Aberystwyth University Introgression of Aegilops

Aberystwyth University

Introgression of Aegilops speltoides segments in Triticum aestivum and theeffect of the gametocidal genesKing, Julie; Grewal, Surbhi ; Yang, Cai-yun; Hubbart Edwards, Stella ; Scholefield, Duncan; Ashling, Stephen;Harper, John; Allen, Alexandra; Edwards, Keith J.; Burridge, Amanda; King, Ian Philip

Published in:Annals of Botany

DOI:10.1093/aob/mcx149

Publication date:2018

Citation for published version (APA):King, J., Grewal, S., Yang, C., Hubbart Edwards, S., Scholefield, D., Ashling, S., Harper, J., Allen, A., Edwards,K. J., Burridge, A., & King, I. P. (2018). Introgression of Aegilops speltoides segments in Triticum aestivum andthe effect of the gametocidal genes. Annals of Botany, 121(2), 229-240. https://doi.org/10.1093/aob/mcx149

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Introgression of Aegilops speltoides segments in Triticum aestivum and the effect of the gametocidal genes

Julie King1,*, Surbhi Grewal1, Cai-yun Yang1, Stella Hubbart Edwards1, Duncan Scholefield1, Stephen Ashling1, John A. Harper2, Alexandra M. Allen3, Keith J. Edwards3,

Amanda J. Burridge3 and Ian P. King1

1Division of Plant and Cop Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK, 2The Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Plas Gogerddan, Aberystwyth

SY23 3EB, UK and 3School of Biological Sciences, University of Bristol, Bristol BS8 1TQ, UK*For correspondence. E-mail [email protected]

Received: 25 May 2017 Returned for revision: 28 August 2017 Editorial decision: 8 September 2017 Accepted: 13 October 2017

• Background and Aims Bread wheat (Triticum aestivum) has been through a severe genetic bottleneck as a result of its evolution and domestication. It is therefore essential that new sources of genetic variation are generated and utilized. This study aimed to generate genome-wide introgressed segments from Aegilops speltoides. Introgressions generated from this research will be made available for phenotypic analysis.• Methods Aegilops speltoides was crossed as the male parent to T. aestivum ‘Paragon’. The interspecific hybrids were then backcrossed to Paragon. Introgressions were detected and characterized using the Affymetrix Axiom Array and genomic in situ hybridization (GISH).• Key Results Recombination in the gametes of the F1 hybrids was at a level where it was possible to generate a genetic linkage map of Ae. speltoides. This was used to identify 294 wheat/Ae. speltoides introgressions. Introgressions from all seven linkage groups of Ae. speltoides were found, including both large and small segments. Comparative analysis showed that overall macro-synteny is conserved between Ae. speltoides and T. aestivum, but that Ae. speltoides does not contain the 4A/5A/7B translocations present in wheat. Aegilops speltoides has been reported to carry gametocidal genes, i.e. genes that ensure their transmission through the gametes to the next generation. Transmission rates of the seven Ae. speltoides linkage groups introgressed into wheat varied. A 100 % transmission rate of linkage group 2 demonstrates the presence of the gametocidal genes on this chromosome.• Conclusions A high level of recombination occurs between the chromosomes of wheat and Ae. speltoides, leading to the generation of large numbers of introgressions with the potential for exploitation in breeding programmes. Due to the gametocidal genes, all germplasm developed will always contain a segment from Ae. speltoides linkage group 2S, in addition to an introgression from any other linkage group.

Key words: Aegilops speltoides, Triticum aestivum, introgression, recombination, comparative synteny, gametocidal, genetic linkage mapping, GISH, chromosome transmission

INTRODUCTION

Bread wheat (Triticum aestivum), one of the world’s leading sources of food, is an allopolyploid (6x = AABBDD = 42) com-posed of the genomes of three different species. The A genome is derived from Triticum urartu (Dvorak et al., 1993), the B from Aegilops speltoides or a closely related species (Sarkar and Stebbins, 1956; Riley et al., 1958; Dvorak and Zhang, 1990; Maestra and Naranjo, 1998; Marcussen et al., 2014) and the D genome from Aegilops tauschii (McFadden and Sears, 1946). The final hybridization event between tetraploid wheat (AABB) and Ae. tauschii (DD) is thought to have occurred only once or twice, ~8000–10 000 years ago. As a result, wheat went through a significant genetic bottleneck. Thus, the significant yield gains achieved by wheat breeders to date have been via the exploitation of genetic variation that has arisen via gene mutation over the last 8000–10 000 years and rare outcross-ing events with tetraploid wheat. In many parts of the world

wheat yields are now starting to plateau and this is thought to be a direct result of a lack of genetic variation compounded by the changing environment at a time of increasing demand for food due to the increasing global population (Charmet, 2011; Grassini et al., 2013; Ray et al., 2013). It is therefore essential that new sources of genetic variation be found that will enable breeders to generate the next generation of high-yielding envi-ronmentally adapted wheat varieties (Tanksley and McCouch, 1997; Haussmann et al., 2004; Tester and Langridge, 2010; McCouch et al., 2013; Warschefsky et al., 2014; Brozynska et al., 2015; Zhang et al., 2017)

Unlike wheat, its progenitor species and wild relatives pro-vide a vast and largely untapped source of genetic variation for most, if not all, traits of agronomic importance. Considerable efforts have been made to exploit the genetic variation within the progenitors and wild relatives, with some noticeable suc-cesses, for example the transfer of a segment of Aegilops

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King et al. — Introgression of Aegilops speltoides in a hexaploid wheat background2

umbellulata to wheat conferring resistance to leaf rust (Sears, 1955), the transfer of a segment from Aegilops ventricosa car-rying resistance to eyespot (Maia, 1967; Doussinault et al., 1983) and its subsequent release in the variety Rendevouz, and the registration of ‘MACE’, a hard red winter wheat carrying a segment from Thinopyrum intermedium conferring resistance to wheat streak mosaic virus (Graybosch et al., 2009). Progress has been severely hindered, however, due to an inability to quickly and accurately identify and characterize interspecific transfers to wheat. However, with the advent of new molecular technologies coupled with specific crossing strategies, we can now systematically exploit the genetic variation available in the progenitors of wheat and its wild relatives. (e.g. Tiwari et al., 2014, 2015; King et al., 2016, 2017).

The transfer of genetic variation to wheat from its progeni-tors/wild relatives occurs via recombination between the chro-mosomes of wheat and those of its wild relative at meiosis in F1 hybrids or in their derivatives, e.g. addition and substitution lines (King et al., 2016, 2017). This results in the generation of interspecific recombinant chromosomes. Once identified, interspecific recombinant chromosomes are then recurrently backcrossed with wheat. The ultimate objective is to generate lines of wheat carrying a small chromosome segment from a progenitor/wild relative with a target gene but few, if any, deleterious genes.

One of the wild relatives of wheat that is of particular interest to researchers is Ae. speltoides, a rich source of genetic variation for resistance to a range of diseases of importance in wheat (Jia et al., 1996; Mago et al., 2009; Klindworth et al., 2012; Deek and Distelfeld, 2014). Aegilops speltoides, or an extinct close relative, has been proposed as the donor of the B genome of wheat (Sarkar and Stebbins, 1956; Dvorak and Zhang, 1990; Friebe and Gill, 1996; Tsunewaki, 1996; Dvorak and Akhunov, 2005; Marcussen et al., 2014). Aegilops speltoides is a facul-tative outbreeder, but can be readily inbred in the glasshouse.

Unlike most of the wild relatives of wheat, Ae. speltoides possesses the ability to suppress the wheat Ph1 locus (Dvorak, 1972; Dvorak et al., 2006). Recombination is normally restricted to homologous chromosomes from the same genome, i.e. although the A, B and D genomes of wheat and chromo-somes from wild relatives are related, recombination can only occur within genomes in the presence of Ph1. However, if Ph1 is removed, related chromosomes (homoeologues) from differ-ent genomes can recombine at meiosis. Aegilops speltoides pos-sesses genes on chromosomes 3S (Su1-Ph1) and 7S (Su2-Ph1) (Dvorak et al., 2006), with the ability to suppress Ph1. Thus, in F1 hybrids between wheat and Ae. speltoides Su1-Ph1 or Su2-Ph1 enables intergenomic homoeologous recombination to occur during meiosis.

Aegilops speltoides also carries gametocidal genes (Gc) (Tsujimoto and Tsunewaki, 1984, 1988; Ogihara et al., 1994), which are preferentially transmitted to the next generation. Individuals heterozygous for Gc gene(s) produce two types of gametes. Both male and female gametes that lack the Gc genes are generally not viable due to chromosome breakage (Finch et al., 1984). Gametes that carry the Gc genes, however, behave normally and thus it is only these gametes that are transmitted to the next generation. Aegilops speltoides carries two alleles of the gametocidal genes. Gc1a and Gc1b, both located on chro-mosome 2S, are transmitted at a very high frequency to the next

generation and give rise to different morphological aberrations. Gc1a causes endosperm degeneration and chromosome aber-rations while Gc1b results in the production of seeds that lack shoot primordia (Tsujimoto and Tsunewaki, 1988). The limita-tions of utilizing species with gametocidal genes for wild rela-tive introgression are: (1) the fertility of hybrid/crossed grain is reduced by at least 50 %; and (2) all introgressions from wild relatives with Gc genes will always carry them and any other genes closely linked to them. Hence it is very difficult, if not impossible, to transfer genes from other regions of the genome in the absence of the Gc genes.

In this paper we investigate (1) the ability of the Ae. spel-toides suppressors of the Ph1 locus located on chromosomes 3S and 7S to induce recombination between the chromosomes of wheat and those of Ae. speltoides, and (2) the frequency of transmission of the Ae. speltoides gametocidal chromosomes through the gametes in F1 hybrids and their derivatives.

MATERIALS AND METHODS

Generation of introgressions

The method used to induce introgressions between wheat and Ae. speltoides is the same as that described by King et al. (2017). In summary, hexaploid wheat (‘Paragon’, obtained from the Germplasm Resources Unit at the JIC; code W10074) was pollinated with Ae. speltoides (accession 2140066 obtained from the Germplasm Resources Unit at the JIC) to produce F1 interspecific hybrids. These hybrids were then grown to ma-turity and backcrossed as the female with the wheat parent to generate BC1 populations. The BC1 individuals and their result-ing progenies were then recurrently pollinated to produce BC2, BC3 etc. populations(Fig. 1).

As a result of the presence of the suppressors of the Ph1 locus, homoeologous recombination was expected to occur at meiosis in the F1 wheat/Ae. speltoides hybrids, leading to the production of interspecific recombinant chromosomes/introgressions. Subsequent recurrent backcrossing of any BC1 progeny to the wheat parent was undertaken to transfer any interspecific recombinant chromosomes generated into a wheat (Paragon) background.

Detection of putative wheat/Ae. speltoides introgressions

A 35K Axiom® Wheat-Relative Genotyping Array (Affymetrix, Santa Clara, CA) was used to detect the presence of putative wheat/Ae. speltoides introgressions in each of the backcross gen-erations (King et al., 2017). In summary, the array is composed of single-nucleotide polymorphisms (SNPs), each showing poly-morphism for the ten wild relatives (under study at the Nottingham/BBSRC Wheat Research Centre), including Ae. speltoides (King et al., 2017). All the SNPs incorporated in this array formed part of the Axiom® 820K SNP array (Winfield et al., 2016). The dataset for the Axiom® 820K SNP array is available from www.cerealsdb.uk.net (Wilkinson et al., 2012, 2016). Table 1 shows the number of putative SNPs between Ae. speltoides and each of the wheat genotypes included on the array. The array has been constructed in such a way that up to 384 lines could be screened at one time. Thus, the array facilitated the high-throughput, high-resolution

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King et al. — Introgression of Aegilops speltoides in a hexaploid wheat background 3

screening of wheat/Ae. speltoides introgressions. Genotyping was performed as described by King et al. (2017) with no modifica-tions (see below).

Genetic mapping of Ae. speltoides

Genetic mapping was as described by King et al. (2017); in summary, DNA was extracted using a CTAB method (Zhang et al., 2013) from individuals of the back-crossed populations, BC1, BC2, BC3, BC4 and BC5 derived from the wheat/Ae. spel-toides F1 hybrids. These populations were genotyped with the Axiom® Wheat-Relative Genotyping Array. Only Poly High Resolution (PHR) SNP markers, which were codominant and polymorphic, with at least two examples of the minor allele were used for genetic mapping (King et al., 2017). The SNP markers that showed (1) heterozygous calls for either parent(s), (2) no polymorphism between the wheat parents and Ae.

speltoides, and/or (3) no calls for either parent(s) were removed using Flapjack™ (Milne et al., 2010; v.1.14.09.24). The resulting markers were sorted into linkage groups (Fig. 2) 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 >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., 2016).

Comparative analysis

Synteny analysis was carried out using sequence information of the markers located on the present map of Ae. speltoides. The sequences of the mapped markers were compared using

Fig. 1. Wheat/wild relative introgression strategy.

Table 1. Number of polymorphic SNPs between Ae. speltoides and hexaploid wheat based on the Affymetrix 35K array. A comparison is made between all calls (all markers showing polymorphism between wheat and Ae. speltoides) and Poly High Resolution (PHR) calls,

which are codominant and polymorphic between wheat and Ae. speltoides with at least two examples of the minor allele

Linkage group Total

1 2 3 4 5 6 7

All calls (% of total) 2770 (12.4) 3992 (17.9) 3358 (15.1) 2798 (12.6) 3646 (16.4) 2530 (11.4) 3164 (14.2) 22 258PHR calls (% of total) 69 (12.7) 78 (14.3) 82 (15.1) 85 (15.6) 90 (16.5) 46 (8.5) 94 (17.3) 544



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, centimorgan (cM) distances on the linkage groups of the present map of Ae. spel-toides were scaled up by a factor of 100 000 to match similar base-pair lengths of the chromosomes of the wheat genome. Figure 3 was visualized using Circos (v. 0.67; Krzywinski et al., 2009) in order to observe synteny between Ae. speltoides (genetic position in cM) and the wheat genome (physical posi-tion in Mb).

Cytogenetic analysis

The protocol for genomic in situ hybridization (GISH) was as described in Zhang et al. (2013), Kato et al. (2004) and King et al. (2017). In summary, genomic DNAs from young leaves of the three putative diploid progenitors of bread wheat, i.e. T. urartu (A genome), Ae. speltoides (B genome) and Ae. tauschii (D genome), were isolated using extraction buffer (0.1 m Tris–HCl pH 7.5, 0.05 m EDTA pH 8.0, 1.25% SDS). Samples were incubated at 65 °C for 1 h before being placed on ice and mixed with ice-cold 6 m NH4C2H3O for 15 min. The samples were then spun down, the supernatant was mixed with

Aegilops speltoides linkage groups

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LG 1S

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AX-9492382428.5AX-9449135129.4AX-9520132931.3AX-9446524731.5AX-9461273837.2AX-9491754238.1AX-9499808138.3AX-95159521 AX-94410787AX-9467005440.2

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76.1

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76.3

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76.8

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AX-94584055 AX-9476875479.1AX-9439060687.6AX-9463156688.2AX-9512322389.3AX-9460174690.3

LG 2S

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AX-951210286.2

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69.1

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AX-94613063 AX-95018583AX-9495505369.5

AX-95137667 AX-94421465AX-9460480669.7

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70.6

AX-9455319371.9AX-94420976 AX-95167864AX-94919444 AX-9510834272.1

AX-9482923390.4AX-9521688998.5

LG 3S

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22.9

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AX-95239425 AX-94400072AX-9520577726.3

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40.2

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LG 4S

Fig. 2. Genetic linkage map of Ae. speltoides.


http://plants.ensembl.org/Triticum_aestivum


isopropanol to pellet the DNA and the isolated DNA was further purified with phenol/chloroform. The genomic DNAs of Ae. speltoides and T. urartu were labelled by nick translation with Chroma Tide Alexa Fluor 488-5-dUTP (Invitrogen, Carlsbad, CA; C11397). Genomic DNA of Ae. tauschii was labelled with Alexa Fluor 594-5-dUTP (Invitrogen; C11400). Genomic DNAs of Ae. speltoides and T. aestivum ‘Chinese Spring’ were fragmented to 300–500 bp in a heat block at 100 °C.

Preparation of chromosome spreads was as described in Kato et al. (2004) and King et al. (2017). Briefly, roots were excised from germinated seeds, treated with nitrous oxide gas at 10 bar for 2 h, fixed in 90 % acetic acid for 10 min and then washed three times in water on ice. Root tips were dissected and digested in 20 µL 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. Root tips were crushed in 70 % ethanol, cells collected by centrifu-gation at 2.5 g for 1 min, briefly dried and then re-suspended in 30–40 µL of 100 % acetic acid prior to being placed on ice. The cell suspension was dropped onto glass slides (6–7 µL per slide) in a moist box and dried slowly under cover.

Slides were initially probed using labelled genomic DNA of Ae. speltoides (100 ng) and fragmented genomic DNA of Chinese Spring (3000 ng) as blocker (in a ratio of 1: 30 per slide) to detect the Ae. speltoides introgressions. The slides were bleached (dipped in 2 × saline–sodium citrate (SSC) to remove the coverslip, transferred to 4 × SSC for 5 min and air-dried in the light) and re-probed with labelled DNAs of T. urartu (100 ng) and Ae. tauschii (200 ng) and fragmented DNA of Ae. speltoides (3000 ng) as blocker in a ratio of 1: 2:30 per

AX-94423707 AX-950945320AX-951816990.2

AX-94823395 AX-946741735.7

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33.9

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35.4

AX-94471678 AX-9520355935.6AX-94576796 AX-9440776736.0AX-9447444736.2AX-95190619 AX-9458626336.4AX-9493395036.6AX-9454052337.7AX-94585720 AX-9460975337.9AX-9465624038.1AX-9478101638.3AX-94456002 AX-9446170738.4AX-9509039839.4AX-94983233 AX-9439941343.3AX-95137082 AX-94957552AX-94503593 AX-94541644AX-95141894

47.8

AX-9445798248.3AX-9474876852.3AX-95073230 AX-95117413AX-9517772952.5AX-9524406256.2AX-95229293 AX-94488894AX-9499985157.9

AX-9476423558.3AX-9444567658.5AX-9476627567.9AX-94710713 AX-94900699AX-9482868269.2

AX-9516553370.8AX-9460067190.1AX-94667744 AX-9466189290.7AX-9519490490.9AX-95245657 AX-9459639391.3AX-94861716 AX-9483700792.0AX-9467164092.2AX-9440747892.9AX-9518297093.6AX-9515415095.7AX-9449738597.4AX-9487079198.2AX-9438536399.9AX-94793000 AX-94573720101.6AX-94694080101.8AX-94826616110.1AX-95098658110.3AX-94847159 AX-94815376110.5

LG 5S

AX-951755290AX-947966725.3AX-9512288613.2AX-94921449 AX-8618643713.9AX-9515479914.9AX-95075504 AX-9472458915.8AX-95251172 AX-9523374816.8AX-94982375 AX-95106079AX-9521821817.9AX-94932402 AX-94679521AX-95150722 AX-9446151418.3

AX-94806979 AX-9483303119.6AX-9518084727.9AX-94946469 AX-9452907128.1AX-9456210828.3AX-95099660 AX-95156927AX-94553420 AX-9510418028.6

AX-94495972 AX-94935112AX-94773412 AX-9447890629.5

AX-95118160 AX-94541273AX-9447696732.6AX-9453951133.8AX-94868359 AX-94420467AX-94841103 AX-9453280434.8

AX-9479882051.6AX-94720384 AX-9525622952.7AX-9484283855.0

AX-9440419160.3AX-9492702663.2AX-9484473363.6

LG 6S

AX-947346190AX-952235124.7AX-951568946.6AX-94731399 AX-9460870211.5AX-94894854 AX-9490525912.7AX-9480283313.7AX-95190773 AX-9459114015.7AX-9448555316.1AX-95127864 AX-9452939316.6AX-94496296 AX-9484230517.6AX-95105411 AX-94887274AX-94499775 AX-9499681318.8

AX-9476792720.2AX-9517342221.4AX-94944928 AX-94961293AX-9455511422.6

AX-94591682 AX-94517409AX-9514086524.6

AX-94951234 AX-94533947AX-9508017729.6

AX-94694924 AX-9465845533.6AX-9440701036.8AX-94849956 AX-95013887AX-94813599 AX-9463988438.9AX-9465727047.4AX-9489930849.3AX-9452630654.4AX-9521538658.1AX-95235263 AX-94823469AX-94525211 AX-94584631AX-94506247

59.1

AX-94631324 AX-95254284AX-9476149763.4

AX-94488789 AX-94887169AX-9466181469.3

AX-94523738 AX-94837654AX-9500576370.1

AX-94870168 AX-94428847AX-9518295272.3

AX-9440428974.5AX-95023020 AX-94934691AX-95106896 AX-94511104AX-95124586

79.8

AX-95232391 AX-95226247AX-95194235 AX-9508131380.0

AX-94987166 AX-94602262AX-95238048 AX-94889553AX-95138960

81.1

AX-94484496 AX-94409864AX-9481136983.9

AX-9488782484.3AX-95157559 AX-94981102AX-94532990 AX-9523905785.5

AX-9512432185.9AX-9466707386.3AX-95191553 AX-9490404386.7AX-95114894 AX-9481339387.8AX-94771731 AX-9449216588.3AX-95122926 AX-9463054689.2AX-9487878889.5AX-9450787091.2AX-9440311692.9

LG 7S

Aegilops speltoides linkage groups

Fig. 2. Continued.



slide to detect the AABBDD genomes of wheat. In both cases, the hybridization mix was made up to 10 µL with 2 × SSC in 1 × Tris/EDTA buffer (TE). Slides were incubated initially at 75 °C for 5 min and then overnight at 55 °C in a closed box

before counterstaining with Vectashield mounting medium with 4',6-diamidino-2-phenylindole,dihydrochloride (DAPI), and analysed using a high-throughput, fully automated Zeiss Axio Imager Z2 upright epifluorescence microscope (Carl Zeiss,

BC1 BC2 BC3 BC4 BC5-5A

BC5-5A

LG 1s

LG 1s

LG 2s

LG 2sLG

3s

LG 3s

LG 4s

LG 4s

LG 6s

LG 6s

LG 7s

LG 7s

LG 5s

LG 5s

LG 1s

LG 2s LG

3s

LG 4s

LG 6s

LG 7s

LG 5s

LG 1s

LG 2s LG

3s

LG 4s

LG 6s

LG 7s

LG 5s

LG 1s

LG 2s LG

3s

LG 4s

LG 6s

LG 7s

LG 5s

LG 1s

LG 2s

LG 3s

LG 4s

LG 6s

LG 7s

LG 5s

A

1S

2S

0 10µm

B

Fig. 3. (A) SNP characterization of Ae. speltoides introgressions in five consecutive generations, i.e. BC1, BC2, BC3, BC4 and BC5. (B) Genomic in situ hybrid-ization image of the BC5 genotype (aneuploid 41-chromosome plant). In the SNP characterization red colour is used to represent the presence of an Ae. speltoides introgression and blue colour represents wheat. It should be noted that these diagrams cannot be used to assess which wheat chromosomes the Ae. speltoides segments have recombined with. The GISH image shows a metaphase spread of BC5-5A probed with labelled genomic DNA of Ae. speltoides. Arrows show Ae.

speltoides introgressions (green).



Oberkochen, Germany) with filters for DAPI (blue), Alexa Fluor 488 (green) and Alexa Fluor 594 (red). Photographs were taken using a MetaSystems Coolcube 1m CCD camera. Further slide analysis was carried out using Meta Systems ISIS and Metafer software (Metasystems, Altlussheim, Germany). This system enabled the fully automated capture of high- and low-power fluorescent images of root tip metaphase spreads. Slides with root tip preparations were automatically scanned and the images downloaded for analysis.

RESULTS

Generation of wheat/Ae. speltoides introgressions

A total of 3890 crosses were made between wheat and Ae. spel-toides and their derivatives, leading to the generation of 9953 crossed seeds and 2029 self-seeds (Fig. 1). The number of seeds germinated, plants crossed and seed set are shown in Table 2. Every ear produced by the F1 was crossed with wheat. The F1 hybrids generated between wheat and Ae. speltoides showed the lowest frequency of germination. Only 15 % germinated com-pared with 100, 73, 70 and 78 % in the BC1, BC2, BC3 and BC4 generations. The F1 hybrids also showed the lowest fertility, with only 29 % of crossed ears producing any seeds compared with crossed ears from the BC1, BC2, BC3 and BC5 generations, which showed 58, 72, 83 and 84 % fertility, respectively.

Of the 20 F1 seeds germinated, only three reached maturity and set seeds. Thus all of the wheat/Ae. speltoides introgres-sions generated in this programme were limited to these three viable F1 hybrids. Table 2 summarizes the number of seeds germinated and seed set for each of the backcross genera-tions. In each generation, the average seed set per crossed ear was very low, i.e. the lowest average seed set per ear was 0.6 (observed in the F1 hybrid), while the highest average seed set per crossed ear was only 3.7 (observed in the BC3). The fertility of plants that had previously been self-fertilized at least once was considerably higher than that of plants in the backcross programme, although again variability was seen, e.g. BC3F1-16A was male-sterile and therefore produced no self-fertilized seed, while seed numbers from crossed ears ranged from 8 to 19. Some plants were also found to be asynchronous for anther and stigma maturity. For these plants with manual pollination, self-fertilized seeds were produced.

Detection of introgressions

Of the SNPs on the Axiom array, 22 258 were polymorphic between Ae. speltoides and wheat (Table 1). The Axiom array was used to screen genomic DNA prepared from 536 samples of BC1 to BC5 lines between wheat and Ae. speltoides. Genotype calls were generated, and the sample call rate (markers working in a particular genotype) ranged from 86.5 to 99.8 %, with an average of 98.8 % for the 536 samples. Affymetrix software classified the scores for each SNP into one of six cluster pat-terns. However, only those classified as Poly High Resolution (PHR) were used for genetic mapping as these are considered to be optimum quality. Linkage group 7 had the highest number of SNPs (17.3 %), while linkage group 6 had the lowest number (8.5 %).

JoinMap® (van Ooijen, 2011) was used to analyse the PHR SNPs and this led to the establishment of seven linkage groups. The genetic map was composed of 544 SNPs and represented the seven chromosomes of Ae. speltoides. Linkage groups 1–7 had 69, 78, 82, 85, 90, 46 and 94 SNPs respectively (Fig. 2). The lengths of linkage groups 1–7 were 70.8, 90.3, 98.5, 68, 110.5, 63.6 and 92.9 cM, respectively, with a total length of 594.6 cM and an average chromosome length of 84.9 cM.

Genomic in situ hybridization

In most cases the number of wheat/Ae. speltoides introgres-sions detected by genomic in situ hybridization (GISH) in BC4 and BC5 individuals corresponded exactly with the number detected via SNP analysis (Table 3 and Fig. 3). One exception was detected in the GISH analysis of BC5-4A. The SNP analysis of this plant showed the only segment present to be a complete 2S chromosome. However, the GISH image showed the pres-ence of a large Ae. speltoides 2S segment (one telomere was missing from the chromosome) and a very small Ae. speltoides segment recombined with a different wheat chromosome.

Syntenic relationship

Figure 4 shows the syntenic relationship between the seven linkage groups of Ae. speltoides and the three genomes of wheat, with large ribbons showing significant synteny. Some

Table 2. Number of seed produced and germinated in relation to the number of crosses carried out for each generation of the introgres-sion programme for Ae. speltoides into wheat

Wheat × Ae. speltoides

F1 BC1 BC2 BC3 BC4 Total

Number of seeds sown NA 20 22 400 187 173 802Number of seeds that

germinated (%)NA 3 (15) 22 (100) 292 (73) 130 (70) 135 (78) 582

Number of plants that set seed (%)

NA 3 (100) 22 (100) 261 (89) 124 (95) 117 (87) 527

Number of seed/total number of crosses (average number of seed set per crossed ear)

127/26 (4.9) 22/35 (0.6) 509/357 (1.4) 5743/2180 (2.6) 3204/876 (3.7) 348/416 (0.8) 9963/3890

Number of crosses producing seed (%)

18 (69) 10 (29) 207 (58) 1579 (72) 730 (83) 350 (84) 2894

Number of self seed produced 0 0 21 520 753 735 2029



gene rearrangements are indicated where single markers cross-map to non-collinear positions on wheat chromosomes. The only major disruption in synteny between the two species is that Ae. speltoides 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). These data demonstrate that there is a close syntenic relationship between the A, B and D genomes of wheat and Ae. speltoides.

Preferential transmission

The data obtained from SNP analysis was used to deter-mine the transmission frequency of chromosome segments from each of the seven Ae. speltoides linkage groups through the female gametes to the BC1, BC2, BC3, BC4 and BC5 gen-erations. Chromosome segments from linkage group 2 were observed in all 536 backcross progeny observed (Table 4).

The smallest segment carried only three SNP markers, AX94631566, AX95123223 and AX94601746, located near one of the terminal ends of the 2S linkage group (Fig. 2). These three SNP markers were found to be present in all the back-cross individuals analysed. In order to determine whether the 2S chromosome was also preferentially transmitted through the male gametes, 41 BC3 and BC4 plants were allowed to self-fertilize and their progenies were analysed using GISH. The BC3/BC4 plants selected each carried a single copy of a 2S chromosome segment. Thus, these plants would produce two types of male and female gametes during gametogenesis: those that carried 2S and those that did not. If the 2S chromosome segment was preferentially transmitted through both the male and female gametes, only those that carried the 2S chromo-some would be viable and as a result all the progeny observed in the BC3/BC4 self-populations would be homozygous for the 2S chromosome segment. Alternatively, if the 2S chromosome segment was not preferentially transmitted through the male or the female gametes, then the progeny would segregate for the presence of either one or two 2S chromosome segments. All progeny observed carried two copies of the 2S chromosome segment (Fig. 5), thus demonstrating that the 2S chromosome segment was preferentially transmitted through both the male and female gametes.

The frequency of transmission of chromosome segments from other linkage groups in each of the backcross progenies was much lower than that observed for linkage group 2. Of the remaining linkage groups, segments from linkage group 5 of Ae. speltoides had the highest frequency of transmission in all the backcross progenies observed (Table 4)

DISCUSSION

Aegilops speltoides is a potentially important source of genetic variation for a range of agronomically important traits (Jia et al., 1996; Mago et al., 2009; Klindworth et al., 2012; Deek and Distelfeld, 2014). In the past, it has been difficult to detect and characterize wheat/wild relative introgressions due to the absence of high-throughput, genome-wide marker technolo-gies. In this paper we have utilized an Affymetrix SNP array (King et al., 2017) complemented by a specific crossing strat-egy and the use of an automated, high-throughput microscope system for GISH image capture.

In this work we generated F1 interspecific hybrids between wheat and Aegilops speltoides and backcrossed these to wheat (Fig. 1). Hybrids were haploid for the A, B and D genomes of

Table 4. Transmission frequencies of the seven linkage groups of Ae. speltoides in the backcross populations (BC1 to BC5) analysed by SNP genotyping

BC1 BC2 BC3 BC4 BC5 Total

Number of plants genotyped 22 252 123 117 22 536Linkage group 1 (%) 18 (82) 124 (49) 36 (29) 13 (11) 3 (14) 194 (36)Linkage group 2 (%) 22 (100) 252 (100) 123 (100) 117 (100) 22 (100) 536 (100)Linkage group 3 (%) 18 (81) 135 (54) 27 (22) 20 (17) 0 (0) 200 (37)Linkage group 4 (%) 15 (68) 89 (35) 23 (19) 7 (6) 0 (0) 134 (25)Linkage group 5 (%) 19 (86) 162 (64) 54 (44) 32 (27) 6 (27) 273 (51)Linkage group 6 (%) 17 (77) 115 (46) 31 (25) 25 (21) 0 (0) 188 (35)Linkage group 7 (%) 13 (59) 80 (32) 15 (12) 9 (8) 0 (0) 117 (22)

Table 3. Number of introgressed segments from Ae. speltoides present in BC4 and BC5 plants as detected by SNP genotyping and GISH. The Ae. speltoides chromosomes have been assigned to link-age groups via the comparative analysis of the SNPs with wheat

Plant accession numbers Number of segments Ae. speltoides linkage group of

segmentsBackcross Number Genotyping GISH

BC4BC5 45A 3 3 2, 5, 645C 1 1 245D 3 3 2, 5, 61A 3 3 2, 4, 51B 1 1 21C 1 1 21D 2 2 2, 52 2 2 2, 53 1 1 14A 1 1 + small

end2,?

5A 2 2 1, 26A 1 1 26B 2 2 1, 26C 1 1 27A 2 2 2, 57C 1 1 28A 1 1 28B 1 1 28C 1 1 28D 1 1 2



wheat and the S genome of Ae. speltoides. Thus, in the absence of homologous chromosomes the only recombination occur-ring at meiosis was between homoeologous chromosomes. This would normally be prevented by the presence of Ph1. However, the presence of the pairing promoters Su1-Ph1 and Su2-Ph1 located on chromosomes 3S and 7S of Ae. speltoides (Chen and Dvorak, 1984; Dvorak et al., 2006) enabled homoeologous re-combination to occur.

Since the interspecific F1 hybrids generated were haploid for the A, B, D and S genomes, their fertility was predicted to be low and this was found to be the case (Table 1). However, their fertility of 29 % was higher than that previously observed in those between wheat and Amblyopyrum muticum, which was 16.2 % (King et al., 2017). The low fertility of the F1 hybrids resulted in the generation of only 22 BC1 seeds that grew to maturity and set seed. Hence, if recombination did not occur in later generations, i.e. in the gametes of the BC1, BC2, BC3 and BC4 generations, then the total number of introgressions that could be generated was limited to the 22 female F1 gametes giv-ing rise to these 22 BC1 plants. However, the level of interspe-cific recombination detected by the genetic mapping was such that it was possible to assemble the seven linkage groups of Ae. speltoides. Using the genetic linkage map of Ae. speltoides,

Aegilops speltoides linkagegroups

Hexaploid wheatchromosomes

Synteny relationships between the 7 linkagegroups of Ae. speltoides and hexaploid wheat.Ae. speltoides does not carry the 4/5/7translocation that is observed for chromosomes4A, 5A and 7B of wheat.

Comparative synteny of Aegilops speltoides and hexaploid wheat

Fig. 4. Synteny of Ae. speltoides (genetic position in cM) with hexaploid wheat (physical position in Mb) [visualized using Circos v. 0.67 (Krzywinski et al., 2009)].

BC3F3-6B

10.0 µm

Fig. 5. GISH of a complete one-cell metaphase spread of a genotype produced by self-fertilization (BC3F3-6B) with labelled genomic Ae. speltoides DNA as probe, showing a homozygous pair of Ae. speltoides linkage group 2 intro-

gressed chromosomes (green).



we estimated that 294 wheat/Ae. speltoides introgressions were generated spanning the entire Ae. speltoides genome. We were able to characterize these introgressions and track them through the backcross generations (Fig. 3). However, while using the genetic map to characterize the introgressions, it was important to treat the cM distances with caution, as the maps were not produced using proper mapping families. Tracking the intro-gressions through the different backcross generations via the SNP data showed that the majority of introgressions generated had occurred due to recombination in the F1 gametes and were therefore present in the BC1 plants.

Validation of introgressions identified by SNP analysis was carried out by GISH analysis. In most cases the number of wheat/Ae. speltoides introgressions detected by the SNP ana-lysis corresponded to the number of introgressions detected by the GISH analysis (Table 3 and Fig. 3). The one exception (BC5-4A) can be explained in two possible ways. The first pos-sibility is that linkage group 2S of Ae. speltoides has recom-bined with a wheat chromosome. Although the telomere is on a separate chromosome, the SNP markers would have detected

the two introgressions as a complete chromosome. The second possibility is that the genetic linkage map of Ae. speltoides is not complete and therefore the smaller introgression has not been detected at all and the large segment has appeared com-plete in the SNP analysis.

Multicolour GISH analysis was carried out to verify which of the wheat genomes were involved in recombination with Ae. speltoides (Fig. 6). The majority of recombination events were shown to have occurred between Ae. speltoides and the B genome of wheat (91 %). Recombination had also occurred between Ae. speltoides and the D genome (18 %) but we found no evidence of recombination between Ae. speltoides and the wheat A genome. As the B genome progenitor of wheat is thought to be Ae. speltoides or a close relative (Sarkar and Stebbins, 1956; Riley et al., 1958; Dvorak and Zhang, 1990; Maestra and Naranjo, 1998), the higher level of recombination between Ae. speltoides and the B genome was expected. The low level of recombination observed with the D genome of wheat would also suggest that Ae. speltoides is more closely related to the D genome than to the A genome. This situation

B

C D

0 25µm 0 25µm

0 25µm 0 25µm

A

Fig. 6. Genomic in situ hybridization showing recombination between Ae. speltoides and the B and D genomes of wheat. (A) GISH analysis of a 41-chromosome BC3F1 individual. Metaphase spread with labelled genomic Ae. speltoides DNA as probe showing Ae. speltoides (green) introgressions (white arrows). (B) Same metaphase spread as (A) with three-colour GISH showing Ae. speltoides recombined with the D genome (red arrows). Enlarged inset shows one of the recombined chromosomes. This individual carries 14 A chromosomes (green), 13 B chromosomes (purple), 12 D chromosomes (red) and two wheat/Ae. speltoides 2S recom-binant chromosomes (the segment of the D genome is red and marked with the red arrow). (C) GISH analysis of a 42-chromosome BC5 individual. Metaphase spread with labelled genomic Ae. speltoides (green) DNA as probe showing one complete Ae. speltoides chromosome (blue arrow) from linkage group 2S and one wheat/Ae. speltoides recombined chromosome (white arrow) from Ae. speltoides linkage group 5S. (D) Same metaphase spread as (C) with three-colour GISH showing Ae. speltoides recombined with the B genome (yellow arrow). Enlarged insert shows the recombined chromosome. This individual carries 14 A chromosomes (green), 12 B chromosomes (purple), 14 D chromosomes (red), one complete Ae. speltoides chromosome (purple) and one wheat/Ae. speltoides

recombinant chromosome (purple).



closely mirrors that observed between wheat and Amblyopyrum muticum, where most of the recombination occurred between the chromosomes of the wild relative and the B and D genomes of wheat (King et al., 2017). In addition, although the genetic location of the Ph1 pairing suppressors located on Am. muticum are not currently known, the fact that both Ae. speltoides and Am. muticum carry suppressors indicates that the two species may have had a common ancestry.

We are currently unable to use the SNP markers to identify which of the chromosomes of wheat have recombined with the Ae. speltoides chromosomes. We are currently developing wheat chromosome-specific Kompetitive Allele Specific PCR (KASP) markers from the SNP markers on the 35K array. These markers will allow the analysis of large numbers of individuals, firstly to tag individual introgressions and track them through the generations (both via backcrossing and selfing) and secondly to identify which wheat chromosome(s) is (are) involved in each introgression once homozygous introgressions have been generated. Although con-siderably more labour-intensive and technically demanding, the latter could also be achieved using fluorescence in situ hybridiza-tion (FISH) with repetitive DNA sequences combined with GISH. This has been successfully demonstrated to identify the recipi-ent wheat chromosomes with introgressions from Thinopyrum bessarabicum (Patokar et al., 2016), Th. intermedium and Secale cereale (Ali et al., 2016) and Aegilops markgrafii, Aegilops triun-cialis and Aegilops cylindrica (Molnár et al., 2015).

In the Ae. speltoides accession used in this study, chromo-some 2S was found to be preferentially transmitted at a fre-quency of 100 % through both the male and female gametes. This demonstrates that the gametocidal genes on chromosome 2S are extremely potent and resemble those in species such as Aegilops sharonensis (King et al., 1991a, b; King and Laurie, 1993). The transmission of the other six linkage groups of Ae. speltoides was also observed in the female gametes, and although linkage group 5 showed a potentially elevated rate of transmission, there is no evidence at this time to suggest that it carries a gene for preferential transmission.

It is important to note that the work undertaken was based on a single accession of Ae. speltoides and thus it is not possible to determine whether all other accessions carry gametocidal genes on chromosome 2S or other linkage groups. However, if they do, then it will be necessary to remove them if introgression lines without them are to be developed. In order to develop lines that lack the 2S gametocidal genes, but still carry introgres-sions from other linkage groups, we are currently undertaking research to mirror the work of Friebe et al. (2003), who success-fully deleted the genes responsible for preferential transmission in Ae. sharonensis.

CONCLUSION

Researchers have been working to transfer genetic variation from its wild relatives into wheat for many years. However, although there have been some notable successes, we have barely scratched the surface of the genetic variation available for exploitation. This has been as a direct result of: (1) the dif-ficulty of inducing wheat/wild relative introgressions and (2) an inability to detect the introgressions. The data obtained here clearly demonstrate that Ae. speltoides chromosomes recom-bine with the chromosomes of wheat at a very high frequency,

giving rise to large numbers of introgressions in derivatives of the F1 hybrid. Considering the importance of the genetic varia-tion identified in Ae. speltoides for a range of agronomic traits (Jia et al., 1996; Mago et al., 2009; Klindworth et al., 2012; Deek and Distelfeld, 2014), wheat/Ae. speltoides introgressions have the potential to play a critical role in the development of superior wheat varieties in the future.

ACKNOWLEDGEMENTS

This work was supported by the Biotechnology and Biological Sciences Research Council as part of the Wheat Improvement Strategic Programme [grant number BB/J004596/1]. The fund-ing body played no role in the design of the study, collection, analysis and interpretation of data and in writing the manuscript.

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Aberystwyth University Introgression of Aegilops