King, Julie and Grewal, Surbhi and Yang, Cai-yun and Hubbart, Stella and Scholefield, Duncan and Ashling, Stephen and Edwards, Keith J. and Allen, Alexandra M. and Burridge, Amanda and Bloor, Claire and Davassi, Alessandro and da Silva, Glacy J. and Chalmers, Ken and King, Ian P. (2016) A step change in the transfer of interspecific variation into wheat from Amblyopyrum muticum. Plant Biotechnology Journal . ISSN 1467-7652

Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/37352/1/King_et_al-2016-Plant_Biotechnology_Journal.pdf

Copyright and reuse:

The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions.

This article is made available under the Creative Commons Attribution licence and may be reused according to the conditions of the licence. For more details see: http://creativecommons.org/licenses/by/2.5/

A note on versions:

The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the repository url above for details on accessing the published version and note that access may require a subscription.

For more information, please contact eprints@nottingham.ac.uk

A step change in the transfer of interspecific variation intowheat from Amblyopyrum muticumJulie King1,*, Surbhi Grewal1, Cai-yun Yang1, Stella Hubbart1, Duncan Scholefield1, Stephen Ashling1,Keith J. Edwards2, Alexandra M. Allen2, Amanda Burridge2, Claire Bloor3, Alessandro Davassi3, Glacy J. da Silva1,4,Ken Chalmers5 and Ian P. King1

1Division of Plant and Crop Sciences, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Loughborough, UK2Life Sciences, University of Bristol, Bristol, UK3Affymetrix UK Ltd, High Wycombe, UK4Federal University of Pelotas, Pelotas, Brazil5School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA, Australia

Received 27 May 2016;

revised 13 July 2016;

accepted 21 July 2016.

*Correspondence (Tel +44 (0)115 9516780;

fax +44 (0) 115 9516343; email Julie.

king@nottingham.ac.uk)

Keywords: Wheat, Introgression, wild

relatives, genotyping, synteny.

SummaryDespite some notable successes, only a fraction of the genetic variation available in wild relatives

has been utilized to produce superior wheat varieties. This is as a direct result of the lack of

availability of suitable high-throughput technologies to detect wheat/wild relative introgressions

when they occur. Here, we report on the use of a new SNP array to detect wheat/wild relative

introgressions in backcross progenies derived from interspecific hexaploid wheat/Ambylopyrum

muticum F1 hybrids. The array enabled the detection and characterization of 218 genomewide

wheat/Am. muticum introgressions, that is a significant step change in the generation and

detection of introgressions compared to previous work in the field. Furthermore, the frequency

of introgressions detected was sufficiently high to enable the construction of seven linkage

groups of the Am. muticum genome, thus enabling the syntenic relationship between the wild

relative and hexaploid wheat to be determined. The importance of the genetic variation from

Am. muticum introduced into wheat for the development of superior varieties is discussed.

Introduction

Hexaploid bread wheat, which is an allopolyploid composed of

three distinct genomes, that is the AA genome from Triticum

urartu, the BB genome from an Aegilops speltoides-like progen-

itor and the DD genome from Aegilops tauschii (Dvorak and

Zhang, 1990; Dvorak et al., 1993; McFadden and Sears, 1946),

evolved only once or at best a few times approximately

10 000 years ago (Charmet, 2011). As a result, wheat has been

through a severe genetic bottleneck with the sum total of genetic

variation present in the species today being a direct result of only

10 000 years of genetic mutation and through possible outcross-

ing events that may have occurred with other species, for

example tetraploid wheat. In addition, the gene pool of modern

cultivated wheat has been further restricted through selection for

specific agronomically important traits, for example free threshing

(Charmet, 2011; Cox, 1997).

Wheat is one of the world’s leading sources of food, and thus,

the narrow gene pool available for the development of superior

varieties is of major concern heightened by increasing global

population predictions. In the past, breeders have had consider-

able success in producing higher yielding varieties with the limited

variation available. However, there is growing evidence that

wheat yields are plateauing and that this is a direct result of the

exhaustion of the available genetic variation compounded by

environmental change (Brisson et al., 2010; Charmet, 2011; Ray

et al., 2013). Thus, there is an urgent need to identify new

sources of genetic variation that can be used to develop superior

wheat varieties.

Wheat is related to a large number of other species many of

which are wild and uncultivated. These wild relatives, unlike

wheat, provide a vast and untapped reservoir of genetic

variation for potentially most, if not all, agronomically impor-

tant traits (Friebe et al., 1996; Jauhar and Chibbar, 1999; Qi

et al., 2007; Schneider et al., 2008). In the past, attempts have

been made to exploit the genetic variation from these wild

species.

Normally, recombination in wheat is restricted to identical

homologous chromosomes from the same genome due to the

presence of the Ph1 locus located on the long arm of chromo-

some 5B of wheat (Al-Kaff et al., 2008; Griffiths et al., 2006;

Riley and Chapman, 1958; Sears, 1976; Sears and Okamoto,

1958). Thus, the Ph1 locus normally has to be removed before

homoeologous recombination between the chromosomes of a

wild relative and wheat can occur (Al-Kaff et al., 2008; Sears,

1977). However, some species such as Amblyopyrum muticum

[(Boiss.) Eig. (Aegilops mutica Boiss.) (2n = 2x = 14; genome TT)]

carry a gene(s) which supresses the Ph1 locus, thus enabling

recombination to occur directly between homoeologous chro-

mosomes in interspecific Am. muticum/wheat F1 hybrids (Dover

and Riley, 1972). Despite the ability to suppress the Ph1 locus in

wheat, very little genetic or trait analysis has been undertaken to

Please cite this article as: King, J., Grewal, S., Yang, C.-Y., Hubbart, S., Scholefield, D., Ashling, S., Edwards, K.J., Allen, A.M., Burridge, A., Bloor, C., Davassi, A., da

Silva, G.J., Chalmers, K. and King, I.P. (2016) A step change in the transfer of interspecific variation into wheat from Amblyopyrum muticum. Plant Biotechnol. J.,

doi: 10.1111/pbi.12606

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.

1

Plant Biotechnology Journal (2016), pp. 1–10 doi: 10.1111/pbi.12606

date on Am. muticum with the exception of some addition and

substitution lines (Iefimenko et al., 2013) and potential resistance

against environmental stresses (Iefimenko et al., 2015) and

fungal diseases such as powdery mildew (Eser, 1998).

Although there have been some notable successes (Garcia-

Olmedo et al., 1977 and Sears, 1955, 1972), only a fraction of

the work has led to the development of new wheat varieties.

The major blocks to the successful large-scale genomewide

exploitation of genetic variation from wild relatives have been

firstly the lack of high-throughput screening technology to

quickly identify and characterize introgressions when they occur

and secondly the apparent low frequency of recombination

between the chromosomes of wheat and many of its wild

relatives.

In the past, the identification of introgressions has relied on

low-throughput analyses with limited success. However, the

development of next-generation sequencing technologies and

single nucleotide polymorphism (SNP) markers provides a mech-

anism for the detection of introgressions into wheat from its wild

relatives as was demonstrated for chromosome 5 of Aegilops

geniculata (Tiwari et al., 2014, 2015). The aim of the research

described here was to use a whole-genome introgression

approach (e.g. King et al., 2013), that is attempt to transfer

chromosome segments from the entire genome of Am. muticum

into hexaploid wheat irrespective of any traits that the wild

relative might carry and then to attempt to detect and charac-

terize the introgressions via a new wheat/wild relative SNP array.

This array was constructed using a subset of the SNPs described

by Winfield et al. (2015) from their ultra-high-density Axiom�

genotyping array.

Results

Generation of introgressions

In total, 1039 crosses (crossed ears) were made (Figure 1)

resulting in the production of 8146 seeds (not including self-

seed). The number of seeds germinated, plants crossed and seed

set, etc. is summarized in Table S1. The F1 interspecific hybrids

between hexaploid wheat cv Paragon and Am. muticum showed

the lowest germination rate—28.6%, as compared to the BC1,

A

B

D

Wheat (Paragon)

X

Amblyopyrum muticum

X

A

B

D

Wheat (Paragon)

High-throughput screening of 1000s of BC1 and subsequent backcross progeny to identify recombinants

Isolation of homozygous introgressions

F1

BC1

SelfAxiom® 35K arrayIdentify introgressions+ KASP- used in later generations

Pairing limited to homoeologouschromosomes during gameteproduction in the F1hybrids

Repeated backcrossing programme to isolate single introgressions

Figure 1 Wheat/wild relative introgression strategy.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Julie King et al.2

BC2 and BC3 generations—52.9%, 77.6% and 60.6%, respec-

tively. In addition, the F1 hybrids also exhibited the highest levels

of infertility (to obtain a broad indication of fertility, the number

of crossed ears that produced seed was recorded for each

generation), that is only 16.2% of F1 crossed ears produced seed

as compared with 79.9%, 88.1% and 98.3% from the crossed

ears of the BC1, BC2 and BC3 generations. A further indication of

the infertility of the F1 was shown by the fact that this generation

set no self-seed in contrast to the other generations. Of the 98 F1seeds germinated, only 11 reached maturity and set seed when

pollinated with Paragon (Table S1). Thus, the total generation of

Am. muticum/wheat introgressions in this program was limited to

these 11 individuals and their derivatives. Of the 34 BC1 seeds

generated from the 11 sexually partially viable F1 plants, 16

survived to maturity and set seed when pollinated with Paragon.

In total, 781 BC2 seeds were obtained from 123 crosses onto

these 16 BC1 plants. One hundred and sixteen of these BC2 seeds

were then selected at random and germinated and subsequent

pollinations with Paragon yielded 4137 BC3 seed. One hundred

and twenty-seven of these BC3 seeds were again selected at

random and crossed to Paragon to generate 2947 BC4 seeds

(Table S1).

Detection of introgressions

An Axiom HD Wheat-Relative Genotyping Array (a full description

of which is provided in the Experimental Procedures) composed of

SNPs showing polymorphism between several wheat varieties and

ten wheat wild relatives [25 487 of the SNPs were polymorphic

between Am. muticum and Paragon (Table 1) with the highest

number for linkage group 2 (17.2%) and the fewest for linkage

group 6 (11.4%) but with a relatively even spread over all seven

linkage groups] was used to screen genomic DNA prepared from

167 samples, which included nine parental lines and 158

backcross lines between wheat and Am. muticum. Genotype

calls were generated, and the sample call rate ranged from 80%

to 97% with an average of 93.3% for the 167 samples. The

lowest call rate was obtained for Am. muticum with an average

of 80.1%.

The scores for the probes were classified into one of six

categories according to the cluster pattern produced by the

Affymetrix software. Only the first group, Poly High Resolution

(PHR), was considered as being optimum quality SNPs for genetic

mapping purposes (see Experimental Procedures).

The PHR SNPs were used in map construction using JoinMap�

(van Ooijen, 2011) and resulted in seven linkage groups repre-

senting the seven Am. muticum chromosomes containing 613

SNP markers [Figure 2 (SNP marker names and cM distances for

each of the seven linkage groups are also shown in Table S2)].

The cM lengths of linkage groups 1–7 were 104.2, 95.3, 75.4,

129.5, 127.9, 93.3 and 103.9, respectively. Hence, the total map

length of this ‘frame’ was 729 cM with an average chromosome

length of 104 cM. SNP markers were again well distributed over

the seven linkage groups.

Genomic in situ hybridization (GISH)

To confirm the SNP analysis, genotyped BC3 individuals were

selected and analysed by multicolour GISH. In each of the

genotypes observed, the number of Am. muticum introgressions

identified by SNP analysis corresponded exactly with the number

of introgressions detected by GISH (Table 2, Figure 3). In all cases,

the introgressions observed involved recombinant events

between T genome chromosomes of Am. muticum and the B

genome chromosomes of wheat (21 recombination events) or the

D genome chromosomes of wheat (18 recombination events)

(Figures 4i–iv). In four unrelated BC3 genotypes, one chromo-

some of Am. muticum was found to have recombined with both

B and D genome chromosomes of wheat. No examples of

recombinant events between the T genome chromosomes of

Am. muticum and A genome chromosomes of wheat have to

date been detected (of 22 genotypes containing introgressions).

GISH also revealed the presence of intergenomic recombinant

events between the A, B and D genomes of wheat (Figure 5). In

the 29 BC3 genotypes analysed, four A/B translocations, ten A/D

translocations and ten B/D translocations were observed.

Syntenic relationship between wheat and Am. muticum

Figure 6 shows the syntenic relationships between the seven

linkage groups of Am. muticum and the seven linkage groups of

each of the three genomes of wheat with large ‘ribbons’ showing

significant synteny. Some gene rearrangements are indicated in

the diagram where usually single markers cross map to non-

collinear positions on the wheat chromosomes. The only major

disruption to the syntenic relationship between these two species

is that Am. muticum does not carry the 4/5/7 translocation

observed for chromosomes 4A, 5A and 7B of wheat (Liu et al.,

1992; Naranjo et al., 1987). Thus, this analysis represents a close

syntenic relationship between Am. muticum and the A, B and D

genomes of wheat.

Discussion

Since the initial discovery of the Ph1 chromosome pairing control

locus in wheat (Riley and Chapman, 1958; Sears and Okamoto,

1958), many attempts have been made to unlock the genetic

variation in wild relatives for wheat improvement. However, while

introgression into wheat has been used successfully, albeit

haphazardly in the past, for example leaf rust resistance transfer

from Aegilops umbellulata (Sears, 1955, 1972), the potential of

wild relatives has remained virtually untapped. The failure for the

systematic exploitation of wild relatives has been the absence of

appropriate high-throughput technologies to screen for, and

specifically identify, introgression events (King et al., 2016). The

Affymetrix wheat/wild relative array used in this present study

enabled the identification and characterization of genomewide

introgressions of various sizes (from large to very small).

The shotgun introgression approach described has resulted in

Table 1 Number of polymorphic SNPs between Am. muticum and hexaploid wheat in total on the Affymetrix 35 K array and used in the linkage

map of Am. muticum

Linkage group 1 Linkage group 2 Linkage group 3 Linkage group 4 Linkage group 5 Linkage group 6 Linkage group 7 Total

All calls (% of total) 3254 (12.8) 4395 (17.2) 3825 (15.0) 3199 (12.6) 4182 (16.4) 2895 (11.4) 3717 (14.7) 25 487

PHR calls (% of total) 80 (13.1) 88 (14.4) 73 (11.9) 74 (12.1) 134 (21.9) 68 (11.1) 96 (15.7) 613

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Wheat/wild relative introgression 3

the generation of a high level of wheat/Am. muticum recombi-

nant chromosomes. The ability to detect these introgressions is a

direct result of using the SNP array, that is large numbers of

markers to detect genomewide introgressions have not been

previously available.

In the work described, recombination between the wheat and

Am. muticum chromosomes, leading to introgressions, was

expected to occur in female gametes of wheat/Am. muticum F1interspecific hybrids (rather than using individual wheat/wild

relative addition or substitution lines that have commonly been

used in the past (King et al., 2016). As these F1 hybrids lack

homologous chromosome pairs, the only recombination that can

occur is between chromosomes from different genomes, that is

between the A, B, D genomes of wheat and the T genome of

Am. muticum. As a result of the absence of homologous

chromosomes, we hypothesized that this strategy might lead to

an enhanced frequency of introgression. However, the drawback

of this approach is that as the frequency of recombination

between chromosomes from different genomes is likely to be very

low, this would lead to significant infertility in the F1, that is low

recombination would result in the failure of normal disjunction of

chromosomes at anaphase I of meiosis leading to the production

of unviable, unbalanced gametes.

The F1 individuals showed very low fertility as predicted, for

example the frequency of seed set per crossed ear of the F1 hybrids

was only 16.2%as compared to 79.67%, 88.09%and 98.25% for

the BC1, BC2 and BC3 generations, respectively (Table S1). As a

result of the low fertility of the F1 hybrids, only 34 BC1 individuals

were generated, and of these, only 16 plants grew to maturity and

set seed. Thus, the total number of introgressions that could be

generated would be limited to the 16 female F1 gametes that gave

rise to these 16 BC1 plants if no further recombination occurred in

later generations, that is in the gametes of the BC1, BC2 and BC3

generations. However, unexpectedly, genetic mapping indicated

that a very high frequency of interspecific recombination had

occurred between the chromosomes of wheat and those of

Am. muticum, that is it was possible to assemble seven linkages

groups—something that has not been possible to achieve previ-

ously in this field of research and thus representing a step change in

the generation, detection and characterization of wheat/wild

relative introgressions. From the genetic mapping of the SNP

markers, it was possible to estimate that we had generated 218

AX-94921536 AX-951619580.0AX-95024675 AX-951032900.6AX-94433776 AX-94882031AX-94536506 AX-945253831.3AX-94879037 AX-944839101.9AX-95088953 AX-946469093.8AX-946585265.1AX-951633526.4AX-9463296216.2AX-95088261 AX-9513543422.9AX-94588067 AX-9493627123.6AX-9439054624.2AX-94606585 AX-94574413AX-94678603 AX-9482226424.8AX-94390150 AX-9519730826.8AX-94528656 AX-94416954AX-9519451027.4AX-9440659628.7AX-94653440 AX-94394839AX-9452173037.7AX-95116095 AX-94802682AX-9511484538.3AX-94489164 AX-94679613AX-9459665239.0AX-94816469 AX-94620619AX-94611444 AX-94571758AX-94883623 AX-94482529

39.6

AX-94449858 AX-9452631740.2AX-9481515642.2AX-94648380 AX-94925965AX-94991357 AX-94610474AX-94578143 AX-94700304

42.8

AX-9458944747.4AX-95184311 AX-95256380AX-94526365 AX-94762019AX-94514056 AX-94549470

52.8

AX-95134864 AX-9448990354.1AX-9479747864.6AX-9507553765.9AX-9461481269.8AX-9472460071.1AX-9465126772.4AX-9492672173.0AX-9463932873.7AX-94910826 AX-9452906474.3AX-9459077778.3AX-9465446678.9AX-94731169 AX-94434693AX-9477751280.8AX-9499913290.6AX-9519906495.9AX-94764182104.2

AX-944869230.0AX-94815994 AX-945545160.6AX-946915215.3AX-944729979.2AX-950840129.9AX-94568077 AX-9563053111.1AX-9448500413.7AX-9445187419.8AX-9463273934.4AX-9474272535.0AX-9492256839.0AX-95172933 AX-94661980AX-95232711 AX-94639854AX-94620995 AX-95103321AX-94575722 AX-94632187AX-94498955 AX-94535299AX-95155530 AX-95217447AX-95217835 AX-94864501

44.3

AX-94606624 AX-94484294AX-94733835 AX-94640360AX-95152427

44.9

AX-9525238246.2AX-94720924 AX-95090395AX-94973643 AX-9511520247.5AX-94896956 AX-94678904AX-9522664950.8AX-94794297 AX-95127627AX-94584055 AX-94600369AX-94410588 AX-94811232AX-95228642 AX-94888719AX-94461212 AX-94948817AX-94982210 AX-94711036AX-94479413 AX-95211211AX-94930973 AX-94512204AX-94724796 AX-94644873AX-95160798 AX-94973159AX-94994927 AX-94493483AX-95021781 AX-94810907

51.4

AX-94519152 AX-95141943AX-9455795852.1AX-94384755 AX-95216110AX-94561964 AX-95097228AX-95109355 AX-94885008AX-95070974

52.7

AX-95086997 AX-9452807556.0AX-9491754258.6AX-95148453 AX-9499808161.2AX-9459214865.1AX-94885681 AX-9468697971.2AX-94705666 AX-94989868AX-9463156675.1AX-9513069088.0AX-9456349392.7AX-9522566295.3

AX-952396710.0AX-95093192 AX-94907381AX-94847911 AX-95237736AX-94494623 AX-94759774AX-94462719 AX-94660277AX-94579237 AX-94576956AX-94650966 AX-94683371AX-94809471 AX-94562576

9.8

AX-94892182 AX-9462917810.4AX-9517220811.7AX-9443480015.6AX-94479254 AX-9449616720.3AX-94626780 AX-94890198AX-94444059 AX-94458317AX-95217472 AX-94858681AX-95137691 AX-95225885AX-95104494 AX-94752954AX-94542560

24.2

AX-95178690 AX-94759841AX-94396324 AX-9441605124.8AX-9469584325.5AX-9502253326.1AX-94507243 AX-9501399626.8AX-95114993 AX-94511622AX-95018583 AX-94417262AX-94406001 AX-94680741AX-95149390 AX-94621359AX-94820220 AX-94534850AX-95091125 AX-95252784AX-95091321 AX-94900238AX-95099983 AX-94955053AX-94894258 AX-95231942AX-94821626

27.4

AX-95175053 AX-94727285AX-9444563934.9AX-94550894 AX-9460480636.2AX-9456392336.8AX-9462406437.4AX-94664855 AX-9448001053.8AX-9487213959.1AX-94942170 AX-9524279566.6AX-9521688974.1AX-9447177975.4

AX-948525290.0

AX-94410667 AX-94650536AX-95232028 AX-948244497.5

AX-9441656612.8AX-94622127 AX-9440036013.5AX-9502562318.1AX-9522219021.4AX-9443350026.0AX-94856207 AX-9448585528.6AX-9485386831.2AX-9512521532.5AX-94728891 AX-9467320633.1AX-94870199 AX-95068915AX-95214129 AX-94737285AX-94456878 AX-94403783AX-94492194 AX-95229154AX-95209170

39.9

AX-9524570059.9

AX-9492342363.8

AX-94423611 AX-9489960773.6

AX-9477764878.9

AX-94696316 AX-9519143398.9AX-94862159 AX-94707138AX-95191192 AX-94978002AX-94673787 AX-94516330AX-95173230

106.4

AX-94460847 AX-94819855AX-95097146 AX-94667360AX-95144713

107.0

AX-94841734111.6AX-94408870 AX-94531979AX-94807329116.3AX-94835958 AX-95239425AX-95138815 AX-95101430AX-95149006 AX-95204560AX-94885415 AX-94608298AX-94398244 AX-95205777AX-94818645

120.9

AX-94830400 AX-95009153AX-94476998 AX-94488340AX-94720805

126.2

AX-94389360 AX-94661431AX-94398079 AX-94727929AX-94538427 AX-95119584

126.9

AX-94564568127.5AX-94946208 AX-94397536129.5

AX-946741730.0AX-945100650.6AX-952534401.3AX-94712036 AX-94521691AX-94734278 AX-94716313AX-95235320 AX-94555123AX-95192654 AX-94499088AX-95001161 AX-94418584AX-95123099 AX-94943750AX-94520182 AX-94480507AX-95159514 AX-95177571AX-95205672 AX-94794859AX-94940030 AX-94820343AX-94439043 AX-94921535

11.8

AX-95181699 AX-94558440AX-94417465 AX-95189309AX-94712402 AX-94623475AX-94537388

12.4

AX-9457014214.4AX-94548714 AX-9486249715.7AX-9443841716.3AX-94500822 AX-94564505AX-94571981 AX-95014404AX-94691689 AX-95140519AX-95141195

16.9

AX-95179109 AX-94653149AX-9470185517.6AX-9507305018.9AX-94394561 AX-94419658AX-94598591 AX-95251447AX-94989255 AX-94589335AX-95166240 AX-95156762AX-94448052 AX-95103103AX-94389275 AX-94490934AX-94461545 AX-94853803AX-94842627 AX-95170787AX-95160169 AX-94459093AX-94437376 AX-94978617AX-94840798 AX-94476567

19.5

AX-9497416220.1AX-94924350 AX-94407767AX-94772679 AX-94677740AX-94995549 AX-95226234

20.8

AX-9507703121.4AX-95245657 AX-9523309222.0AX-95203559 AX-94536550AX-9489276422.7AX-94398093 AX-9458626325.3AX-9461415326.6AX-94988614 AX-9469149228.5AX-94551616 AX-94607914AX-9476627535.3AX-9516580336.5AX-9458468139.8AX-9481137542.4AX-94552837 AX-9490069943.7AX-9507194747.0AX-94444505 AX-94420820AX-9517438649.6AX-94635862 AX-94671640AX-9496919250.2AX-94412298 AX-9521733853.5AX-94842449 AX-9513708254.1AX-9514189454.7AX-95206774 AX-9488266055.4AX-95166588 AX-94441736AX-94450794 AX-95153922AX-95241429 AX-94687848AX-95247927

56.0

AX-94432510 AX-94637377AX-94456002 AX-9455129956.6AX-9449825459.9AX-94861348 AX-94515524AX-9496026062.5AX-9489523170.7AX-9460965372.7AX-9469408076.0AX-95199123 AX-9497505179.2AX-9455472894.7AX-94750909101.4AX-94490226123.3AX-95170123124.6AX-94695574127.9

AX-95008221 AX-952168210.0AX-952044056.8AX-94591770 AX-95175529AX-952564078.7AX-94836047 AX-9445033710.0AX-9525117210.6AX-9492491114.6AX-94679663 AX-94593555AX-9483151622.8AX-95154799 AX-94724589AX-94786288 AX-9507550423.4AX-9446855724.1AX-95218218 AX-94808907AX-94806979 AX-94880647AX-95120832 AX-94682982

24.7

AX-95202819 AX-95145180AX-94735225 AX-94454883AX-94461514 AX-95142323AX-94520215

25.4

AX-9509930626.6AX-9523374827.9AX-94776871 AX-94478906AX-94664517 AX-9492031830.5AX-95012505 AX-95248020AX-94920652 AX-95104180AX-94747714 AX-95180847AX-94562499 AX-94541273AX-94823659 AX-94562688AX-94975749 AX-94476517AX-94761183 AX-94946469

33.1

AX-94536363 AX-9508433733.8AX-9519875634.4AX-9501612535.0AX-9447696736.3AX-9452907136.9AX-94773412 AX-9455342057.9AX-9521027563.9AX-94919784 AX-9439225075.2AX-9459918088.1AX-94823100 AX-9517885290.7AX-9492702692.7AX-94688239 AX-9476878493.3

AX-95018981 AX-950988010.0AX-95199774 AX-944496943.3AX-94977562 AX-9521609318.7AX-95227859 AX-94755373AX-94726646 AX-9475335122.0AX-94504660 AX-9454078325.3AX-9484952825.9AX-95162927 AX-94648397AX-94873933 AX-94519563AX-94445791

26.5

AX-95127864 AX-94504244AX-94451749 AX-95021932AX-94813599 AX-95193916AX-94711793 AX-95184301AX-94581390 AX-94499775AX-94392514 AX-94465607AX-94936772

27.8

AX-9523780628.5AX-94945788 AX-9444098529.1AX-94639975 AX-95249077AX-9518272029.7AX-9494492830.4AX-9464206031.0AX-9469492432.3AX-9502066934.2AX-94457524 AX-94468508AX-94887274 AX-9479558136.2AX-9523873836.8AX-9481571140.1AX-94889874 AX-9458217144.0AX-9514344345.3AX-9469218949.2AX-9518589452.5AX-94453227 AX-9458061154.4AX-94486291 AX-9507921057.7AX-94943283 AX-94463478AX-94701057 AX-94699229AX-94984908 AX-94668679

62.4

AX-9463738463.6AX-95226247 AX-94606716AX-94487259 AX-94471470AX-94874646 AX-94478741AX-94525211 AX-94507010AX-94526306 AX-94506247AX-94468312 AX-94712104AX-95124586 AX-94433754

64.3

AX-9448275364.9AX-95182952 AX-94488789AX-94445159 AX-9486773065.5AX-94563557 AX-9464968868.8AX-9438470771.4AX-94630546 AX-9508174776.1AX-9492533978.0AX-9447699584.0AX-9523677986.6AX-9453889893.4AX-95076674 AX-9449748596.0AX-94484496 AX-9498716699.3AX-95088552103.9

LG 1T LG 2T LG 3T LG 4T LG 5T LG 6T LG 7T

Figure 2 Genetic linkage map of Am. muticum. SNP marker names and cM distances for each of the seven linkage groups are also shown in supporting

Table S2.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Julie King et al.4

wheat/Am. muticum introgressions spanning the entire genomeof

the wild relative. The genetic map also allowed us to characterize

and track the introgressions as can be seen in Figure 3. However, it

should be noted that the germplasmused to generate these linkage

maps did not constitute proper mapping families and in fact we

combined different generations in order to have sufficient num-

bers. Therefore, the cM distances should be treated with consid-

erable caution. Analysis of the SNP data revealed that the majority

of introgressions generated were present in the 16 BC1 individuals,

that is significant levels of recombination occurred in the gametes

of the F1 hybrids. However, SNP analysis also indicated that further

recombination between wheat/Am. muticum chromosomes was

also continuing to occur in later generations (Figure 7).

In summary, the very high frequency of introgression detected

via the SNP array offsets any problems in the low fertility observed

in the F1 interspecific hybrids, and hence, this low fertility can be

simply overcome by increasing the number of crosses made to the

F1 individuals in an introgression programme.

SNP markers from the array are being converted into KASP

markers, thus allowing us to track individual introgressions

through the process of backcrossing and selfing as we are able

to ‘tag’ the introgressions with selected markers. The advantage

of this approach as opposed to the one used by Wendler et al.

(2014, 2015) to detect Hordeum bulbosum introgressions in

H. vulgare, that is that of applying genotyping by sequencing or

exome capture re-sequencing and mapping SNP variation to a

reference genome, is that we are able to use the one dedicated

array to identify and characterize introgressions from all ten of the

wild relative species we are currently working with, without the

need for a reference genome. To date, we have screened

approximately 2500 individual genotypes via the array.

The presence of wheat/Am. muticum introgressions that were

identified via SNP analysis and genetic mapping was confirmed by

multicolour GISH (Figure 4i–iv). In addition, intergenomic recom-

binants between the A, B and D genomes of wheat were also

observed (Figure 5). Thus, as part of the ongoing programme, we

are backcrossing lines to obtain individuals with single introgres-

sions but which have lost the A, B and D intergenomic

recombinants. SNP analysis revealed a close syntenic relationship

between all three genomes of wheat and the T genome of

Am. muticum with the exception that the latter does not carry

the 4A/5A/7B translocation seen in wheat (Figure 6). However, to

date, GISH has only identified Am. muticum introgressions

between the B and D genomes of wheat. None have been

observed with the A genome of wheat. The observation that

Am. muticum preferentially pairs with B and D genome chromo-

somes indicates that the T genome of the wild relative is more

closely related to the progenitors of the B (thought to be

Ae. speltoides or a close relative – Dvorak and Zhang, 1990) and

D genomes (Ae. tauschii – McFadden and Sears, 1946) than to

the A genome donor (T. urartu – Dvorak et al., 1993; King et al.,

1994). A further indicator of a potentially close relationship

between Am. muticum and Ae. speltoides is the fact that both

species carry an inhibitor of the Ph1 locus (Bennett et al., 1974;

Chen et al., 1994; Dover and Riley, 1972; Dvorak, 1972) a

phenomenon which is extremely rare. The present SNP analysis

does not reveal which wheat chromosomes have been involved in

introgressions with Am. muticum. Thus, it is presently not

possible to determine whether introgressions between the A

genome of wheat and the T genome of Am. muticum have

occurred other than by GISH. However, a future aim of the

programme is to produce lines that are homozygous for each of

the introgressions generated. Once generated, wheat chromo-

some-specific markers will be used to determine which wheat

chromosome(s) is (are) involved in each of the introgressions.

In the work described, homoeologous recombination was

induced by the Ph1 suppressor action of Am. muticum genes.

Previous work postulated that the suppression of Ph1 pairing

control by Am. muticum involved two gene loci with two

different allelic variants (Bennett et al., 1974; Dover and Riley,

1972). In this work, the Ph1 suppressors resulted in a high

frequency of homoeologous recombination during gametogen-

esis in the F1 hybrids. However, homoeologous recombination

between the chromosomes of Am. muticum and those of wheat

was also observed in later generations. We are presently trying to

determine the genetic control of the Am. muticum Ph1 suppres-

sion system. This will provide information on whether the genes

that suppress the Ph1 locus were present in lines that underwent

further recombination in the BC1, BC2 and BC3 generations.

The wild relatives of wheat provide a vast reservoir of genetic

variation for agronomically important traits such as plant

production (e.g. photosynthetic capacity), tolerance to abiotic

stresses (e.g. heat, drought and salinity) and biotic stresses (e.g.

Table 2 Number of introgressed segments from Am. muticum

present in BC3 plants as detected by SNP genotyping and genomic

in situ hybridization (GISH). The Am. muticum linkage group of each

introgression is based on the SNP marker positions in wheat

Accession

numbers of

BC3 plants

Number of segments Am. muticum

linkagegroup

of segmentGenotyping GISH

157C 0 0

157D 0 0

157E 0 0

159F 2 2 LG3, LG4

159G 1 1 LG4

159H 0 0

163C 0 0

163D 0 0

163E 0 0

172D 1 1 LG5

172E 3 3 LG1, LG3, LG5

177C 2 2 LG2, LG4

177D 1 1 LG4

177E 2 2 LG2, LG4

178C 1 1 LG7

178D 1 1 LG3

181C 1 1 LG6

182F 0 0

182G 3 3 LG1, LG2, LG7

182H 1 1 LG2

187E 1 1 LG2

238A 2 2 LG6, LG7

238B 2 2 LG6, LG7

240A 3 3 LG4, LG5, LG7

241A 3 3 LG4, LG5, LG7

242A 2 2 LG5, LG7

243A 2 2 LG1, LG5

243B 3 3 LG1, LG5, LG6

243C 2 2 LG1, LG5

246 4 4 LG3, LG4, LG5, LG6

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Wheat/wild relative introgression 5

fungal diseases and insect attack). With the lack of diversity

within wheat itself, wild relatives may prove to be the primary

means by which to increase wheat yields above the current

plateau. In this work, we have shown that for the first time, we

can systematically unlock the genomewide reservoir of genetic

variation available in a wild relative for utilization in wheat

breeding. However, for the full value of the material to be

recognized, it is essential that the germplasm generated is fully

phenotypically characterized.

Experimental procedures

Plant material

In order to generate introgressions, hexaploid wheat (variety,

Paragon) was pollinated with Am. muticum (accessions 2130004,

2130008, 2130012 obtained from JIC stock centre) to produce F1interspecific hybrids (Figure 1). Introgression of genetic variation

from Am. muticum into wheat occurs when the chromosomes of

the two species recombine during gametogenesis in these

interspecific F1 hybrids. This results in the production of gametes

which carry Am. muticum/wheat recombinant chromosomes (the

subsequent transmission of these recombinant chromosomes to

their progeny leads to the generation of Am. muticum/wheat

introgressions).

The hybrids were then grown to maturity and backcrossed as

the female with the wheat parent to generate BC1 populations.

The BC1 individuals and their resulting progenies were then

recurrently pollinated with the wheat parent to produce BC2, BC3

populations, etc. (Figure 1).

Identification of introgressions via an Affymetrix SNParray

The Nottingham/BBSRCWheat Research Centre (WRC) is presently

engaged in the genomewide introgression of genetic variation

from ten wild relatives into wheat, that is Am. muticum, Ae. spel-

toides, Aegilops caudata, Triticum timopheevii, T. urartu, Secale

cereale, Thinopyrum bessarabicum, Thinopyrum elongatum,

Thinopyrum intermedium and Thinopyrum ponticum. To detect

introgressed chromosomes and chromosome segments from these

wild relatives into wheat, an array of circa 35K SNPs has been

developed. In summary, the array is composed of SNPs each

showing polymorphism for the ten wild relatives relative to the

wheat genotypes understudy. [All the SNPs incorporated in this

array formed part of the Axiom� 820K SNP array (Winfield et al.,

2015). The data set for the Axiom� 820K array is available from

www.cerealsdb.uk.net (Wilkinson et al., 2012)]. Table 1 shows the

number of putative SNPs between Am. muticum and each of the

wheat genotypes included on the array. The array has been

constructed in such a way that up to 384 lines can be screened at

one time. Thus, the array facilitates the high-throughput, high-

resolution screening of introgressions that are being generated

from any of the ten wild relatives, including Am. muticum.

Figure 3 SNP characterization of Am. muticum introgressions in three consecutive generations, that is BC1, BC2 and BC3 and genomic in situ

hybridisation image of the BC3 genotype. In the SNP characterization, red colour is used to represent the presence of an Am. muticum introgression, blue

colour wheat. It should be noted that these diagrams cannot be used to assess which wheat chromosomes the Am. muticum segments have recombined

with. The GISH image shows a metaphase spread of BC3 159F probed with labelled genomic DNA of Am. muticum. Arrows show Am. muticum

introgressions (green).

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Julie King et al.6

Genotyping

The Axiom� Wheat-Relative Genotyping Array was used to

genotype 167 samples using the Affymetrix GeneTitan� system

according to the procedure described by Affymetrix (Axiom� 2.0

Assay Manual Workflow User Guide Rev3). Allele calling was

carried out using the Affymetrix proprietary software packages

Affymetrix Power Tools (APT) and SNPolisherTM (http://

www.affymetrix.com/estore/partners_programs/programs/devel-

oper/tools/devnettools.affx). A custom software pipeline ADAP

(Axiom� Data Analysis Pipeline) was written in perl to simplify the

data analysis, following the Axiom� Best Practices Genotyping

Workflow (http://media.affymetrix.com/support/downloads/man-

uals/axiom_genotyping_solution_analysis_guide.pdf). A variant

call rate threshold of 80% was used instead of the default value

(97%) to account for the lower call rates typically obtained from

hybridising wheat relatives and progenitors to the array. The apt-

probeset-genotype program within Affymetrix Power Tools

determines genotype calls from Affymetrix SNP microarrays.

Following this, the SNPolisher R package calculates SNP perfor-

mance metrics, such as call rate, cluster separation and deviation

from expected cluster position. It then classifies the SNPs into

performance categories. These categories were as follows: (i)

‘Poly High Resolution’ (PHR), which were codominant and

polymorphic, with at least two examples of the minor allele; (ii)

‘No Minor Homozygote’ (NMH), which were polymorphic and

dominant, with two clusters observed; (iii) ‘Off-Target Variant’

(OTV), which had four clusters, one representing a null allele; (iv)

‘Mono High Resolution’ (MHR), which were monomorphic; (v)

‘Call Rate Below Threshold’ (CRBT), where SNP call rate was

below threshold but other cluster properties were above thresh-

old; and (vi)’ Other’, where one or more cluster properties were

below threshold. For genetic mapping purposes, only the PHR

SNPs were used as they provide good cluster resolution where

each SNP essentially behaves like a diploid.

Figure 4 i–iv. Genomic in situ hybridization

(GISH) showing recombination between

Am. muticum and the B and D genomes of

wheat. (i) GISH of complete one-cell metaphase

spread with labelled genomic Am. muticum as

probe showing Am. muticum (green)

introgressions (white arrows). (ii) Same metaphase

spread as (i) with three-colour GISH showing one

Am. muticum introgression recombined with the

B genome (purple) of wheat and the second

introgression recombined with both the B (purple)

and D (red) genomes of wheat. (iii) GISH of

complete one-cell metaphase spread with labelled

Am. muticum as probe showing an Am. muticum

(green) introgression (white arrow) [also shown in

the magnified inset chromosome]. (iv) Same

metaphase spread as (iii) with three-colour GISH

showing recombination between Am. muticum

and the D (red) genome of wheat.

Figure 5 GISH image showing intergenomic recombination. The

metaphase spread shows a 41 chromosome cell with 12 A genome

chromosomes (green), 13 B genome chromosomes (purple) and 12 D

genome chromosomes (red). There are also two Am. muticum

introgressions (white arrows), one A/D recombinant chromosome (yellow

arrow) and one B/A/D recombinant chromosome (red arrow).

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Wheat/wild relative introgression 7

Figure 6 Synteny of Am. muticum (genetic

position in cM) with hexaploid wheat (physical

position in Mb) [visualized using Circos v. 0.67;

Krzywinski et al., 2009].

Figure 7 SNP analysis of Am. muticum

introgressions in two consecutive generations

(BC1 and BC2) showing recombination (involving

linkage group 2) has occurred during

gametogenesis in the BC1 genotype. Red colour

shows the presence of Am. muticum

introgressions, and blue colour shows wheat

chromosomes.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Julie King et al.8

Genetic Mapping of Am. muticum chromosomes

Individuals from a backcross population between T. aestivum

and Am. muticum were genotyped with the Axiom� Wheat-

Relative Genotyping Array. Along with triplicates of the three

parental lines, 158 lines comprising BC1, BC2 and BC3 popula-

tions of Am. muticum were genotyped altogether. As stated

above, only the PHR SNP markers were used for genetic

mapping. SNP markers which showed (i) heterozygous calls for

either parent(s), (ii) no polymorphism between the wheat

parents and Am. muticum and/or (iii) no calls for either parent

(s) were removed using FlapjackTM (Milne et al., 2010;

v.1.14.09.24). The resulting markers were sorted into linkage

groups in JoinMap� 4.1 (van Ooijen, 2011) with a LOD score of

20 and a recombination frequency threshold of 0.1 using the

Haldane mapping function (Haldane, 1919). All markers that did

not show any heterozygous call or were unlinked were ignored

and only the highest ranking linkage groups with more than 30

markers were selected for map construction. These were

exported and assigned to chromosomes using information from

the Axiom� Wheat HD Genotyping Array (Winfield et al., 2015).

Where chromosomes were split into multiple linkage groups,

these were re-formed into a single linkage group and reordered.

Erroneous markers that had more than 20% missing data or

showed a unique pattern of segregation that was either not

observed in the previous backcross generation or not consistent

with the recombination of neighbouring markers in the group,

in different samples, were also removed. The long and short arm

of each chromosome was identified from the IWGSC wheat

survey sequence (The International Wheat Genome Sequencing

Consortium, 2014), and groups were orientated to have the

short arm above the long arm. Final map reordering was

conducted with JoinMap 4.1 and genetic maps produced

through MapChart 2.3 (Voorrips, 2002). In some cases, physical

map information was employed to order loci. Graphical geno-

type visualization was performed using Graphical GenoTypes 2.0

(GGT; van Berloo, 2008).

Comparative analysis

Synteny analysis was carried out using sequence information

of the markers located on the present map of Am. muticum.

The sequences of the mapped markers were compared using

BLAST (e-value cut-off of 1e-05) against the wheat genome

(http://plants.ensembl.org/Triticum_aestivum) to obtain the

orthologous map positions of the top hits in the A, B and

D genomes of wheat. To generate the figures, cM distances

on the linkage groups of the present map of Am. muticum

were scaled up by a factor of 100 000 to match similar base

pair lengths of the chromosomes of the wheat genome.

Figure 6 was visualized using Circos (v. 0.67; Krzywinski

et al., 2009) to observe synteny between Am. muticum

(genetic position in cM) and the wheat genome (physical

position in Mb).

Cytogenetic analysis

The protocol for genomic in situ hybridisation (GISH) was as

described in Zhang et al. (2013) and Kato et al. (2004). Genomic

DNA was isolated using a CTAB method (Zhang et al., 2013) from

young leaves of the three putative diploid progenitors of bread

wheat, that is T. urartu (A genome), Ae. spltoides (B genome)

and Ae. tauschii (D genome), and from Am. muticum. The

genomic DNA of Am. muticum and T. urartu was labelled by

nick translation with Chroma Tide Alexa Fluor 488-5-dUTP

(Invitrogen; C11397). Genomic DNA of Ae. tauschii was labelled

with Alexa Fluor 594-5-dUTP (Invitrogen; C11400). Genomic

DNAs of Ae. speltoides and T. aestivum cv. Chinese Spring were

fragmented to 300–500 bp in boiling water.

Preparation of chromosome spread was as described in Kato

et al. (2004), with modifications. Roots from each germinated

introgression line were excised and treated with nitrous oxide gas

at 10 bar for 2 h. Treated roots were fixed in 90% acetic acid for

10 min and then washed three times in water on ice. The root tip

was dissected and digested in 20 lL of 1% pectolyase Y23 and

2% cellulase Onozuka R-10 (Yakult Pharmaceutical, Tokyo)

solution for 50 min at 37 °C and then washed three times in

70% ethanol. The root tips were crushed in 70% ethanol, and

the cells collected by centrifugation at 3000 9 g for 1 min,

briefly dried and then re-suspended in 30–40 lL of 100% acetic

acid before being placed on ice. The cell suspension was dropped

onto glass slides (6–7 lL per slide) in a moist box and dried slowly

under cover.

Slides were initially probed using labelled genomic DNA of

Am. muticum 10(0 ng) and fragmented genomic DNA of Chi-

nese Spring (3000 ng) as blocker to detect the Am. muticum

introgressions. Probe to block was in a ratio of 1–30 (the

hybridization solution was made up to 10 lL with 2 9 SSC in

1 9 TE). The slides were then bleached and re-probed with

labelled DNAs of T. urartu (100 ng) and Ae. taushii (200 ng) and

fragmented DNA of Ae. speltoides (5000 ng) as blocker in the

ratio 1–2 to 50 to detect the AABBDD genomes of wheat. All

slides were counterstained with DAPI and analysed using a Leica

DM5500B epifluorescence microscope (Leica Microsystems, Wet-

zlar, Germany) with filters for DAPI (blue), Alexa Fluor 488 (green)

and Alexa Fluor 594 (red). Photographs were taken using a Leica

DFC 350FX digital camera.

Acknowledgements

This work at the Nottingham/BBSRC Wheat Research Centre

(http://www.nottingham.ac.uk/wisp/index.aspx) is part of the

BBSRC-funded Wheat Improvement Strategic Programme (WISP)

(http://www.wheatisp.org). The authors declare no conflict of

interest.

References

Al-Kaff, N., Knight, E., Bertin, I., Foote, T., Hart, N., Griffiths, S. and Moore, G.

(2008) Detailed dissection of the chromosomal region containing the Ph1

locus in wheat Triticum aestivum: with deletion mutants and expression

profiling. Ann. Bot. 101, 863–872.

Bennett, M.D., Dover, G.A. and Riley, R. (1974) Meiotic duration in wheat

genotypes with or without homoeologous meiotic chromosome pairing.

Proc. R. Soc. Lond. B, 187, 191–207.

van Berloo, R. (2008) GGT 2.0: versatile software for visualization and analysis

of genetic data. J. Hered. 99, 232–236.

Brisson, N., Gate, P., Gouache, D., Charmet, G., Oury, F.-X. and Huard, F.

(2010) Why are wheat yields stagnating in Europe? A comprehensive data

analysis for France. Field Crops Res. 119, 201–212.

Charmet, G. (2011) Wheat domestication: lessons for the future. C. R. Biol.

334, 212–220.

Chen, P.D., Tsujimoto, H. and Gill, B.S. (1994) Transfer of PhI genes promoting

homoeologous recombination from Triticum speltoides to common wheat.

Theor. Appl. Genet. 88, 97–101.

Cox, T.S. (1997) Deepening the wheat gene pool. J. Crop Prod. 1, 1–25.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Wheat/wild relative introgression 9

Dover, G.A. and Riley, R. (1972) Variation at two loci affecting homoeologous

meiotic pairing in Triticum aestivum x Aegilops mutica hybrids. Nature New

Biol. 235, 61–62.

Dvorak, J. (1972) Genetic variability in Aegilops speltoides

affecting homoeologous pairing in wheat. Can. J. Genet. Cytol. 14,

371–380.

Dvorak, J. and Zhang, H.B. (1990) Variation in repeated nucleotide sequences

sheds light on the phylogeny of the wheat B and G genomes. Proc. Natl Acad.

Sci. USA, 87, 9640–9644.

Dvorak, J., di Terlizzi, P., Zhang, H.B. and Resta, P. (1993) The evolution of

polyploid wheats – Identification of the A-genome donor species. Genome,

36, 21–31.

Eser, V. (1998) Characterisation of powdery mildew resistant lines derived from

crosses between Triticum aestivum and Aegilops speltoides and Ae. mutica.

Euphytica, 100, 269–272.

Friebe, B., Jiang, J., Raupp, W.J., McIntosh, R.A. and Gill, B.S. (1996)

Characterization of wheat-alien translocations conferring resistance to

disease and pests: current status. Euphytica, 91, 59–87.

Garcia-Olmedo, F., Delibes, A. and Sanchez-Monge, R. (1977) Transfer of

resistance to eyespot disease from Aegilops ventricosa to wheat. Proceedings

of the 8th Congress of Eucarpia 91–97.

Griffiths, S., Sharpe, R., Foote, T.N., Bertin, I., Wanous, M., Reader, S., Colas, I.

et al. (2006) Molecular characterization of PH1 as a major chromosome

pairing locus in polyploid wheat. Nature, 439, 749–752.

Haldane, J.B.S. (1919) The probable errors of calculated linkage values, and the

most accurate method of determining gametic from certain zygotic series. J.

Hered. 8, 291–297.

Iefimenko, T.S., Antonyuk, M.Z. and Martynenko, V.S. (2013) Development of

alien-substitution and alien-addition Triticum aestivum/Aegilops mutica lines.

In Factors of Experimental Evolution of Organisms: Collected Scientific Papers,

pp. 114–118. Kyiv: Logos.

Iefimenko, T.S., Fedak, Y.G., Antonyuk, M.Z. and Ternovska, T.K. (2015)

Microsatellite analysis of chromosomes from the fifth homoeologous group in

the introgressive Triticum aestivum/Amblyopyrum muticum wheat lines.

Cytol. Genet. 49, 183–191.

Jauhar, P.P. and Chibbar, R.N. (1999) Chromosome-mediated direct gene

transfers in wheat. Genome, 42, 570–583.

Kato, A., Lamb, J.C. and Birchler, J.A. (2004) Chromosome painting using

repetitive DNA sequences as probes for somatic chromosome identification in

maize. Proc. Natl Acad. Sci. USA, 101, 13554–13559.

King, I.P., Purdie, K.A., Liu, C.J., Reader, S.M., Pittaway, T.S., Orford, S.E. and

Miller, T.E. (1994) Detection of interchromosomal translocations within the

Triticeae by RFLP analysis. Genome, 37, 882–887.

King, J., Armstead, I., Harper, J., Ramsey, L., Snape, J., Waugh, R., James, C.

et al. (2013) Exploitation of interspecific diversity for monocot crop

improvement. Heredity, 110, 475–483.

King, J., Gustafson, P., Allen, A. and King, I.P. (2016) Exploitation of

interspecific diversity in wheat. In World Wheat Book, Vol. 3 (Bonjean,

A., van Ginkel, M. and Angus, B., eds), pp. 1125–1139. Paris: Lavoisier

ch. 4.

Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D.,

Jones, S.J. et al. (2009) Circos: an information aesthetic for comparative

genomics. Genome Res. 19, 1639–1645.

Liu, C.J., Devos, K.M., Chinoy, C.N., Atkinson, M.D. and Gale, M.D. (1992)

Non-homoeologous translocations between group 4, 5 and 7 chromosomes

in wheat and rye. Theor. Appl. Genet. 83, 305–312.

McFadden, E. and Sears, E.R. (1946) The origin of Triticum spelta and its free-

threshing hexaploid relatives. J. Hered. 37, 81–107.

Milne, I., Shaw, P., Stephen, G., Bayer, M., Cardle, L., Thomas, W.T.B., Flavell,

A.J. et al. (2010) Flapjack – graphical genotype visualisation. Bioinformatics,

26, 3133–3134.

Naranjo, T., Roca, A., Goicoecha, P.G. and Giraldz, R. (1987) Arm homoeology

of wheat and rye chromosomes. Genome, 29, 873–882.

van Ooijen, J.W. (2011) Multipoint maximum likelihood mapping in a full-sib

family of an outbreeding species. Genet. Res. 93, 343–349.

Qi, L., Friebe, B., Zhang, P. and Gill, B.S. (2007) Homoeologous recombination,

chromosome engineering and crop improvement.Chromosome Res. 15, 3–19.

Ray, D.K., Mueller, N.D., West, P.C. and Foley, J.A. (2013) Yield trends are

insufficient to double global crop production by 2050. PLoS ONE, 8,

e66428.

Riley, R. and Chapman, V. (1958) Genetic control of the cytologically diploid

behaviour of hexaploid wheat. Nature, 182, 713–715.

Schneider, A., Moln�ar, I. and Moln�ar-L�ang, M. (2008) Utilisation of Aegilops

(goatgrass) species to widen the genetic diversity of cultivated wheat.

Euphytica, 163, 1–19.

Sears, E.R. (1955) An induced gene transfer from Aegilops to Triticum.

Genetics, 40, 595.

Sears, E.R. (1972) Agropyron-wheat transfers through induced homoeologous

pairing. Can. J. Genet. Cytol. 14, 736.

Sears, E.R. (1976) Genetic control of chromosome-pairing in wheat. Annu. Rev.

Genet. 10, 31–51.

Sears, E.R. (1977) An induced mutant with homoeologous pairing in common

wheat. Can. J. Genet. Cytol. 4, 585–593.

Sears, E.R. and Okamoto, M. (1958) Intergenomic chromsome relationships in

hexaploid wheat. In proceedings of the Xth International Congress on

Genetics, Vol. 2: August 20–27, 1958, (Sewall, Wright, ed). McGill University,

Montreal: University of Toronto Press. pp. 258–259.

The International Wheat Genome Sequencing Consortium. (2014) A

chromosome-based draft sequence of the hexaploid bread wheat (Triticum

aestivum) genome. Science, 345, 1251788-1–1251788-11.

Tiwari, V.K., Wang, S., Sehgal, S., Vr�ana, J., Friebe, B., Kubal�akov�a, M.,

Chhuneja, P. et al. (2014) SNP discovery for mapping alien introgressions in

wheat. BMC Genom. 15, 273.

Tiwari, V.K., Wang, S., Danilova, T., Koo, D.H., Vr�ana, J., Kubal�akov�a, M.,

Hribova, E. et al. (2015) Exploring the tertiary gene pool of bread wheat:

sequence assembly and analysis of chromsome 5Mg of Aegilops geniculata.

Plant J. 84, 733–746.

Voorrips, R.E. (2002) MapChart: software for the graphical presentation of

linkage maps and QTLs. J. Hered. 93, 77–78.

Wendler, N., Mascher, M., Noh, C., Himmelbach, A., Scholz, U., Ruge-Wehling,

B. and Stein, N. (2014) Unlocking the secondary gene-pool of barley with

next-generation sequencing. Plant Biotechnol. J. 12, 1122–1131.

Wendler, N., Mascher, M., Himmelbach, A., Johnston, P., Pickering, R. and

Stein, N. (2015) Bulbosum to go: a toolbox to utilize Hordeum vulgare/

bulbosum introgressions for breeding and beyond. Mol. Plant, 8, 1507–1519.

Wilkinson, P.A., Winfield, M.O., Barker, G.L.A., Allen, A.M., Burridge, A.,

Coghill, J.A. and Edwards, K.J. (2012) CerealsDB 2.0: an integrated resource

for plant breeders and scientists. BMC Bioinform. 13, 219.

Winfield, M.O., Allen, A.M., Burridge, A.J., Barker, G.L.A., Benbow, H.R.,

Wilkinson, P.A., Coghill, J. et al. (2015) High-density SNP genotyping array

for hexaploid wheat and its secondary and tertiary gene pool. Plant

Biotechnol. J. 13, 733–742.

Zhang, H., Bian, Y., Gou, X., Zhu, B., Xu, C., Qi, B., Li, N. et al. (2013) Persistent

whole-chromosome aneuploidy is generally associated with nascent

allohexaploid wheat. Proc. Natl Acad. Sci. USA, 110, 3447–3452.

Supporting information

Additional Supporting Information may be found online in the

supporting information tab for this article:

Table S1. Number of seed produced and germinated in relation

to number of crosses carried out for each generation of the

introgression program for Am. muticum into wheat.

Table S2. SNP marker names and cM position on the genetic

linkage map of Am. muticum shown in Fig. 2.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–10

Julie King et al.10