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2016-04-27 Presentation
1. Mapping Maize Gene Mutations
with Bulked Segregant Analysis
and Next Generation Sequencing
David Huizinga
2. Carbon Partitioning Mutants: Background & Reasoning
• The transport of carbon compounds, especially carbohydrate
nutrients, is a critical part of maize development. Sugars are
produced in source leaves and then moved to sink tissues and
organs by way of a complex network of transport molecules and
developmental cues. We aim to reveal novel genes that are
necessary for the regulation and maintenance of this process.
• Affects: stress tolerance, plant development, seed production,
food production, biofuels, and carbon sequestration.
3. Carbon Transport: From Source to phloem to Sink
• Symplasmic: sugars diffuse directly through
plasmodesmata
• Apoplasmic: sugars are pumped out of cells into
the apoplasm via transporters
Background
Braun, 2012, Plant Science
4. Breeding Plan
M0 Mutate IL1 pollen with EMS. [Pollinate IL1 with EMS pollen.]
M1 Heterozygous mutations in IL1. [Self cross.]
M2 Segregating mutations in IL1. [Plant 20. Identify mutants.]
[Random inter-mating within family.]
M3 1:1 mutant allele frequency in IL1. [bulk up seed: collect 1000 kernels.]
[Plant 20. Cross mutants to IL2 – 3 crosses.]
F1 Heterozygous across genome. [Self cross 40 plants.]
F2 Segregating. [Self cross 40 non-mutants: 75% of family.]
{Reduces IL1 background near mutation.}
F3 2/3 rows mutants; 1/3 rows wild type.
Genome heterozygous for IL1 & IL2, favoring IL1 in vicinity of mutation.
[Plant 1600, expect ~267 mutants and 1333 non-mutants per family.]
5. Starch Staining
• Plants produce sugars during the day, then transport them out of
the leaves at night.
• Leave that still have starch accumulation at the end of the night
must have some problem with this process.
• By staining with iodine, these mutant leaves will turn black.
• Basic Method:
• Cut leaves before dawn.
• Clear out pigments using heated ethanol (well ventilated!)
• Treat with iodine solution.
7. Starch Staining Results
CPD LINE INBRED PHENOTYPE Starch 2013 GH Starch 2013 FD Starch 2015 FD
100 04IAB73PS*074B9 B73/MO17 reduced height, red leaves
101 04IAB73PS*080E2 B73/MO17 small plant, withered tassel, chlorosis, red tips 0 2 2
104 04IAB73PS*105D3 B73/MO17 small plant, zebra banding 2 1 0
106 04IAB73PS*049D6 B73/MO17 small plant, red leaves 3 3 2
107 04INW22CW*07643 W22/B73 small plant, red margins, chlorotic sectors 1 3
108 04IAB73PS*174G06 B73/MO17 chlorotic, red leaves 3 3
109 04IAB73PS*058A10 B73/MO17 small plant, interveinal chlorosis 1 2 1
110 04CAB73SH*203 B73/MO17 small plant 1 0
114 04IAB73PS*160C2 B73/MO17 small plant, interveinal chlorosis 1 1 2
115 04IAB73PS*116F6 B73/MO17 small plant, interveinal chlorosis 2 2 2
117 04IAB73PS*156C2 B73/MO17 small plant, interveinal chlorosis 1 1 1
118 04INW22CW*11916 W22/B73 zebra banding
119 04IAB73PS*151B1 B73/MO17 small plant, chlorosis, necrotic sectors 1 2 2
120 04CAB73SH*244 B73/MO17 small plant, zebra banding 1
122 04CAB73SH*504 B73/MO17 small plant, chlorosis, red margins 2
123 04IAB73PS*064H9 B73/MO17 small plant 1 3
124 04IAB73PS*061G4 B73/MO17 small plant, chlorotic striping, red leaves 1 2 1
125 04INW22CW*09071 W22/B73 small plant, red margins 0 1
126 04IAB73PS*043A3 B73/MO17 reduced height, red margings 1 2
127 04IAB73PS*114C3 B73/MO17 small plant, chlorosis, necrotic margins 0 2
129 04IAB73PS*030A2 B73/MO17 small plant, chlorosis 0 0
130 04INW22CW*08484 W22/B73 small plant, interveinal chlorosis, red leaves
131 04INW22CW*11100 W22/B73 red leaves
132 04INW22CW*11348 W22/B73 small plant, chlorosis, red leaves
135 04INW22CW*10901 W22/B73 small plant, chlorosis, red leaves
136 04INW22CW*11169 W22/B73 small plant, chlorosis, red leaves
137 04INW22CW*11502 W22/B73 small plant, chlorosis, red margins, zebra banding
138 04IAB73PS*172D4 B73/MO17 reduced height, zebra banding, interveinal chlorosis 2
142 PW03P*1339 B73/MO17 small plant, red margins 3
144 04IAB73PS*063H6 B73/MO17 small plant, chlorosis, red margins 2
145 04IAB73PS*069G1 B73/MO17 small plant, chlorosis, red leaves 0 2 2
146 04INW22CW*11186 W22/B73 chlorosis
153 04IAB73PS*077D6 B73/MO17 small plant, chlorosis, red margins 1
160 04IAB73PS*008F4 B73/MO17 small plant, chlorotic, tattered leaves
165 04INW22CW*1103 W22/B73 gnarled, striped leaves
0 = none
1 = light
2 = moderate
3 = heavy
8. DNA Extraction Method
• Collect 2 leaf punches per plant and pool them.
• Grind Frozen Leaf Tissue and incubate in CTAB solution:
o Grinding disrupts cell walls, CTAB degrades cell membranes and denatures
proteins
• Add chloroform, mix and spin:
o removes protein, cell walls, membranes
• Treat with RNase
o Breaks down RNA
• Ethanol Precipitation
o Removes sugars, salts, and other contaminants
• Test DNA integrity by running on an agarose get
o Degraded DNA with have a band of small fragments, intact DNA will have a band
at the top of the gel. Also test with a nanodrop.
10. BSA – SNP Selection
• Illumina Maize SNP50 beadchip microarray measures the allele
frequency of 56,110 Single Nucleotide Polymorphisms (SNPs), each
with 2 possible alleles.
• We run each original inbred line on the SNP50 chip.
• Filter the SNPs:
oFilter: SNPs with probes that map to a single locus in B73 v3
oFilter: SNPs that Genome Studio reports with 90% confidence of being
homozygous in each line
oFilter: SNPs that are not monomorphic between the inbred lines (Example,
for one SNP, the allele A is homozygous in one inbred line, and homozygous
for B in the other.
• 26,112 valid SNPs for B73/Mo17
• 18,466 valid SNPs for W22/B73
11. BSA Results
• For SNPs identified as informative, measure the frequency (from 0 to 1) of the B73
allele in both the mutant and wild type sibling pooled DNA
• For the SNPs where the frequency of the B73 (original inbred) allele in the mutant
DNA pool is 0.5 greater than the B73 allele frequency in the WT pool, plot the
chromosome and position on the X axis, and the following ratio on the Y axis:
Frequency of the IL1 allele in the mutant DNA
Frequency of the IL1 allele in the non-mutant DNA
• If the mutant selection was correct and plants are segregating, then Y is expected
to approach 5.0 in the vicinity of the mutation, if only rows with mutants were
sampled, then Y will approach 3.0.
15. Exome Capture
1. Genomic DNA: SeqCap EZ Oligo pool is made against
target regions in the genome.
2. Library Preparation: Standard shot-gun sequencing
library is made from genomic DNA.
3. Hybridization: The sequencing library is hybridized to
the SeqCap EZ Oligo pool.
4. Bead Capture: Capture beads are used to pull down
the complex of capture oligos and genomic DNA
fragments.
5. Washing: Unbound fragments are removed by
washing.
6. Amplification: Enriched fragment pool is amplified by
PCR.
7. Enrichment QC: The success of enrichment is
measured by qPCR at control loci.
8. Sequencing-Ready DNA: The end product is a
sequencing library enriched for target regions, ready
for high throughput sequencing.
2015 Roche NimbleGen, Inc.
16. Sonication
• Covaris Sonicator located in Biochemistry Building.
• With maize genomic DNA, use the following settings to shear the DNA into 200 bp
fragments:
a) Peak Incident Power: 200
• a measure of the instantaneous ultrasonic power applied to the sample
b) Duty Factor: 10%
• percentage of time that the ultrasound signal is applied to the sample
c) Cycles per Burst: 200
• the number of cycles (sinusoidal bursts) of ultrasonic energy to deliver during the on portion
of the duty cycle
d) Time in seconds: 150
• Time that the sample is under a treatment
• Quantify the DNA with a Qubit fluorimeter. It is more accurate than a Nanodrop
spectrophotometer.
17. Library Prep Method
• Bind 200 bp fragments to magnetic beads.
• Wash with ethanol.
• Blunt the ends of the DNA and with an enzymatic reaction.
• Wash with ethanol.
• Add an A-Tail with an enzymatic reaction.
• Wash with ethanol.
• Ligate Illumina adapters with an enzymatic reaction.
• Use PEG/NaCl SPRI solution to filter out any fragment that are not ~300 bp.
• Wash with ethanol then elute DNA from beads.
• Ligation Mediate (LM) PCR to amplify DNA. Protocol called for 7 cycles, but I
found that 11 cycles were necessary to achieve a high enough concentration.
• Bind 300 bp fragments to magnetic beads.
• Wash with ethanol.
18. Exome Capture Method
• To pool multiple libraries, use equal amounts of DNA from each
library, as measured with a Qubit.
• Add oligo blockers to prevent the probes from binding to the
adapters and to non-specific regions of DNA.
• Allow probes to anneal to the DNA for 72 hours at 42°C.
• As with the library prep, perform LM-PCR for 14 cycles.
• Use magnetic beads to wash with ethanol.
19. Exome Capture Analysis
• Trim all sequences to remove adapter sequence and low quality
reads.
• Compare Mo17 to B73 to find all variants.
• Align sample sequences to the B73 genome.
• Find all variants between your sample and B73.
• Compare these variants to other samples and to those from
Mo17.
20. Exome Capture Analysis
• Remove all variants that known differences between Mo17 and
B73
• Remove all variants that are common to multiple lines.
• Remove all variants that are not G to A or C to T changes from
the B73.
• Focus on variants where the G to A or C to T change causes a
stop codon or alters an intron splice site. Otherwise, look at
variants that cause a significant amino acid change.
• Look first at the genes near the peak from the BSA, then look at
the rest of the genome.
• Focus on genes that when disrupted, could cause the phenotype
observed.
21. Exome Capture Results
Coverage of the genome was incomplete, so something went wrong.
Possibilities:
• Probes did not bind to DNA adequately, perhaps because PCR machine did not
hold the correct temperature, or there were too few probes in the mixture.
• PCR amplification favored some DNA fragments over others. Especially since I
added extra cycles to the first LM-PCR.
• GRMZM2G156599
• Iron-phytosiderophore transporter yellow stripe 1 (ZmYS1)
• GRMZM2G365319
• ATP binding protein (post-translational modification)
22. Confirmation of Genes Identified
In order to verify that the gene we identify is truly the gene responsible for
the mutation, several tests can be performed:
• Perform NGS on a single mutant plant and a single wild type sibling plant.
The causative SNP should be 100% in the mutant, but only 0 or 50% in the
wild type. (We do not have the money to do this.)
• Use PCR to amplify the gene from genomic DNA from a mutant plant and a
wild type sibling. Again, only the mutant DNA should have 100% mutation
frequency for the mutant plant.
• Cross plants to known knockout lines to do an allelism test. If the causative
mutation in the test line is in the same gene as the known knockout, then
the offspring will show the phenotype. If the gene is not correct, then the
offspring will be wild type. It is possible some these crosses can occur this
summer.
23. Future Steps
• We are working on a paper that describes the methods above and the
genes that were uncovered.
• This summer, all the lines from the first cohort of CPD plants (100-165)
will be planted to observe and photograph phenotypes. Starch stain
testing would be nice too, but there may not be the resources to do
this. 1 in 3 rows will not have mutants.
• The second cohort (166-223) will be planted this summer as well,
again to observe the phenotypes but also to do crosses within the line
to generate the F3 mapping population. NOTE: DO NOT DO ANY
CROSSES ON ROWS WITHOUT MUTANT PHENOTYPES. EACH
SEGREGATING ROW SHOULD HAVE ABOUT 5 MUTANTS.
24. Clifford Weil Lab
Roselyn Hatch
Kin Lau
Jacquee Blodgett
David Schlueter
NSF Grant
David Braun
University of Missouri
Karen Koch
Byung-Ho Kang
University of Florida
Mark Lubkowitz
Saint Michael’s College
Clifford Weil
Rebecca Doerge
Purdue University
Jiri Adamec
University of Nebraska
Past Lab Members
Moriah Massafaro
Ellen Murchie
Meghan Ahearn
Nicole Francis
John Reiser
Lauren Miranda
St. Michael’s College
Jamie Woodcock
Yang Wang
Xiqing Ma
Acknowledgements and Thanks
