Mapping Maize Gene Mutations with Bulked

Segregant Analysis and Next Generation Sequencing

David Huizinga

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.

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

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.]

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.

Starch Staining Images

CPD-106 from FieldCPD-104 from Greenhouse

Starch Staining ResultsCPD LINE INBRED PHENOTYPE Starch 2013 GH Starch 2013 FD Starch 2015 FD100 04IAB73PS*074B9 B73/MO17 reduced height, red leaves 101 04IAB73PS*080E2 B73/MO17 small plant, withered tassel, chlorosis, red tips 0 2 2104 04IAB73PS*105D3 B73/MO17 small plant, zebra banding 2 1 0106 04IAB73PS*049D6 B73/MO17 small plant, red leaves 3 3 2107 04INW22CW*07643 W22/B73 small plant, red margins, chlorotic sectors 1 3 108 04IAB73PS*174G06 B73/MO17 chlorotic, red leaves 3 3109 04IAB73PS*058A10 B73/MO17 small plant, interveinal chlorosis 1 2 1110 04CAB73SH*203 B73/MO17 small plant 1 0114 04IAB73PS*160C2 B73/MO17 small plant, interveinal chlorosis 1 1 2115 04IAB73PS*116F6 B73/MO17 small plant, interveinal chlorosis 2 2 2117 04IAB73PS*156C2 B73/MO17 small plant, interveinal chlorosis 1 1 1118 04INW22CW*11916 W22/B73 zebra banding 119 04IAB73PS*151B1 B73/MO17 small plant, chlorosis, necrotic sectors 1 2 2120 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 1125 04INW22CW*09071 W22/B73 small plant, red margins 0 1126 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 0130 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 2142 PW03P*1339 B73/MO17 small plant, red margins 3144 04IAB73PS*063H6 B73/MO17 small plant, chlorosis, red margins 2145 04IAB73PS*069G1 B73/MO17 small plant, chlorosis, red leaves 0 2 2146 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 = none1 = light2 = moderate3 = heavy

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 RNaseo Breaks down RNA

• Ethanol Precipitationo Removes sugars, salts, and other contaminants

• Test DNA integrity by running on an agarose geto 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.

DNA Extraction Gel

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:

o Filter: SNPs with probes that map to a single locus in B73 v3o Filter: SNPs that Genome Studio reports with 90% confidence of being homozygous

in each lineo Filter: 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

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.

Sample BSA Peak – CPD-101

BSA ResultsCPD LINE INBRED PHENOTYPE MUT POOL WT POOL BSA CHR BSA START BSA PEAK BSA MEDIAN BSA END100 04IAB73PS*074B9 B73/MO17 reduced height, red leaves 398 100 1 179,626,570 211,584,486 218,762,557 254,020,578101 04IAB73PS*080E2 B73/MO17 small plant, withered tassel, chlorosis, red tips 264 100 1 236,447,060 281,731,919 273,221,884 290,274,714104 04IAB73PS*105D3 B73/MO17 small plant, zebra banding 318 100 9 9,398,848 53,408,546 16,482,743 53,408,546106 04IAB73PS*049D6 B73/MO17 small plant, red leaves 354 100 6 164,174,013 168,639,584 166,807,390 169,215,559107 04INW22CW*07643 W22/B73 small plant, red margins, chlorotic sectors 338 100 10 11,760,399 55,599,709 51,367,933 105,299,259108 04IAB73PS*174G06 B73/MO17 chlorotic, red leaves 329 100 10 635,378 3,165,277 2,766,848 5,878,157109 04IAB73PS*058A10 B73/MO17 small plant, interveinal chlorosis 367 100 5 23,381,744 202,875,583 184,177,706 204,573,951110 04CAB73SH*203 B73/MO17 small plant 278 100 7 56,051,304 56,051,304 122,442,729 158,503,256114 04IAB73PS*160C2 B73/MO17 small plant, interveinal chlorosis 286 100 4 11,904,574 58,981,408 24,809,860 58,981,408115 04IAB73PS*116F6 B73/MO17 small plant, interveinal chlorosis 157 100 5 175,507,053 188,575,650 179,974,728 204,644,709117 04IAB73PS*156C2 B73/MO17 small plant, interveinal chlorosis 571 24 7 163,098,440 173,322,123 171,015,700 176,191,229118 04INW22CW*11916 W22/B73 zebra banding 310 24 119 04IAB73PS*151B1 B73/MO17 small plant, chlorosis, necrotic sectors 275 24 7 173,020,935 174,192,477 174,723,753 176,182,206120 04CAB73SH*244 B73/MO17 small plant, zebra banding 291 100 10 19,596,064 62,170,120 45,278,046 67,441,179122 04CAB73SH*504 B73/MO17 small plant, chlorosis, red margins 272 24 4 4,249,253 58,981,408 77,237,220 174,495,572123 04IAB73PS*064H9 B73/MO17 small plant 190 100 1 18,990,004 93,400,170 69,311,054 103,315,765124 04IAB73PS*061G4 B73/MO17 small plant, chlorotic striping, red leaves 324 100 1 211,671,928 236,447,060 222,182,239 236,447,060125 04INW22CW*09071 W22/B73 small plant, red margins 75 100 1 91,092,573 113,087,333 120,387,045 147,170,136126 04IAB73PS*043A3 B73/MO17 reduced height, red margings 214 100 5 80,997 1,250,179 1,636,043 4,312,814127 04IAB73PS*114C3 B73/MO17 small plant, chlorosis, necrotic margins 209 24 129 04IAB73PS*030A2 B73/MO17 small plant, chlorosis 111 100 5 434,908 3,548,306 1,678,955 4,254,273130 04INW22CW*08484 W22/B73 small plant, interveinal chlorosis, red leaves 404 100 5 174,159,019 195,472,161 190,717,070 204,645,214131 04INW22CW*11100 W22/B73 red leaves 267 100 1 211,671,928 228,779,859 241,222,750 255,758,727132 04INW22CW*11348 W22/B73 small plant, chlorosis, red leaves 244 100 2 1,464,442 2,808,326 3,115,986 5,075,189135 04INW22CW*10901 W22/B73 small plant, chlorosis, red leaves 318 100 3 216,675,004 226,349,989 225,642,793 232,132,496136 04INW22CW*11169 W22/B73 small plant, chlorosis, red leaves 202 100 9 110,950,740 139,961,240 133,765,653 142,191,630137 04INW22CW*11502 W22/B73 small plant, chlorosis, red margins, zebra banding 402 100 1 211,671,928 249,380,498 245,036,329 279,185,192138 04IAB73PS*172D4 B73/MO17 reduced height, zebra banding, interveinal chlorosis 183 100 5 179,974,728 179,974,728 188,023,416 204,644,709142 PW03P*1339 B73/MO17 small plant, red margins 90 24 5 15,564,828 157,184,160 84,505,627 168,713,620144 04IAB73PS*063H6 B73/MO17 small plant, chlorosis, red margins 158 100 5 80,997 3,548,306 1,854,064 4,312,814145 04IAB73PS*069G1 B73/MO17 small plant, chlorosis, red leaves 316 100 5 1,250,179 3,548,306 3,047,497 5,249,766146 04INW22CW*11186 W22/B73 chlorosis 333 24 153 04IAB73PS*077D6 B73/MO17 small plant, chlorosis, red margins 215 24 1 34,410,183 34,410,183 36,994,283 39,253,157160 04IAB73PS*008F4 B73/MO17 small plant, chlorotic, tattered leaves 246 24 165 04INW22CW*1103 W22/B73 gnarled, striped leaves 165 24

Next-Generation Sequencing

Brown, 2012, Next Generation Sequencing

• End Repair• A-Tailing• Adapter Ligation• LM-PCR

• Sonication• 200 BP Fragments

Exome Capture1. 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.

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 sampleb) Duty Factor: 10%

• percentage of time that the ultrasound signal is applied to the samplec) 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.

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.

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.

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.

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.

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)

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.

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.

Clifford Weil LabRoselyn HatchKin LauJacquee BlodgettDavid Schlueter

NSF GrantDavid Braun University of MissouriKaren KochByung-Ho Kang University of FloridaMark Lubkowitz Saint Michael’s CollegeClifford WeilRebecca Doerge Purdue UniversityJiri Adamec University of Nebraska

Past Lab MembersMoriah MassafaroEllen MurchieMeghan AhearnNicole FrancisJohn ReiserLauren Miranda

St. Michael’s CollegeJamie WoodcockYang WangXiqing Ma

Acknowledgements and Thanks