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

Summary

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.

Abstract

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.

Introduction

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 sp....

Protocol

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 unique probes are spaced approximately every 150 base pairs (bp) in exonic regions and 261 bp in intronic regions of the M. truncatula genome.

Results

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 .......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medicago truncatula genome array, 1 x 1 M	Agilent	G4123A
Medicago truncatula FN6191 (mutant)	In house	FN6191
Medicago truncatula Jemalong A17 (reference)	In house	A17
Sulfuric acid	Sigma-Aldrich	320501
DNeasy Plant Mini Kit	Qiagen	69104
Nanodrop Spectrophotometer	Thermo Scientific	1000D
SureTag DNA Labeling Kit	Agilent	5190-3400
Random primer	Agilent	5190-3399
Acetonitrile	Sigma-Aldrich	271004-1L
Thermocycler	MJ research	PTC-200
Centrifuge	Labnet international Inc	Spectrafuge 24D
Stabilization and Drying Solution	Agilent	5185-5979
Oligo aCGH/ChIP-on-chip Hybridization Kit	Agilent	5188-5380
Hybridization Chamber gasket slides	Agilent	G2505
Human Cot-1 DNA	Agilent	5190-3393
Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2	Agilent	5188-5221
Hybridization Chamber, stainless	Agilent	G2534A
Hybridization oven	Agilent	G2545A
Purification Columns	Agilent	5190-3391
Laser scanner	Roche	MS200
NimbleScan 2.6	Roche Nimblegen	5225035001
Signal Map 1.9	Roche Nimblegen	Signalmap1.9