Funding of this work is provided in part by a grant from NSF Plant Genome Research (IOS-1127155).

NOTE: Figure 1 shows the five steps for the array CGH protocol. They are: 1) Preparation of plant materials; 2) Isolation of high quality DNA samples; 3) Labeling and purification of DNA samples; 4) Hybridization, washing, and scanning of whole genome arrays; and 5) CGH data analysis. M. truncatula whole genome tiling arrays contain a total of 971,041 unique oligo probes targeting more than 50,000 genes or gene models in the genome (See Table of Materials). The uniq...

<ol>
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We have developed an array-based CGH platform for the detection and characterization of fast neutron bombardment (FNB)-induced mutants in M. truncatula cv. Jemalong A17. To demonstrate the use of the array CGH method in detecting gene mutations, we performed aCGH analysis of the mutant FN6191, which exhibited a hyper-nodulation phenotype in contrast to wild type plants, when inoculated with S. meliloti Sm1021. For segmentation analysis, a segment was deemed significant if the log2 ratio mean ...

Legumes (Fabaceae) are the third largest family of flowering plants, with many economically important species such as soybean (Glycine max) and alfalfa (Medicago sativa). Legume plants can interact with nitrogen-fixing soil bacteria, generally called Rhizobia to develop root nodules in which the atmospheric dinitrogen is reduced to ammonia for use by the host plant. As such, cultivation of legume crops requires little input of nitrogen fertilizers and thus contributes to sustainable agriculture. Legume crops produce leaves and seeds with high protein content, serving as excellent forage and grain crops. However, cultivated legume species generally have complex genome structures, making functional studies of genes that play key roles in legume-specific processes cumbersome. Medicago truncatula has been widely adopted as a model species for legume studies primarily because (1) it has a diploid genome with a relatively small haploid genome size (~550 Mbp); (2) plants can be stably transformed for gene functional studies; and (3) it is closely related to alfalfa (M. sativa), the queen of forages, and many other economically important crops for translational studies. Recently, the genome sequence of M. truncatula cv Jemalong A17 has been released1,2. Annotation of the genome shows that there are more than 50,000 predicted genes or gene models in the genome. To determine the function of most of the genes in the M. truncatula genome is a challenging task. To facilitate functional studies of genes, a comprehensive collection of mutants in the range of over 150,000 M1 lines has been generated using fast neutron bombardment (FNB) mutagenesis in M. truncatula cv Jemalong A173,4. Fast neutron, a high energy ionization mutagen, has been used in generating mutants in many plant species including Arabidopsis5,6, rice (Oryza sativa)7, tomato (Solanum lycopersicum), soybean (Glycine soja; G. max)8,9, barley (Hordeum vulgare), and Lotus japonicus10. A large portion of mutations derived from FNB mutagenesis are due to DNA deletions that range in size from a few base pairs to mega base pairs9,11. Many phenotype-associated genes have been successfully identified and characterized4,12,13,14,15,16,17,18,19. Previously, molecular cloning of the underlying genes from FNB mutants relied on a map-based approach, which is time consuming and limits the number of mutants to be characterized at the molecular level. Recently, several complimentary approaches including transcript-based methods, genome tiling array-based comparative genomic hybridization (CGH) for DNA copy number variation detection, and whole genome sequencing, have been employed to facilitate the characterization of deletion mutants in diverse organisms including animals and plants20,21,22,23,24,25,26,27,28,29,30,31.
To facilitate the characterization of FNB mutants in M. truncatula, a whole-genome array-based comparative genomic hybridization (CGH) platform has been developed and validated. As reported in animal systems, the array-based CGH platform allows detection of copy number variations (CNVs) at the whole genome level in M. truncatula FNB mutants. Furthermore, lesions can be confirmed by PCR and deletion borders can be identified by sequencing. Overall, the array CGH platform is an efficient and effective tool in identifying lesions in M. truncatula FNB mutants. Here, the array CGH procedure and PCR characterization of deletion borders in an M. truncatula FNB mutant are illustrated.
The following protocol provides experimental steps and information about reagents, equipment and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants. As an example, Medicago truncatula FN6191 mutant was used to identify deletion regions and candidate genes associated with mutant phenotypes. M. truncatula FN6191 mutant, originally isolated from a fast neutron bombardment-induced deletion mutant collection32 (see Table of Materials), exhibited a hyper-nodulation phenotype after inoculation with the soil bacterium, Sihorhizobium meliloti Sm1021, in contrast to wild type plants.

<table>
	<tbody>
		<tr>
			<td>Medicago truncatula genome array, 1 x 1 M</td>
			<td>Agilent</td>
			<td>G4123A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula FN6191 (mutant)</td>
			<td>In house</td>
			<td>FN6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula Jemalong A17 (reference)</td>
			<td>In house</td>
			<td>A17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>1000D</td>
			<td></td>
		</tr>
		<tr>
			<td>SureTag DNA Labeling Kit</td>
			<td>Agilent</td>
			<td>5190-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Random primer</td>
			<td>Agilent</td>
			<td>5190-3399</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>MJ research</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labnet international Inc</td>
			<td>Spectrafuge 24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and Drying Solution</td>
			<td>Agilent</td>
			<td>5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td>
			<td>Agilent</td>
			<td>5188-5380</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber gasket slides</td>
			<td>Agilent</td>
			<td>G2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Agilent</td>
			<td>5190-3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2</td>
			<td>Agilent</td>
			<td>5188-5221</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber, stainless</td>
			<td>Agilent</td>
			<td>G2534A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Agilent</td>
			<td>G2545A</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Columns</td>
			<td>Agilent</td>
			<td>5190-3391</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanner</td>
			<td>Roche</td>
			<td>MS200</td>
			<td></td>
		</tr>
		<tr>
			<td>NimbleScan 2.6</td>
			<td>Roche Nimblegen</td>
			<td>5225035001</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal Map 1.9</td>
			<td>Roche Nimblegen</td>
			<td>Signalmap1.9</td>
			<td></td>
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an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

Figure 2 shows the distribution of normalized log2 ratios of mutant versus WT signals across the whole genome. Analysis of CGH data revealed an approximate 22 kb deletion on chromosome 4 that encompasses the entire SUNN gene33 and several other annotated genes in FN6191 mutant (Figure 2, Figure 3). The candidate deleted region was covered by 73 consecutive ...

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An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

The overall goal of this array-based comparative genomic hybridization protocol is to rapidly identify copy number variations in fast neutron bombardment induced mutants of the legume plant Medicago truncatula. This method helps to answer key questions in the field of legume biology. Such as, facilitating identification of empathogens on low site involved in regulating nodule development in legumes.The main advantage of this technique is that it is of highest repute and sensitive in detecting copy number variations such as deletion mutations in the neutron bombardment mutants of the legume plant Medicago truncatula. Demonstrating this procedure will be myself, and Hongcheng Wang, Host Doctor Fellows from Genetic Lab. Quickly freeze one gram of young leaf tissue collected from single plants in liquid nitrogen.Then, use a mortar and pestle and grind the frozen leaf tissue to fine powder within the liquid nitrogen. Extract the wild type and mutant genomic DNA samples from the fine powder using a DNA isolation kit. Use 500 microliter centrifuge tubes to dilute one microgram of each wild type and mutant genomic DNAs with double distilled water to a final volume of 20 microliters.Then, add five microliters of random primer to the centrifuge tubes. Quickly vortex the tubes, and after a quick spin down place them in a thermocycler for 10 minutes to denature the DNA samples at 98 degrees Celsius. After 10 minutes remove the tubes from the thermocycler, and instantly place them on ice water for five minutes.Then, prepare two labeling mixes and add 25 microliters of labeling mix as one and two to the wild type and mutant DNA tubes respectively. Pipette the DNA and the labeling mix three times, and then briefly spin the tubes. After spinning, incubate the tubes in the thermocycler at 37 degrees Celsius for two hours, and then at 65 degrees Celsius for 20 minutes to inactivate the exo Klenow enzyme.Then, add 430 microliters of One X TE Buffer, and mix the contents in each tube. Next, briefly spin the tubes and transfer the solution in the tubes to purification columns with two milliliter collection tubes. Centrifuge the purification columns at 14, 000 times G for 10 minutes and discard the flow through.Add 480 microliters of One X TE Buffer to each column, and again centrifuge at 14, 000 times G for 10 minutes. Discard the flow through. Transfer each of the wild type and mutant labeled DNAs from the bottom of the column to new centrifuge tubes.After measuring the volume of each of the labeled DNAs with the pipette adjust the final volume to 80 microliters with One X TE Buffer, then measure the concentration of the labeled DNAs in a spectrophotometer. Next, mix equivalent volumes of mutant and wild type DNAs to hybridize probes on a micro array chip for comparative hybridization. Bring the final volume to 160 microliters with One X TE Buffer.Next, prepare the hybridization solution and pipette it three times to mix three agents well with mixed DNA. After briefly spinning the tubes incubate them in a thermocycler at 98 degrees Celsius for 10 minutes, and at 37 degrees Celsius for 20 minutes. Remember to set the temperature to 67 degrees Celsius to preheat the hybridization oven four hours before hybridization.Then, place a gasket slide on the base of the hybridization chamber and load 490 microliters of the hybridization solution onto it after 20 minutes of incubation at 37 degrees Celsius in the thermocycler. To form the hybridization chamber cover the gasket slide with Medicago truncatula genome micro array chip, then cover the hybridization chamber and securely tighten it with the clamps. Incubate the assembled hybridization chamber in the hybridization oven at 67 degrees Celsius for 40 to 48 hours.Meanwhile, prepare two slide washing dishes with 250 milliliters of Washing Buffer One kept at room temperature. Then, make one slide washing dish with Washing Buffer Two kept at 37 degrees Celsius. Then, prepare one slide washing jar with 70 milliliters of acetonitrile, and another slide washing jar with 70 milliliters of stabilization and drawing solution.Place both the slide washing jars in a fume hood at room temperature. After incubation remove the hybridization chamber from the hybridization oven and loosen the chamber clamps to open the cover. Then, remove the micro array chip in the gasket slide and place them on slide washing dish with Wash Buffer One.Isolate the micro array chip from the cover slide. Next, transfer the micro array chip to the second slide washing dish with Wash Buffer One. Using a stir bar, gently stir the solution on a magnetic stir plate at room temperature for five minutes.Place the micro array chip on the slide washing dish with pre-warmed Washing Buffer Two, then stir with the stir bar to wash the solution at 37 degrees Celsius for one minute. Remove the micro array chip and place it in the acetonitrile containing slide washing jar in a fume hood to wash for 30 seconds. Transfer the micro array chip to the slide washing jar with stabilization and drying solution and wash again for 30 seconds.Carefully remove the chip and dry for one minute inside the fume hood. Scan the micro array chip using a scanner under two micrometer resolutions. A signal mapping software was used as an interface to analyze the distribution of normalized log two ratios of mutant versus wild type signals across eight chromosomes.For putative deletions value of log two ratio equal to or less than minus two point five standard deviation is considered. Segmentation analysis of the software demonstrates an estimated 22 kilobase deletion region on chromosome four shown in the graph. Analysis of the deletion borders flanked by the micro array probes show a reduced mean normalized log two ratio.The graph also shows six other annotated genes including the son gene encompassing the deleted region. Son gene is responsible for controlling nodulation in Metecago truncatula. Next, PCR amplification done to confirm the deletion borders shows a one point five kilobase product amplified from FN6191 mutant, but not in the wild type.DNA sequencing was done to show the deletion junction in FN6191 mutant shown in the black arrow. Sequencing was also done that confirmed the location of the deletion borders. Once mastered, this technique can be completed in 72 hours if it is performed properly.To obtain high quality data, remember to keep the ozone concentration low in the room where performing the procedure. Following this procedure, other measures such as polymerase chain reactions of all genome sequencing can be performed to answer additional questions like the size and the copy number variations in the genome. Event and validation of this technique has paved the way for researchers to explore copy number variation they induce to mutants and the natural variants in plant species that are not amenable to insertional mutagenesis such as Garden P.Don't forget that working with acetonitrile, stabilization and drying solution can be extremely hazardous.Precautions, such as wearing eye proctection, avoiding direct contact with the agents, and working careful is absolutely essential.

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7.7K visualizações.  Noble Research Institute. The overall goal of this array-based comparative genomic hybridization protocol is to rapidly identify copy number variations in fast neutron bombardment induced mutants of the legume plant Medicago truncatula. This method helps to answer key questions in the field of legume biology. Such as, facilitating identification of empathogens on low site involved in regulating nodule development in legumes.The main advantage of this technique is that it is of highest repute and sensitive in de...

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