Hexaploid wheat, 2-fold pooling - Gene Target …...TILLINGfor Mutation Discovery in Rice, Wheat,...

Dayna Loeffler, Jessica Mullenberg, Aaron Holm Arcadia Biosciences, Inc., 410 W. Harrison St., Suite 150, Seattle, WA 98119 Deepak Dibya, Jeremy Kenseth Advanced Analytical Technologies, Inc., 2711 South Loop Dr., Suite 4150, Ames, IA 50010 ABSTRACT: TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput platform for uncovering SNPs (single nucleotide polymorphisms) in genes of interest. These SNPs can be introduced using chemical mutagens or can be uncovered by comparing different varieties or populations of plants and animals (EcoTILLING, Comai et al., 2004). At Arcadia Biosciences, we have mutagenized DNA libraries in many crops including tetraploid and hexaploid wheat, rice, lettuce, soybean, castor and tomato. Since 2002, we have discovered more than 4,600 SNPs in multiple crops using the Li-Cor 4200 platform for mutation detection. Here we have evaluated a new platform that is high-throughput, robust and easily amenable as well as having a low cost per sample analyzed. Advanced Analytical’s AdvanCE™ FS96 system has been shown to separate enzymatically cleaved DNA fragments in various ratios of mutant to wild type DNA in a 96-well high-throughput format in less than an hour per run. We evaluated the ability of the AdvanCE™ FS96 system to detect over 100 different induced mutations in 5 different crops with a variety of pooling strategies. We found the AdvanCE™ FS96 system to be very effective at identifying SNPs. RESULTS: 6-fold pooling, diploid organisms Rice: The same mutation, on the left heterozygous and on the right homozygous Hexaploid wheat, 2-fold pooling Hexaploid wheat, 4-fold pooling Soybean: 2 identical genes amplified (equal to 12-fold pooling) Lettuce: 2 mutations, both homozygous Li-Cor & FS96 COMPARISON: Tomato, 6-fold pooled heterozygous SNP Li-Cor 700 channel Li-Cor 800 channel Advanced Analytical gel view, one capillary Advanced Analytical, capillary view Full length PCR Lower marker Upper marker SNP: cut 1 520bp SNP: cut 2 650bp The 700 nm and the 800 nm Li-Cor images show 6 lanes of an assay. The Advanced Analytical images show one capillary, which was loaded with PCR product that corresponds to the lane in the Li-Cor image that contains the highlighted SNP. The upper and lower markers in the Advance Analytical images are used to normalize the capillaries. The full length PCR product for these samples is 1200bp. Each lane has a SNP. The sizes are listed in the chart above. cut 1 (bp) cut 2 (bp) F01 900 300 F02 900 300 F03 630 570 F04 545 655 F05 850 350 F06 250 950 F07 400 800 F08 465 735 F09 465 735 F10 770 430 F11 920 280 F12 500 700 The image to the left shows the gel view, and the image above the capillary view for a 4-fold pooled, hexaploid, wheat sample with two SNPs. Cut fragments are expected at roughly 125 &1075bp and 290 & 910bp. The faint peak at 620bp is unexpected. Peaks annotated with peak height METHODS: Normalized DNA samples are pooled. Pooling is determined for each species based on the maximum amount possible that will still allow for detection of the majority of mutations. PCR and heteroduplex formation is performed according to published protocols. For wheat, homoeolog specific primers are used. Next, a portion of PCR product is subjected to enzymatic digestion, followed by dilution with a buffer and subsequently electrophoresed for less than 1 hour on AdvanCE™ FS96 system. TILLING for Mutation Discovery in Rice, Wheat, Lettuce, Tomato and Soybean Using the AdvanCE™ FS96 High-Throughput Capillary Electrophoresis System P101 CONCLUSIONS: The AdvanCE™ FS96 system is capable of detecting SNPs in multiple species, with a sensitivity equal to or greater than the Li-Cor platform. The AdvanCE™ FS96 system offers multiple advantages over the Li-Cor 4200 system, including a shorter assay time and a smaller environmental footprint with no need for acrylamide gels or formamide.

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Transcript of Hexaploid wheat, 2-fold pooling - Gene Target …...TILLINGfor Mutation Discovery in Rice, Wheat,...

Page 1: Hexaploid wheat, 2-fold pooling - Gene Target …...TILLINGfor Mutation Discovery in Rice, Wheat, Lettuce, Tomato and Soybean Using the AdvanCE!"FS96 High-Throughput Capillary Electrophoresis

ABSTRACT: TILLING (Targeting Induced Local Lesions IN Genomes)is a high-throughput platform for uncovering SNPs (single nucleotidepolymorphisms) in genes of interest. These SNPs can be introducedusing chemical mutagens or can be uncovered by comparing differentvarieties or populations of plants and animals (EcoTILLING, Comai etal., 2004). At Arcadia Biosciences, we have mutagenized DNAlibraries in many crops including tetraploid and hexaploid wheat, rice,lettuce, soybean, castor and tomato. Since 2002, we have discoveredmore than 4,600 SNPs in multiple crops using the Li-Cor 4200 platformfor mutation detection. Here we have evaluated a new platform that ishigh-throughput, robust and easily amenable as well as having a lowcost per sample analyzed. Advanced Analytical’s AdvanCE™ FS96system has been shown to separate enzymatically cleaved DNAfragments in various ratios of mutant to wild type DNA in a 96-wellhigh-throughput format in less than an hour per run. We evaluated theability of the AdvanCE™ FS96 system to detect over 100 differentinduced mutations in 5 different crops with a variety of poolingstrategies. We found the AdvanCE™ FS96 system to be very effectiveat identifying SNPs.

RESULTS:6-fold pooling, diploid organisms

Rice: The same mutation, on the left heterozygousand on the right homozygous

Hexaploid wheat, 2-fold pooling

Hexaploid wheat, 4-fold pooling

Soybean:2 identical genesamplified (equal to12-fold pooling)

Lettuce:2 mutations, bothhomozygous

Li-Cor & FS96 COMPARISON: Tomato, 6-fold pooled heterozygous SNP

Li-Cor700

channel

Li-Cor800

channelAdvanced Analytical

gel view, one capillary

Advanced Analytical, capillary view

Full length PCR

Lower marker Upper marker

SNP: cut 1520bp

SNP: cut 2650bp

The 700 nm and the 800 nm Li-Corimages show 6 lanes of an assay.The Advanced Analytical imagesshow one capillary, which was loadedwith PCR product that corresponds tothe lane in the Li-Cor image thatcontains the highlighted SNP. Theupper and lower markers in theAdvance Analytical images are usedto normalize the capillaries.

The full length PCR product for thesesamples is 1200bp. Each lane has a SNP.The sizes are listed in the chart above.

cut 1(bp)

cut 2(bp)

F01 900 300F02 900 300F03 630 570F04 545 655F05 850 350F06 250 950F07 400 800F08 465 735F09 465 735F10 770 430F11 920 280F12 500 700

The image to the left shows the gel view, and theimage above the capillary view for a 4-fold pooled,hexaploid, wheat sample with two SNPs. Cut fragmentsare expected at roughly 125 &1075bp and 290 & 910bp.The faint peak at 620bp is unexpected.

Peaks annotated withpeak height

METHODS: Normalized DNA samples are pooled.Pooling is determined for each species based onthe maximum amount possible that will still allowfor detection of the majority of mutations. PCRand heteroduplex formation is performedaccording to published protocols. For wheat,homoeolog specific primers are used. Next, aportion of PCR product is subjected to enzymaticdigestion, followed by dilution with a buffer andsubsequently electrophoresed for less than 1 houron AdvanCE™ FS96 system.

TILLING for Mutation Discovery in Rice, Wheat, Lettuce, Tomato andSoybean Using the AdvanCE™ FS96 High-Throughput CapillaryElectrophoresis System

P101

CONCLUSIONS:The AdvanCE™ FS96 system is capable of detecting SNPs in multiplespecies, with a sensitivity equal to or greater than the Li-Cor platform.The AdvanCE™ FS96 system offers multiple advantages over the Li-Cor4200 system, including a shorter assay time and a smaller environmentalfootprint with no need for acrylamide gels or formamide.