Development and validation of Thinopyrum elongatum expressed molecular 1

markers specific for the long arm of chromosome 7E 2

3

Lulu Gou, Jiro Hattori, George Fedak, Margaret Balcerzak, Andrew Sharpe, Paul 4

Visendi, David Edwards, Nicholas Tinker, Yu-Ming Wei, Guo-Yue Chen and Thérèse 5

Ouellet* 6

Jiro Hattori, George Fedak, Margaret Balcerzak, Nicholas Tinker, Thérèse Ouellet 7

Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 8

Carling Ave, Ottawa, ON K1A 0C6, Canada; 9

Lulu Gou, Yuming Wei, Guoyue Chen 10

Triticeae Research Institute, Sichuan Agricultural University, 211 Huimin Road, 11

Wenjiang, Chengdu, Sichuan 611130, P.R.China; 12

Andrew Sharpe 13

Wheat Improvement Flagship, National Research Council of Canada, 110 Gymnasium 14

Place, Saskatoon, SK S7N 0W9; 15

Paul Visendi 16

School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 17

4072 Australia. 18

David Edwards 19

School of Plant Biology, University of Western Australia, WA 6009, Australia 20

Received ---------------. *Corresponding author (therese.ouellet@agr.gc.ca). 21

1

22

Abbreviations: BC1F2, backcross-one second filial; CS, wheat variety Chinese Spring; 23

CS-7EL, Chinese Spring ditelocentric addition line containing the chromosome 7EL; 24

FHB, fusarium head blight. 25

2

ABSTRACT 26

27

The ditelocentric addition line CS-7EL of the spring wheat (Triticum aestivum) cultivar 28

Chinese Spring (CS) contains the long arm of the chromosome 7E from Thinopyrum 29

elongatum (CS-7EL), which confers high resistance to fusarium head blight. It is of 30

great interest to breeders to integrate the resistance locus (loci) from Th. elongatum 31

into commercial wheat varieties. The objectives of this study were to identify 32

candidate genes expressed from the 7EL chromosome of CS-7EL, to develop 33

7EL-specific molecular markers, and to validate their usefulness to characterize 34

recombination between one of the group 7 chromosomes of wheat and Th. 35

elongatum. High-throughput sequencing of Fusarium graminearum-infected and 36

control CS and CS-7EL cDNA libraries was performed using RNA-Seq. A stepwise 37

bioinformatics strategy was applied to assemble the sequences obtained from 38

RNA-Seq and to create a conservative list of candidate genes expressed from the 39

foreign chromosome 7EL. PCR primer pairs were designed and tested for 135 40

candidate genes. A total of 48 expressed molecular markers specific for the 41

chromosome 7EL were successfully developed. Screening of progenies from two 42

BC1F2 families from the cross CS-7E(7D)×2*CSph1b showed that these markers are 43

useful to characterize recombination events between the chromosomes 7D from 44

wheat and 7E from Th. elongatum. 45

46

3

INTRODUCTION 47

Many accessions of tall wheatgrass Thinopyrum elongatum (Host) D.R. Dewey (also 48

called Lophopyrum elongatum, Agropyron elongatum and Elytrigia elongatum) 49

(2n=14, EE genome) tolerate many abiotic and biotic stresses (Sharma et al., 1989; 50

Taeb et al., 1993; Friebe et al., 1996; Bai and Shaner, 2004; Lammer et al., 2004; 51

Shen et al., 2004; Colmer et al., 2006; Garg et al., 2009; Hussein et al., 2014). Since 52

the last century, successful crosses of E-genome containing species with wheat have 53

facilitated the transfer of desired traits and genes to wheat for genetic improvement 54

(Dvořák and Knott, 1974; Mujeeb-Kazi et al., 2008; Wang et al., 2011). Up to now, Th. 55

elongatum has been used as a donor to transfer genes to bread wheat for 56

improvement of seed quality (Lammer et al., 2004; Garg et al., 2009), as well as 57

resistance to different stresses, including salinity (Colmer, et al., 2006; Hussein et al., 58

2014), waterlogging (Taeb et al., 1993), and diseases such as barley yellow dwarf, 59

leaf rust, and Fusarium head blight (FHB) (Sharma et al., 1989; Friebe et al., 1996; 60

Shen et al., 2004; Zhang et al., 2005) . 61

Fusarium head blight is a devastating disease, caused mainly by the fungus 62

Fusarium graminearum Schwabe (Gibberella zeae (Schwein.) Petch). Fusarium head 63

blight leads to a reduction in grain production in wheat and other small grain cereals, 64

lower grain quality and accumulation of mycotoxins, including deoxynivalenol, 65

potentially making the grain toxic as food and feed (McMullen et al., 1997; Bai and 66

Shaner, 2004). The long arm of chromosome 7E of Th. elongatum has been shown 67

4

to carry the major determinant(s) of resistance to FHB (Shen et al., 2004; Shen and 68

Ohm, 2006; Shen and Ohm, 2007; Wang et al., 2010; Miller et al., 2011; Fu et al., 69

2012). 70

Genetic markers developed for wheat, including genomic- and expressed 71

sequence tags (EST)-derived single sequence repeats (SSRs) and cleaved amplified 72

polymorphic sequences (CAPS), have been useful for mapping traits on Th. 73

elongatum chromosome 7E (You et al., 2003; Shen et al., 2004; Shen and Ohm, 2006; 74

Hu et al., 2012). More recently, 7E specific markers were designed based on specific 75

length amplified fragment sequencing (SLAF) of Th. elongatum genomic DNA 76

fragments (Chen et al., 2013). However, more efforts are needed to develop a 77

sufficient number of Th. elongatum-specific markers to allow genetic mapping and 78

introgression of loci for the desired traits present in Th. elongatum, including 79

resistance to FHB. 80

RNA-Seq is a technology based on high-throughput sequencing which can allow 81

an entire transcriptome to be surveyed in a deep and quantitative manner. However, 82

RNA-Seq data analysis is challenging and costly, requiring specialised bioinformatics 83

software and large computational resources. This is especially true when no 84

reference genomic sequence is available for use as an assembly template (Garg and 85

Jain, 2013). For T. aestivum, which is composed of three large subgenomes and 86

contains more than 80% repetitive sequence, a stepwise assembly process was 87

recently proposed (Duan et al., 2012). However, such analysis is still a large 88

5

undertaking for laboratories with limited computational resources. The 89

bioinformatics strategy presented here for sequence assembly relied on modest 90

computer capacity and on software available in most molecular biology laboratories. 91

Our objectives were to identify differentially expressed candidate gene 92

sequences associated to the Th. elongatum chromosome 7EL through RNA-Seq 93

analysis of heads from spring wheat variety Chinese Spring (CS) compared to those 94

from the ditelocentric addition line CS-7EL when treated with either F. graminearum 95

or water, and to develop, from these sequences, molecular markers diagnostic of the 96

presence of the 7EL fragment. We propose that such markers would improve the 97

accuracy of gene discovery and introgression. 98

99

6

MATERIALS AND METHODS 100

Plant materials 101

Genetic stocks used in this work (Table 1) included Chinese Spring (CS), a CSph1b 102

mutant (Sears, 1977), CS-Th. elongatum ditelocentric addition lines, CS-Th. 103

elongatum disomic addition lines, and CS-Th. elongatum disomic substitution lines 104

(Dvořák and Knott, 1974; Dvořák, 1979; Dvořák, 1980). A cross was made between 105

the CSph1b mutant (confirmed homozygous for ph1b) and the substitution line 106

CS-7E(7D) using pollen from a single plant of each genotype. F1 plants were 107

backcrossed with the CSph1b mutant and 556 BC1F1 (backcross-one first filial) plants 108

were obtained; those were screened with the marker PSR574 (specific for ph1b; 109

Roberts et al., 1999) and 286 plants were identified as homozygous recessive for 110

ph1b. The homozygous recessive plants were point-inoculated with F. graminearum 111

(Somers et al., 2003) and 124 had a FHB-resistance phenotype. Meiosis was 112

examined in the pollen mother cells of the BC1F1 plants with an FHB-resistance 113

phenotype for normal chromosome pairing, which was indicative of an induction of 114

chromosome pairing between the homoeologous chromosomes 7E and 7D. Ten 115

plants were selected on the basis of normal chromosome pairing and best scores for 116

FHB resistance. BC1F2 progenies (47 individuals) from two of those plants, 58-5 and 117

64-8, were used in this work. 118

119

Inoculation of plants with F. graminearum 120

7

A highly virulent isolate of F. graminearum, DAOM 180378 (Canadian Collection of 121

Fungal Cultures, Agriculture and Agri-Food Canada, Ottawa, Canada) was used for 122

plant inoculations. For spore production, a 3 mm plug of F. graminearum was 123

extracted from a fresh SNA (Spezieller-Nährstoffar Agar) plate culture and 124

transferred to 50 mL of CMC (carboxymethylcellulose) medium (Cappellini and 125

Peterson, 1965); cultures were shaken at 28 °C in the dark for 3 d and conidia were 126

harvested by passing the culture through sterile Miracloth (Calbiochem) to separate 127

mycelium from spores. Macroconidia were washed with sterile water three times by 128

centrifugation at 4,000 rpm for 10 min. 129

Plants from CS, a moderately FHB-susceptible variety, and CS-7EL, a 130

FHB-resistant ditelocentric addition line which contains the long arm of chromosome 131

7E from Th. elongatum (Wang et al., 2010), were grown in a controlled-environment 132

cabinet under a 16 h light (20 °C, about 750 μmol photons/m2 x s) and 8 h dark (16 °C) 133

cycle until mid-anthesis. 3 plants were grown per pot, in 5 in fiber pots, and the soil 134

used was a mix of 1 part top soil, 1 part sand and 1 part Pro-Mix. Fertilizer 135

(20-20-20, 2 g/L) was applied once a week from the 2-leaf stage until the plants were 136

3 to 4 in tall, then fed twice a week until plants were in boot stage; between the 137

start of boot stage and mid-anthesis, the plants were fed with a 20-5-35 fertilizer (2 138

g/L) once a week, then the 20-20-20 fertilization was resumed once a week. The 139

plants at mid-anthesis were transferred to a growth room at 25 °C, and 10 µL of 140

either water (mock inoculation treatment as a control) or a F. graminearum 141

8

macroconidial spore suspension at 1x105 spores/mL was point-inoculated with a 142

micropipette between the lemma and palea of two basal florets of each fully 143

developed spikelet of each head. Ten to twelve heads were inoculated per biological 144

replicate and three replicates were done per treatment. Following inoculation, 145

plants were transferred into a growth room where they were misted overhead for 30 146

s every 1 h during the light period for 2 d. Following this, they were moved to a 147

non-misted bench in the same growth room maintained at 75% humidity. Day-length 148

in the growth room was maintained at 16 h, and temperature was maintained at a 149

constant 25 °C. At 4 d after inoculation, rachis segments from the inoculated 150

portion of each head (spikelets removed) were harvested, immediately frozen in 151

liquid nitrogen and stored at -80 °C. 152

153

RNA isolation, cDNA library preparation and sequencing 154

Plant total RNA from rachis tissues was extracted using the TRIzol (Invitrogen Canada 155

Inc., Burlinton, Ontario) method. Isolated RNAs were treated with an RNase-free 156

DNase (Qiagen, Mississauga, Canada) and cleaned using RNeasy Mini Kits (Qiagen) 157

according to manufacturer’s instructions. RNA quality and quantity was 158

determined using a 2100 BioAnalyzer (Agilent Technologies Canada Inc, Mississauga, 159

ON, Canada). 160

Multiplexed cDNA libraries for sequencing were prepared from total RNA using 161

the TruSeq RNA Sample Preparation v2 kit (Illumina, Inc, San Diego, CA) and MID tags 162

9

as described by the Manufacturer. The twelve libraries were sequenced using the 163

short-read paired-end protocol (2 x 100 b/read) in one lane on an Illumina (San 164

Diego, USA) HiSeq 2500 System using the Illumina HiSeq Cluster kit and following the 165

manufacturer’s instructions. 166

167

Bioinformatics analyses of RNA-Seq sequences 168

Illumina reads from the six CS and six CS-7EL cDNA libraries were assembled 169

following a two-step strategy to identify candidate expressed genes originating from 170

the Th. elongatum 7EL chromosome arm in the CS ditelocentric addition line CS-7EL. 171

At the time of the analysis, only a draft genome sequence was available for T. 172

aestivum and very limited genomic sequences were available for Th. elongatum. 173

The focus of the bioinformatics analysis was to identify enriched CS-7EL expressed 174

genes that were absent in the CS samples (Part A below), and which did not show 175

homology or homoeology with other available T. aestivum genomic and cDNA 176

sequences, but did show putative orthology with T. aestivum group 7 chromosomes 177

sequences (Part B). 178

Part A: assembly to identify CS-7EL-enriched sequences. 179

This step is illustrated in Fig. 1, with detailed parameters presented in 180

Supplementary Table 1. All assemblies were performed using SeqMan NGen 4.0.0 181

(DNASTAR, Madison, WI) with the option ‘de novo’ for the first two steps and 182

SeqMan NGen 3.1.2 (DNASTAR) with the option “small-templated” for the 3rd step. 183

10

In a first step, each of the twelve samples was assembled separately. Unassembled 184

reads (referred to as singletons) were then assembled across treated and 185

non-treated samples within each germplasm; remaining singletons were discarded. 186

In a second step, for CS-7EL, the de novo assembly was based on all contigs from the 187

sub-assemblies in the first step. For CS, all contigs from step 1 were co-assembled 188

with publicly available EST sequences from CS (NCBI; http://www.ncbi.nlm.nih.gov/ ), 189

to provide a broader reference base of expressed T. aestivum sequences. The 190

resulting contigs in both assemblies were scanned for F. graminearum contaminating 191

sequences using the F. graminearum genome database (FGDB) v3.2 open reading 192

frames (ORFs; Wong et al., 2011) obtained from the Munich Information Center for 193

Protein Sequences (http://mips.helmholtz-muenchen.de/genre/proj/FGDB/), and 194

matching contigs were removed. In a third step, the CS contigs and singletons from 195

the second step, together with F. graminearum ORFs, were used as a reference 196

template onto which second-step contigs from CS-7EL were assembled. The CS-7EL 197

sequences that assembled with CS or F. graminearum sequences were discarded. 198

The CS-7EL sequences that did not assemble onto the reference template are 199

referred to as ‘CS-7EL-enriched singletons’; in addition to Th. elongatum 7EL 200

candidate genes, these singletons could also include T. aestivum sequences that 201

were not present in the public database at the time of analysis. Further enrichment 202

and characterization of these “singletons” was performed in Part B of the analysis. 203

Part B: Identification of 7EL orthologs. 204

11

RNA-Seq reads from all 12 cDNA libraries were independently mapped to a 205

combined reference template including T. aestivum genomic sequence for 206

chromosomes 7A, 7B, 7D and 4AL (Berkman et al., 2011; Hernandez et al., 2012; 207

Lorenc et al., 2012; Berkman et al., 2013) and F. graminearum genomic sequence 208

using Tophat (version 2.0.4) (Kim et al., 2013), to identify among the T. aestivum 209

genomic contigs originating from the group 7 chromosomes of wheat those that 210

contains sequences expressed in our samples. We then proceeded in two steps to 211

identify CS-7EL-enriched singletons that matched T. aestivum genomic sequences 212

from the group 7 chromosomes at a predicted range of orthology (see below). First, 213

the CS-7EL-enriched singletons from step 3 of Part A were BLAST-aligned against the 214

genomic sequences from the group 7 chromosomes of T. aestivum that contained 215

expressed sequences, and singletons within the estimated range of orthology were 216

kept. Those singletons were then blasted against a full draft genome sequence 217

assembly (5x coverage) of Chinese Spring ( Brenchley et al., 2012) and those showing 218

similarity in a range estimated for homoeology between wheat subgenomes were 219

discarded. Finally, the longest (>500bp) of the remaining singletons were further 220

blasted to a local T. aestivum EST database containing ESTs from all T. aestivum 221

varieties (Hattori et al., 2005) and to the raw Wheat Survey Sequence database 222

(International Wheat Genome Sequencing Consortium repository hosted at Unité de 223

Recherche Génomique, http://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST; 224

12

accessed between February 2013 and March 2014), to further eliminate probable T. 225

aestivum sequences. 226

Appropriate thresholds to distinguish homology and orthology were estimated 227

as follows. Firstly, the level of sequence similarity between Th. elongatum and T. 228

aestivum sequences was estimated from BLAST alignments between 369 Th. 229

elongatum sequences (obtained from NCBI) and their closest sequence match 230

among the contigs from our CS assembly. Secondly, the average level of similarity 231

between hexaploid T. aestivum homoeologous gene sequences was estimated from 232

CS ESTs that assembled into distinct triplet sets. This step allowed calibration of the 233

similarity on a cDNA basis, and avoided uncertainty related to genome assembly and 234

gene prediction, both of which were unavailable for T. aestivum at the time of 235

analysis. The CS-7EL-enriched singletons retained, referred to as candidate 7EL 236

expressed genes, were mapped onto the complete Brachipodium distachyon 237

genome (http://www.plantgdb.org/BdGDB/ cgi-bin/blastGDB.pl) using sequence 238

similarity detected by BLAST. 239

240

DNA extraction and PCR analysis 241

Total genomic DNA was extracted from young leaves of CS, CS-7EL and CS-7ES using 242

a modified cetyl trimethylammonium bromide (CTAB) extraction protocol (Doyle, 243

1990). DNA quality was checked on 1.0% agarose gels, and concentration was 244

13

determined using a NanoDrop-1000 spectrometer (NanoDrop Technologies, DE, 245

USA). 246

Up to three PCR primer pairs were designed for each of 135 candidate 7EL 247

expressed genes using the PrimerSelect application from the software Lasergene 248

(DNASTAR) and synthesized by Sigma Biology (Oakville, Canada). Regions of 249

maximum difference between the 7EL candidate genes and the most similar T. 250

aestivum sequences were favored for primer design. PCR amplifications were 251

performed in a 20 µL reaction mix consisting of 1×PCR buffer II (Roche, Branchburg, 252

New Jersey, USA), 2.0 mM MgCl2, 0.6 mM dNTPs, 1.0 mM oligonucleotide primers, 253

20 ng DNA solution from CS, CS-7EL or CS-7ES genomic DNA, and 1 U AmpliTaq Gold 254

polymerase (Roche). Reactions were performed in an MJ Research Thermal Cycler, 255

with conditions set to 94 ℃ for 5 min, followed by 35 cycles consisting of 94 ℃ for 30 256

s, 50-60 ℃ for 30 s, 72 ℃ for 40 s, with a final extension at 72 ℃ for 10 min.. PCR 257

products were resolved on 2% agarose gels containing ethidium bromide and 258

visualized with a UV transilluminator. 259

Using the best pair of primers for each 7EL-specific molecular markers, 260

specificity of amplification was further tested on genomic DNA from a series of 261

disomic addition lines, with each line containing one of the seven chromosomes 262

from Th. elongatum in CS background: CS-1E to CS-7E, as well as on DNAs from the 263

CS-Th. elongatum disomic substitution lines CS-7E(7A), CS-7E(7B), CS-7E(7D), where 264

one of the chromosome 7 of T. aestivum (the one mentioned in bracket in the name) 265

14

is substituted by the chromosome 7E from Th. elongatum (Table 1; Dvořák and Knott, 266

1974; Dvořák, 1980). 267

To assess the utility of the 7EL-specific molecular markers, they were also used 268

to characterize 47 progeny from two BC1F2 families selected from the cross 269

CS-7E(7D)x 2*CSph1b (Table 1). The data obtained was analysed with the maximum 270

likelihood algorithm in JoinMap 4.0 (Kyazma, Wageningen, Netherlands) to produce 271

a preliminary order for our 7EL-specific molecular markers. 272

15

RESULTS 273

Candidate 7EL expressed genes identified from RNA-Seq analysis 274

Twelve rachis samples from the moderately FHB-susceptible CS and the 275

FHB-resistant ditelocentric addition line CS-7EL, which contains the long arm of 276

chromosome 7E from Th. elongatum, were sequenced using RNA-Seq; 8.8 to 11.6 M 277

reads were obtained per sample. The stepwise bioinformatics strategy used to 278

identify candidate 7EL expressed genes from the RNA-Seq analysis is described in 279

details in Material and Methods and the assembly part of it (Part A) is illustrated in 280

Fig. 1. Our assembly strategy identified a set of about 1,229,000 CS-7EL-enriched 281

“singletons” (sequences not assembled in the last assembly step); those needed to 282

be further characterized for additional enrichment. 283

To estimate the degree of orthology between Th. elongatum and T. aestivum, 284

we compared 369 Th. elongatum sequences with their closest match among the 285

contigs from our CS assembly. Seventy percent of the Th. elongatum sequences 286

showed greater than 84% similarity to CS contigs (Supplementary Fig. 1). To estimate 287

the degree of homoeology between T. aestivum subgenomes, triplet sequences from 288

low copy genes in the CS EST collections were aligned; this showed that most 289

homoeologs within T. aestivum differed by less than 4% (data not shown). Based 290

on this information, we selected thresholds of 85% to 95% (for orthology) and 291

greater than 95% (for homoeology) as conservative choices for further enrichment of 292

the collection of CS-7EL enriched “singletons” to a more conservative group of 9,494 293

16

candidate 7EL expressed genes. Most (78%) of the candidate 7EL expressed genes 294

mapping to B. distachyon genomic sequence showed a best ortholog on Bd 1 or Bd 3. 295

296

7EL-specific molecular markers 297

After additional BLAST-alignment of candidate 7EL expressed genes with any 298

available T. aestivum sequences and removal of those with similarity within the 299

range estimated for homoeology, a conservative set of 135 candidate 7EL expressed 300

genes with length greater than 500 b were selected for validation as candidate genes 301

from the chromosome 7EL of Th. elongatum. Primer pairs for 87 candidate 7EL 302

expressed genes produced non-polymorphic bands between CS, CS-7EL and/or 303

CS-7ES in our assay conditions. Fully diagnostic Th. elongatum 7EL 304

chromosome-specific molecular markers were identified for 48 candidate 7EL 305

expressed genes. The primer sequences for all of the 7EL-specific molecular markers 306

are presented in Table 2. Of the 48 candidate expressed genes from which 307

7EL-specific molecular markers were designed, the majority (28) had a best homolog 308

on chromosome 1 of B. distachyon, nine matched to either chromosome 2, 3 or 4, 309

and 11 had no significant similarity with B. distachyon genes (Table 2). Full sequences 310

and additional information on the 48 candidate 7EL expressed genes are provided in 311

Supplementary Table 2. Examples of PCR amplification results with some of the 312

7EL-specific molecular markers are presented in Fig. 2, showing specific amplification 313

with CS-7EL only. Further amplification tests showed that all of the markers were 314

17

specific to the 7E chromosome and did not amplify any band from the 6 other E 315

chromosomes nor unexpected bands from the CS-Th. elongatum disomic 316

substitution lines CS-7E(7A), CS-7E(7B), CS-7E(7D),( see example with molecular 317

marker M7EL-1 in Fig. 3). 318

319

Recombination in F2 families from the cross CS-7E(7D)× 2*CSph1b 320

The 7EL-specific molecular markers were used to characterize 47 progeny from two 321

BC1F2 families selected from the cross CS-7E(7D)x 2*CSph1b (Table 1), which allowed 322

recombination between the chromosomes 7D (of T. aestivum) and 7E (of Th. 323

elongatum) . Figure 4 shows an example of the results obtained with the molecular 324

marker M7EL-1. Results of all 7EL-specific molecular markers on all lines from the 325

two families are summarized in Supplementary Table 3 (in tab “Marker results 326

unordered”). Our results showed that only the CS genotype was observed in eight 327

out of 19 individuals from the 64-8 family and in 14 out of 28 individuals from the 328

58-5 family, while four individuals of the 58-5 family (none in 64-8) carried all 329

markers tested for 7EL. In all other individuals of both families, a variable subset of 330

the 7EL-specific markers was absent. Using the above results, we estimated that 331

recombination between the long arms of 7E and 7D occurred in 32% of the progeny 332

in family 58-5 and in 58% of the progeny from family 64-8. However, since all T. 333

aestivum scores are marked by the null PCR allele, we cannot exclude that some null 334

scores represent misamplifications of the dominant Th. elongatum allele. This is 335

18

most likely to be the case for progeny in which only a single null allele was observed. 336

A more conservative estimation of the recombination frequency between 7EL and 337

7DL would be of 25% and 53% for the families 58-5 and 64-8, respectively. 338

339

19

DISCUSSION 340

A RNA-Seq dataset comparing expression in two wheat lines, CS and CS-7EL, was 341

used to identify candidate expressed genes specific to the Th. elongatum 7EL 342

chromosome arm. The bioinformatics strategy that we have tested, using modest 343

computer capacity and software available in most molecular biology laboratories, 344

has allowed the identification of over 9,000 candidate 7EL expressed genes. Some 345

of the stepwise assembly methods (e.g. assembly of individual samples) could be 346

simplified by single assembly steps if resources (e.g. computer memory) allowed. 347

However, the stepwise methods also have the advantage of providing intermediate 348

sub-assemblies that may be useful in other investigations. 349

The assignment of candidate genes to the 7EL-specific group was based on 350

current understanding of the relationship between T. aestivum and Th. elongatum 351

genomes and comprehensive use of publicly-available databases. Recently published 352

studies corroborate our choices of orthology and homoeology thresholds. 353

Bioinformatics analysis of transcriptome from Th. elongatum under water deficit 354

stress has shown that 47% of the unique transcripts profiled had a significant 355

similarity to T. aestivum genes, with most of them showing 80% or more similarity 356

and the largest number of transcripts being 95 to 98% similar to T. aestivum (Shu et 357

al., 2015). Our selected threshold for exclusion of homoeologous sequences (96 to 358

100% similarity) is supported by a recent study that reported an average of 97% 359

gene sequence similarity between subgenomes A, B and D of T. aestivum (The 360

20

International Wheat Genome sequencing Consortium, 2014). Our selected 361

thresholds will exclude highly conserved Th. elongatum genes (e.g., ubiquitin, 362

histones) and also genes with no T. aestivum counterparts. Discovery of those 363

genes would require the sequence of the Th. elongatum genome, or a concerted 364

effort to test and map more PCR amplicons by trial and error. Consequently, the set 365

of 7EL-specific candidate genes obtained is considered to be a conservative but 366

useful set of reference genes, and it is expected that additional genes specific to 7EL 367

will be identified when extensive genomic sequence data becomes available for Th. 368

elongatum. 369

Mapping of the candidate 7EL expressed genes to B. distachyon genomic 370

sequence showed a best ortholog on Bd 1 or Bd 3, the two B. distachyon 371

chromosomes known to be syntenic to T. aestivum chromosomes from the group 7 372

(International Brachypodium Initiative, 2010; Kumar et al., 2012). This is consistent 373

with the close relationship observed between the E genome of Th. elongatum and 374

the A, B and D subgenomes of T. aestivum (Dvořák and Knott, 1974; Liu et al., 2007; 375

Hu et al., 2012). 376

We were able to develop 7EL-specific molecular markers for 48 of the 135 377

candidate 7EL expressed genes used for validation. Our result represents a success 378

rate of 36% for the development of specific markers from characterized sequences. 379

This is higher than the success obtained when using sequences from SLAF-Seq 380

technology (Chen et al., 2013), where 26% of the genomic sequences led to the 381

21

development of specific markers for the same chromosome arm. The method 382

presented here also shows a considerable improvement in the rate of discovery of 383

specific markers when compared to other methods used to develop 7E-specific 384

markers, such as RAPD, SSR, SSH, ESTs and PLUG (You et al., 2003; Shen et al., 2004; 385

Shen and Ohm, 2006; Fu et al., 2012; Hu et al., 2012). Our greater success rate can 386

be explained at least in part by the increasing availability of Th. elongatum and T. 387

aestivum genomic sequences, allowing for a more precise design of species-specific 388

molecular markers. 389

Spontaneous translocations and substitutions between Thinopyrum E and T. 390

aestivum D genomes have been observed in other studies, and Southern 391

hybridizations with genomic DNAs has shown that the E genome of Thinopyrum 392

species was more closely related to the D genome than the A and B subgenomes of T. 393

aestivum (Liu et al., 2007). It has also been shown that homoeologous pairing and 394

recombination between the D and E genomes can be facilitated by the use of the 395

wheat mutant line CSph1b (Qi et al., 2007; Niu et al., 2011; Niu et al., 2014). In our 396

crossing experiment, the ph1b mutation in CSph1b (Sears 1977) allowed 397

homoeologous pairing between the Th. elongatum chromosome 7E in the 398

substitution line CS-7E(7D), which is missing 7D, and the 7D chromosome present in 399

CSph1b. The molecular markers developed in this study allowed us to estimate the 400

recombination frequency between 7D (T. aestivum) and 7E (Th. elongatum) in two 401

BC1F2 families from the cross CS-7E(7D)x 2*CSph1b. No 7EL-specific markers were 402

22

observed in 22 of the progeny analysed, suggesting that no recombination occurred 403

between the long arms of the 7E and 7D chromosomes in those individuals and that 404

only the long arm of 7D remained in these lines. Four individuals carried all markers 405

tested for 7EL and may contain a full version of the long arm of 7E. In all other 406

individuals of both families, a variable subset of the 7EL-specific markers was absent, 407

suggesting that recombination between the long arms of 7E and 7D had occurred. 408

The recombination frequency of 25% to 53% that we have observed in the two BC1F2 409

families is intermediate to other reports. Niu et al. (2014) results suggested about 410

80% recombination between chromosome 7D (T. aestivum) and a large segment of 411

7E (whole 7EL+7ES half proximal to the centromere) from Th. ponticum. Qi et al. 412

(2007) obtained 2% recombination between the short arms of chromosomes 4D (T. 413

aestivum) and 4Ai of Th. intermedium. 414

A genetic order and graphical genotypes produced using the 7EL-specific 415

molecular marker data for the BC1F2 families revealed putative sites of 416

recombination (Tab “Markers ordered” of Supplementary Table 3). However, 417

because this result is based on a very small number of progeny, it is very 418

approximate; data will need to be collected on a larger number of progenies and 419

families before meaningful mapping could be performed. 420

There has been substantial interest in transferring traits from Thinopyrum 421

species to wheat, particularly those conferring resistance to disease. However, it has 422

been difficult to develop reliable molecular markers specific to Thinopyrum 423

23

chromosomes. This is in part due to the high level of sequence similarity between T. 424

aestivum and Thinopyrum species (Liu et al., 2007; Hu et al., 2012), and to the 425

paucity of sequence information, especially from Thinopyrum species. In the 426

present study, we have tested with success a bioinformatics strategy to analyse 427

RNA-Seq data and publically available sequences in the identification of candidate 428

expressed genes from the Th. elongatum 7EL chromosome arm introgressed into the 429

ditelocentric addition line CS-7EL. Furthermore, we have used these candidate 7EL 430

expressed genes to develop reliable PCR-based markers, and have demonstrated the 431

utility of those markers in identifying recombination events between a native and an 432

introgressed chromosome fragments. These markers will provide identification of 433

specific fragments that may allow refinement and isolation of genes for desired 434

traits. 435

436

437

24

Acknowledgements 438

We would like to thank Darrin Klassen, Danielle Wolfe–Deshaies and Dawn Chi for 439

providing the plant material used in this study. The financial support of Agriculture 440

and Agri-Food Canada’s Canadian Crops Genomics Initiative and A-base are 441

gratefully acknowledged. L.G. was supported by the China Scholarship Council under 442

the MOE-AAFC PhD Research Program. D.E. would like to acknowledge funding 443

support from Australian Research Council (Projects LP0882095 and LP0883462). 444

Support is also acknowledged from the Australian Genome Research Facility (AGRF). 445

P.V. would also like to thank the Australian Department of Foreign Affairs and Trade 446

(DFAT) for funding support. 447

448

25

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637 34

Figure captions 638

Figure 1. Strategy used to assemble the Illumina reads from the six CS and six CS-7EL 639

cDNA libraries and create a pool of sequences enriched in 7EL candidate genes. CS, 640

Chinese Spring; CS-7EL, Chinese Spring ditelocentric addition line containing the 641

chromosome arm 7EL; Fg, F. graminearum treatment; W, water treatment; Fg ORFs, 642

open reading frames from F. graminearum; R1 to R3: biological replicate samples 1 643

to 3 of a given treatment; contigs, groups each containing two or more assembled 644

sequences with 97 to 100% similarity, either from sequencing reads or from the 645

previous assembly step; singletons: unassembled sequences from a given assembly 646

step. 647

648

Figure 2. Example of PCR amplification with 7EL-specific markers using genomic 649

DNAs from CS and the CS addition lines CS-7EL (7EL) and CS-7ES (7ES). M: DNA 650

ladder. 651

652

Figure 3. PCR amplification using marker M7EL-1 and genomic DNAs from the CS-Th. 653

elongatum disomic addition lines CS-1E to CS-7E, and the CS-Th. elongatum disomic 654

substitution lines CS-7E(7A), CS-7E(7B) and CS-7E(7D). M: DNA ladder. 655

656

35

Figure 4. PCR amplification using marker M7EL-1 and DNA from progeny of BC1F2 657

families 64-8 (A) and 58-5 (B), from the cross CS-7E(7D) x 2*CSph1b. M: DNA ladder; 658

1-17: BC1F2 progeny; 18: CS-7E(7D); 19: CSph1b 659

660

Supplementary Figure 1. Sequence similarity between Th. elongatum and wheat 661

contigs. The values on the X-axis indicate the maximum value for each interval of 2%. 662

Th. elongatum mRNA sequences were obtained from NCBI; contigs obtained after 663

step three of our assembly (in Part A) of CS reads were used as wheat sequences. 664

665

Supplementary Table 1. Parameters and statistics for the different steps of the 666

assembly strategy. 667

668

Supplementary Table 2. Sequences and additional information on the 48 candidate 669

7EL expressed genes used to design the molecular markers. 670

671

Supplementary Table 3. Tab “Marker results unordered”: summary of the results 672

obtained with all 7EL-specific molecular markers for the characterization of 47 673

progeny from two BC1F2 families selected from the cross CS-7E(7D) x 2*CSph1b. 674

Tab “Markers ordered”: naïve ordering of 7EL-specific markers performed using 675

JoinMap on all progeny that showed heterogeneous marker scores. Presence or 676

absence of a 7EL-specific band was scored as 1 or 0, respectively. 677

36

Table 1. Experimental material used in this publication. 678

679 Description of materials Abbreviated names Chinese Spring and Chinese Spring with ph1b mutation

CS and CSph1b

Chinese Spring-Th. elongatum ditelocentric addition lines (long and short arm)

CS-7EL, CS-7ES

Chinese Spring-Th. elongatum disomic addition lines (chromosomes 1E to 7E)

CS-1E, CS-2E, CS-3E, CS-4E, CS-5E, CS-6E, CS-7E

Chinese Spring-Th. elongatum disomic substitution lines (with 7E replacing 7A, 7B or 7D)

CS-7E(7A), CS-7E(7B), CS-7E(7D)

CS-7E(7D) x 2*CSph1b, progeny from two BC1F2 families

64-8-N and 58-5-N series

680 681

682

37

Table 2. 7EL–specific molecular markers developed from candidate 7EL expressed 683 genes. 684 685

Marker name

Sequence of the PCR primer pairs (5’-3’) Original

contigs

Chromosome location of homolog in

B. distachyon

Forward Reverse

M7EL-1 AGTGCTGGCTAGGTACATTTTTCT CGAGCGCGAGGTTGAGG Contig98334 Chr 1

M7EL-2 GGTGGTACAGATTCAGTCAAAA GCAGAGGTAGCAGCAGTAGTAGTC Contig2061459 Chr 1

M7EL-3 TGTCGCGTGGTATCCCAT TCCCTGTATAGCCAAATGAAGAGT 1274 Chr 1

M7EL-4 CGCCGCGACATTTTGAG CTGTAAGATTTGCTGGCGGGTTTT Contig917019 Chr 1

M7EL-5 TTACCTATGGGCCTATGCTTCCTT TGTCTATTCTTGCCACGATTGT Contig319660 Chr 1

M7EL-6 ACCGGGTAGCAGTTGTA GGTTCCCCCATTTTCTAT Contig311687 Chr 1

M7EL-7 TGTTGGGTCCTATCCTTTCACTTT ACGCCGATCTCCCCATACATT Contig1096359 Chr 1

M7EL-8 TGGTGGTGGGCGACAGTG TCCCCAGCCTTAGAAATCAAAAT Contig184838 Chr 1

M7EL-9 GGTTTTCTATATTTGATTACAG AACAAATCTTAGGTCCTCATACTG Contig861043 Chr 1

M7EL-10 GTAATTCTGCTGCTGCTGTTCTTG GCCACGTTTCGGGTCATTTG Contig412195 Chr 1

M7EL-11 GCTTGCACACGGCGATTTATTG CTGGCGGCGATGAGGGAGAAGA Contig269170 Chr 1

M7EL-12 TATATCTAAGAACAAGTAAAA GTCGCCCCACAGCAGGAA Contig610325 Chr 1

M7EL-13 CCGCGAGGAGTTCAAGCAGA TGAAAAGCAATCCAGCAAACACAT Contig413980 Chr 1

M7EL-14 GCGGCGGCAGCATCAATCA ACGCATCGAGTGGTGTTCTAT Contig313982 Chr 1

M7EL-15 ATGGATGGATCGTCAAGGTG CTAAGAACAAGTAAAAGAAAAA Contig892764 Chr 1

M7EL-16 GCCGTGGTGCTCGTCGTC GCACCACGGCTAACCTCTCAG Contig274134 Chr 1

M7EL-17 ATACGCCATTACATTACAG TGACCTCGATACAGCATAC Contig174400 Chr 1

M7EL-18 CGGCTCGATCGACAGACACC GGCAAGACCGTTTCCTACAGC Contig373357 Chr 1

M7EL-19 ACAAACCATCATCATCATCCTCTG ATTTGGCAACTGCTGACATTTTCT Contig384336 Chr 1

M7EL-20 AGGATCTCGCCAAAAAG CGCGAGCGACGGTGTTC Contig430201 Chr 1

38

M7EL-21 GAGGGAAGACGGGAGGAGGAAGAT GTGATCGACAATGAACCAAC Contig588646 Chr 1

M7EL-22 CACTAGAATAAAGACAAAAATGGT TAAATGGAACTGTGGGTAAGATA Contig583775 Chr 1

M7EL-23 TCACATCGATCGAAACCTAATA GAACCGCTCGCACTCCTCGTC Contig169654 Chr 1

M7EL-24 TCGCCAGCCCCCTACAGCAGAC AGCCATCATCGTCCCTCCTTCCTT Contig736571 Chr 1

M7EL-25 AGTGCTGGCTAGGTACATTTTTCT CGAGCGCGAGGTTGAGG Contig923004 Chr 1

M7EL-26 GCACACCCCAGGCTAAAGTTC TAAGTCATGGCAGTATCC Contig139646 Chr 1

M7EL-27 TCACCACAGATGTTCAGAGATA AAGCCGAGGCCACTAAGGAATA Contig1117864 Chr 1

M7EL-28 AGGGCATGCACTGTCTGG ATGCCCTAAATGATACCCTGAG Contig196989 Chr 1

M7EL-29 CTCCAGCAGGTCGTCGTTTTC GGGCCTCTTCCGCTTACCTC Contig661732 Chr 3

M7EL-30 GCCCAACAAAATCATCACTA TCGCTTATTTTGGGACAGA Contig357536 Chr 3

M7EL-31 GTCTCGGGCTCCACTCC TGGCTCCGATGCACTCTCACTTTC Contig414085 Chr 3

M7EL-32 TCGTTTTGCTCCGTATGTAGTCTG CTGCTGCAACCCAAGTCTGAA Contig519891 Chr 2

M7EL-33 GTTCTCCTCGACGACCCTCTG CCCCGCCGGCGACCACAA Contig306667 Chr 2

M7EL-34 CGTCAAGTCGAGCGTGTGC ATGGGAAGGATGGGAGGAGAC Contig32077 Chr 2

M7EL-35 CATCGTCGGGCCTGTTAGC TAGTGCAATGGGATGATGATGAGA Contig977836 Chr 2

M7EL-36 TATAAATTTAGGGTTCGTTCT CTCTCGGGATGCTCTGT Contig71253 Chr 4

M7EL-37 TGGTCGACAGGGGAAAATG AACTGGCTGGATAACAAAAGGAAG Contig196962 Chr 4

M7EL-38 TGGATTATCGCATGGTGTGACTA AACTTGGCAGATCGGAGAGGAA Contig1225302 -

M7EL-39 GCACCGAGCGAGCCACCTG GCGCGACCTTTCCCTTCCTCT Contig202087 -

M7EL-40 GAGATCAGTGGCTTGGGGTTGG GTTTGGACTTTTGGTTGC Contig322987 -

M7EL-41 CAAGGAAATTAATCAAGAACTACT CTGCTCAGCTGGGTGTGG Contig263095 -

M7EL-42 GAGTTTCCGTGCTTCTGG AAAAACACAACTAAACCCTAACGA Contig1455493 -

M7EL-43 GATGGTTATTTTGCGTTTCAC CTCCCTCCGATCCATATTACTTGT Contig1052811 -

M7EL-44 ATGAGTTTTCCAGTTATTACG ATCATCCATCTCTTGTCCT Contig1305214 -

M7EL-45 GAAATTTTTAGGTATACTGTCAAC GATAATTCTCCCTCGTCCTCTA Contig1930629 -

39

M7EL-46 CTGGCCAGATGAAAGTAGTT AAGCCAGTGACCCGGACAATAA Contig1809051 -

M7EL-47 CTTGCATCAGAATACCCTTTTACT CATCACGATCTACTATTCTACTTG Contig1901674 -

M7EL-48 TGATGAAGATGGCGACACAAA TTCGGCTTAATCAACTCCAT Contig1375954 -

686 687 688 689

40

CS-7EL+Fg R1 R2 R3

CS-7EL+W R1 R2 R3

CS+Fg R1 R2 R3

CS+W R1 R2 R3

reference templates

Fg ORFs

CS ESTs

contigs singletons contigs singletons contigs singletons contigs singletons

contigs contigs contigs contigs

contigs singletons contigs

12 separate assemblies

Assembly of singletons

Assembly with Fg scan Removed Fg sequences and wheat sequences highly similar to Fg

Templated assembly of CS-7EL on CS+FG Singletons contain: 7EL candidates genes (and wheat sequences not in the templates, contaminants, bad or misassembled sequences)

singletons

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