Creation and Genomic Analysis of Irradiation Hybrids in Populus

Matthew Zinkgraf1, Kathy Haiby2, Meric C. Lieberman3, Luca Comai3, Isabelle M. Henry3, Andrew Groover4

1 Department of Computer Science, University of California, Davis, California, 2 Greenwood Resources, Portland, Oregon, 3 Genome Center, University of California Davis, California, 4 Department of Plant Biology, University of California, Davis, California
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20025
Online Posting Date:  June, 2016
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Abstract

Establishing efficient functional genomic systems for creating and characterizing genetic variation in forest trees is challenging. Here we describe protocols for creating novel gene‐dosage variation in Populus through gamma‐irradiation of pollen, followed by genomic analysis to identify chromosomal regions that have been deleted or inserted in each F1 individual. Irradiated pollen is used in a controlled, interspecific cross to create F1 progeny that carry deletions and insertions of chromosomal regions. These insertions and deletions result in novel changes in gene dosage that in turn affect both qualitative and quantitative phenotypic traits. The protocols described here outline the processes involved in optimizing irradiation levels and performing controlled crosses, sowing seed and propagating seedlings, and genome‐wide sequence‐based analysis of deletions and insertions in the F1 progeny. The same approach could be applied to other vegetatively propagated species. © 2016 by John Wiley & Sons, Inc.

Keywords: forest trees; gene dosage; genomics; mutagenesis; Populus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Pollen Irradiation and Performing Controlled Crosses
  • Basic Protocol 2: Collecting, Cleaning, and Sowing Seed
  • Basic Protocol 3: Detection of Large‐Scale Insertion and Deletion Mutations
  • Basic Protocol 4: Genotyping of Mutations
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pollen Irradiation and Performing Controlled Crosses

  Materials
  • Fertile male and female branches from individuals to be crossed
  • Dip‐N'Grow (http://www.dipngrow.com/)
  • Potting soil (Sunshine Mix 4; http://sunshineadvanced.com/)
  • FloraLife FS1107 (http://www.floralife.com/)
  • Five‐gallon buckets, to hold male branches during pollen collection process
  • Glassine bags, 6 × 3.5 × 13‐in. (Brown Paper Goods, Waukegan, IL)
  • Desiccator and desiccant
  • False‐bottom five‐gallon buckets for rooting female branches (preferably opaque, to allow visualization of root development)
  • Temperature probes (placed inside each female bucket to monitor temperatures)
  • Pail heaters (Grainger, cat. no. SDH5120‐3; http://www.grainger.com/)
  • Trash bags
  • Pollen‐tight breeding enclosures
  • Scintillation vials (Fisher Scientific, cat. no. 03‐227‐4)
  • Gamma‐irradiation source
  • Pollen applicators or atomizers

Basic Protocol 2: Collecting, Cleaning, and Sowing Seed

  Materials (also see protocol 1)
  • Fertilized female branches (from protocol 1)
  • Potting soil (Sunshine Mix #4)
  • Subdue MAXX Fungicide (Syngenta) Mefenoxam is the active ingredient. Follow manufacturer's instructions for application, hazards, required personal protective equipment, and disposal.
  • Gnatrol biological larvicide (active ingredient Bacillus thuringiensis)
  • Glassine bags, 6 × 3 ½ × 13‐in. (Brown Paper Goods, Waukegan, IL)
  • Soil sieves
  • Yellow Cone‐tainers and leach racks (SC10; https://www.stuewe.com/products/rayleach.php)
  • Misting bench

Basic Protocol 3: Detection of Large‐Scale Insertion and Deletion Mutations

  Materials
  • Software
  • Linux or MacOS, Unix‐based computer system
  • Python2.6 or Python2.7
  • Allprep (if necessary)
  • BWA version 0.5.7 (http://bio‐bwa.sourceforge.net/)
  • SAMtools version 1.18‐dev or 1.19 (http://SAMtools.sourceforge.net/)
  • BWA‐DoAll (http://comailab.genomecenter.ucdavis.edu/index.php/Bwa‐doall)
  • Bin‐by‐sam (http://comailab.genomecenter.ucdavis.edu/index.php/Bin‐by‐sam)
  • Data
  • High‐quality demultiplexed FASTQ read sequence files (“.fq” files).
  • Reference genomic file in FASTA format

Basic Protocol 4: Genotyping of Mutations

  Materials
  • Software
  • Linux or MacOS, Unix‐based computer system
  • Python2.6 or Python2.7
  • Mpileup generation and parsing tools (http://comailab.genomecenter.ucdavis.edu/index.php/Mpileup)
  • CallAllelesAB (http://comailab.genomecenter.ucdavis.edu/index.php/Call_Alleles)
  • indel‐allelic‐ratio.py (http://comailab.genomecenter.ucdavis.edu/index.php/Indel_allelic_ratio)
  • Data
  • SAMtools sorted BAM files (.sorted.bam; see protocol 3)
  • Reference genome file in FASTA format (.fa)
  • List of parental SNP alleles
  • List of indels (identified in protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
  Bissof, S. and Gullberg, U. 1996. Poplar breeding and selection strategies. National Research Council of Canada, Ottawa, Ontario, Canada.
  Dickmann, D.I. and Isebrand, J.G. 2001. Poplar clones: An introduction and caution. National Research Council of Canada, Ottawa, Ontario, Canada.
  Eckenwalder, J.E. 2001. Keys to species and main crosses. National Research Council of Canada, Ottawa, Ontario, Canada.
  Henry, I.M., Zinkgraf, M.S., Groover, A.T., and Comai, L. 2015. A system for dosage‐based functional genomics in poplar. Plant Cell 27:2370‐2383 doi: 10.1105/tpc.15.00349.
  Li, H. and Durbin, R. 2009. Fast and accurate short read alignment with Burrows‐Wheeler transform. Bioinformatics 25:1754‐1760. doi: 10.1093/bioinformatics/btp324.
  Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., and Durbin, R. 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25:2078‐2079. doi: 10.1093/bioinformatics/btp352.
  Mock, K.E., Callahan, C.M., Islam‐Faridi, M.N., Shaw, J.D., Rai, H.S., Sanderson, S.C., Rowe, C.A., Ryel, R.J., Madritch, M.D., Gardner, R.S., and Wolf, P.G. 2012. Widespread triploidy in Western North American aspen (Populus tremuloides). PLoS One 7:e48406. doi: 10.1371/journal.pone.0048406.
  Riemenschneider, D.E., Stanton, B.J., Vallee, G., and Perinet, P. 2001. Poplar breeding strategies. National Research Council of Canada, Ottawa, Ontario, Canada.
  Stanton, B and Villar, M. 1996. Controlled reproduction of Populus. In Biology of Populus and its implications for management and conservation. Stettler, Bradshaw, Heilman, and Hinckley, Eds. NRC Research Press, Ottawa, Ontario, Canada.
  Stettler, R.F., Zsuffa, L., and Wu, R. 1996. The role of hybridization in the genetic manipulation of Populus. NRC Press, Ottawa, Ontario, Canada.
Internet Resources
  https://www.python.org/
  Python language website.
  http://samtools.sourceforge.net/
  SAMtools software website.
  http://bio‐bwa.sourceforge.net/
  BWA software website.
  http://comailab.genomecenter.ucdavis.edu/index.php/Bwa‐doall
  BWA‐DoAll software website.
  http://comailab.genomecenter.ucdavis.edu/index.php/Bin‐by‐sam
  Bin‐by‐sam software website.
  http://comailab.genomecenter.ucdavis.edu/index.php/Mpileup
  Mpileup generation and parsing tools website.
  http://comailab.genomecenter.ucdavis.edu/index.php/Call_Alleles
  CallAllelesAB software website.
  http://comailab.genomecenter.ucdavis.edu/index.php/Indel_allelic_ratio
  Indel‐allelic‐ratio software website.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library