere to the end of the locus arm.
Cytological representation of the knobs in the first sample on chromosomes 1-10
given TA Kato Y’s data.
B73 RefGen_v1- reference genome assembly for this research, version 1
assembly of the B73 maize genome (Schnable et al., 2009).
This graph is based on the IBM2 2008 Neighbors Map from
MaizeGDB (cM). This is a genetic map showing where knobs
might be using genetic data rather than cytological
measurements. The IBM2008 map is a very common genetic map
used in maize genetics This representation was created by
drawing the genetic length of the chromosome, finding the
repeat in MaizeGDB and reporting the genetic coordinates,
then centromeres were added. Although only knob locations
are shown here, many other loci including genes are
available on the full IBM2 2008 Neighbors map.
MaizeGDB- Maize Genetics and Genomics Database, used for data and
visual analysis (Sen et al., 2009).
BLAST- Used to align knob repeat sequence on B73 genome assembly
(Altschul et al., 1997).
Graph 2
Graph 3
CViT- Used for creating map images (Cannon and Cannon, 2005).
GBrowse- A genome browser used for viewing features on genomic sequence
(Stein et al., 2002).
Locus Lookup- Searches for genomic coordinates of a locus (Andorf et
al., 2010).
IBM2 2008 Neighbors map- Provided genetic coordinates for knobs
(Schaeffer et al., 2008).
180 bp and TR-1- repeat sequence used to identify knob regions
(GenBank).
Results:
Figure 1
Sequence map (bp) created using MaizeGDB and Locus Pair
Lookup. Locus Pair Lookup finds knobs on the different
chromosomes and determines ranges for their locations
based upon probe locations on the genome assembly and/or
loci that are on either side of the knob on genetic maps.
Those coordinates are used to approximate where the knob
might be. The red are the ranges of where a knob might be
and the black are the centromeres.
Karyotype from T.A. Kato Y showing knob positions on chromosomes 1-10.
Sequence map (bp) with full BLAST results. Repeats within 500 bp of
each other are collapsed into regions. This map was created using the
BLAST software. We took the sequence for the 180 base pair repeat and
the TR-1 repeat and used the command line version of BLAST to find
matches across the entire maize genome assembly. From this we massaged
the data based upon percent identity and only show matches that were 90%
identical or higher. We then converted those results into a gff file.
The red regions are the BLAST hits and the black are the centromeres.
Sequence map (bp) generated using GBrowse and BLAST to estimate
where knobs are located. We used BLAST to search for clusters of
knob repeats and uploaded hits to MaizeGDB’s instance of
Gbrowse, which is a common software package for genome
visualization. In this representation there could be errors
because during the sequencing process and again for assembly,
the Maize Genome Sequencing Consortium intentionally removed
repetitive elements. This was done for sequencing because the
goal was to sequence only genic regions of the genome. For
assembly, repetitive elements were removed because it is
difficult to correctly assemble highly repetitive regions
correctly. The red regions are the knob hits that were found and
the black regions represent centromeres.
References:
1. Chughtai, S.R. and Steffensen, D.M. (1987) Maize Genetics Cooperation
News Letter 61, 98-99.
2. Creighton, H., and McClintock, B. 1931 A correlation of cytological
and genetical crossing-over in Zea mays. PNAS 17:492–497.
3. Durbin, R., Eddy, S., Krough, A., and Mitchison, G. (1998).
Biological Sequence Analysis: Probabilistic Models of Protein and
Nucleic Acid. Cambridge University Press, New York,NY.
4. Kato, Takeo Angel. Cytological Studies Of Maize [Zea Mays L.] and
Teosinte [Zea Mexican (Scrader) Kuntze] In Relation to Their Origin
And Evolution. Massachusetts: University of Massachusetts, 1975.
5. Lawrence, Carolyn J., Seigfried, Trent E., Bass, Hank W., and
Anderson, Lorinda K.(2006). Predicting Chromosomal Locations of
Genetically Mapped Loci in Maize Using the Morgan2McClintock
Translator. Genetics Society of America.
6. Moll, R.G., Hansen, W.D., Levings, C.S., and Ohta, Y. (1972) Crop
Sci. 12, 585-589.
7. Peacock, W.J. Dennis, E.S., Rhoades, M.M., and Pryor, A.J. Highly
Repeated DNA Sequence Limited to Knob Heterochromatin in Maize. PNAS
USA 1981 pg. 4490.
8. Sen, TZ, Andorf, CM, Schaeffer, ML, Harper, LC, Sparks, ME, Duvick,
J, Brendel, VP, Cannon, E, Campbell, DA, Lawrence, CJ. (2009)
MaizeGDB becomes 'sequence-centric' Database. 2009:Vol. 2009:bap020.
9. Wellhausen, E.J. and Prywer, C. (1954) Agron J. 46,507-511.
10.Sen, TZ, Andorf, CM, Schaeffer, ML, Harper, LC, Sparks, ME, Duvick,
J, Brendel, VP, Cannon, E, Campbell, DA, Lawrence, CJ. (2009)
MaizeGDB becomes 'sequence-centric' Database. 2009:Vol. 2009:bap020.
11.Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang
Z, Miller W, Lipman DJ Nucleic Acids Res. 1997 Sep 1;25(17):3389-402.
12.Cannon, EK; Cannon SB. (2005) CViT: Chromosome Visualization Tool,
Unpublished
13.The generic genome browser: a building block for a model organism
system database. Stein LD, Mungall C, Shu S, Caudy M, Mangone M, Day
A, Nickerson E, Stajich JE, Harris TW, Arva A, Lewis S. Genome Res.
2002 Oct;12(10):1599-610.
14.The Locus Lookup tool at MaizeGDB: identification of genomic regions
in maize by integrating sequence information with physical and
genetic maps. Andorf CM, Lawrence CJ, Harper LC, Schaeffer ML,
Campbell DA, Sen TZ. Bioinformatics. 2010 Feb 1;26(3):434-6.
15.Schaeffer (Polacco), ML; Sanchez-Villeda, H; Coe, E. (2008) IBM2
2008 Neighbors, Unpublished
16.GenBank accessions: DQ186871.1, M32528.1
Acknowledgments:
As a Native American Outreach Program participant, I am very grateful
to the sponsors that have made this program possible. A special thanks
to my Mentors and Graduate Student Mentors who have put the time and
effort into helping me grow as researcher and student. List of Sponsors:
Carolyn Lawrence, Ethalinda Cannon, Trent Moore, Mary De Baca, Aurelio
Curbelo, Jovaughn Barnard, Dustin Thunder Hawk, Ranelle White Buffalo,
George Washington Carver Internship, National Science Foundation, USDAARS, and Iowa State University.