RPViewDoc2.pdf

190

NOTE / NOTE

Attempts to induce homoeologous pairing

between wheat and Agropyron cristatum genomes

Mélanie Jubault, Anne-Marie Tanguy, Paulette Abélard, Olivier Coriton,

Jean-Claude Dusautoir, and Joseph Jahier

Abstract: Agropyron cristatum (2 n =4 x = 28, PPPP) possesses potentially valuable traits that could be used in wheat

( Triticum aestivum ) improvement through interspecific hybridization. Homoeologous pairing between wheat chromo-

somes and P chromosomes added to wheat in a set of wheat – A. cristatum addition lines was assessed. First, the Ph -

suppressing effect of P chromosomes (except 7P) was analyzed. It was concluded that this system is polygenic with no

major gene, and consequently, has no prospect in the transfer of alien genes from wild relatives. In a second step, the

potential of the deletion ph1b of the Ph1 gene for inducing P–ABD pairing was evaluated. Allosyndetic associations

between P and ABD genomes are very rare. This very low level of pairing is likely due to divergence in the repeated

sequences between Agropyron and wheat genomes. Development of translocation lines using ionizing radiation seems

to be a more suitable technique than homoeologous recombination to exploit the A. cristatum genome in wheat im-

provement.

Key words: Triticum aestivum , Agropyron cristatum , addition line, GISH, Ph1 gene.

Résumé : Agropyron cristatum (2 n =4 x = 28, PPPP) possède des caractères agronomiques potentiellement intéressants

qui pourraient être utilisés dans les programmes d’amélioration du blé tendre ( Triticum aestivum ) par hybridation in-

terspécifique. L’appariement homéologue entre les chromosomes de blé et les chromosomes P ajoutés dans un en-

semble de lignées d’addition blé – A. cristatum a été évalué. L’effet suppresseur du système Ph des chromosomes P (à

l’exception du chromosome 7P) a tout d’abord été analysé. De nature polygénique, il ne contient aucun gène majeur et

n’offre par conséquent aucune perspective pour le transfert de gènes provenant d’espèces sauvages apparentées. Dans

un second temps, le potentiel de la délétion ph1b du gène Ph1 pour l’induction d’appariement P-ABD a été évalué.

Les associations allosyndétiques entre les génomes P et ABD sont très rares. Ce très faible niveau d’appariement est

certainement lié à une divergence des séquences répétées entre les génomes d’ Agropyron et de blé. Le développement

de lignées de translocation par radiations ionisantes semble être une technique plus adaptée que la recombinaison ho-

méologue pour exploiter le génome d’ Agropyron dans l’amélioration du blé tendre.

Mots clés : Triticum aestivum , Agropyron cristatum , addition line, GISH, Ph1 gene.

Introduction

Agropyron is a perennial genus of the Triticeae consisting

of 10 species that occur at 3 ploidy levels, diploid (2 n = 14),

autotetraploid (2 n = 28), and autohexaploid (2 n = 42), and

that are built on 1 basic genome, P (Dewey 1984). In addi-

tion to being economically important forages, Agropyron

species have been found to possess potentially valuable traits

for wheat ( Triticum aestivum ) improvement, including toler-

ance to drought (Dewey 1984; Asay and Johnson 1990) and

low temperature (Limin and Fowler 1987), and resistance to

diseases such as barley yellow dwarf virus (BYDV) (Sharma

et al. 1984).

Chen et al. (1989) successfully hybridized Triticum aes-

tivum ‘Chinese Spring’ (CS) (2 n =6 x = 42, AABBDD) and

tetraploid Agropyron cristatum (2 n =4 x = 28, PPPP). The

analysis of the meiotic behaviour in the F 1 hybrids showed

the occurrence in the P genome of a genetic system that in-

terferes with the wheat Ph1 gene, which is recognized as the

main factor responsible for the cytological diploid-like be-

haviour of polyploid wheats because it suppresses homoeol-

ogous pairing (Chen et al. 1989, 1991). In the backcross

progenies, 6 disomic addition lines (1P to 6P) and 5 ditelo-

somic addition lines (2PS, 2PL, 4PS, 5PL, and 6PS) were

extracted and identified through the use of a set of RFLP

Jubault et al. 193

Received 6 February 2004. Accepted 16 June 2005. Published

on the NRC Research Press Web site at http://genome.nrc.ca

on 1 February 2006.

Corresponding Editor: J.P. Gustafson.

M. Jubault, 1 A.-M. Tanguy, P. Abélard, O. Coriton, and

J. Jahier. I.N.R.A., UMR APBV, Domaine de la Motte, BP

35327, 35653 Le Rheu CEDEX, France.

J.-C. Dusautoir. I.N.R.A., UMR DGPC, Domaine de

Melgueil, 34130 Mauguio, France.

1 Corresponding author (e-mail:

Melanie.Jubault@rennes.inra.fr).

Genome 49 : 190–193 (2006)

doi:10.1139/G05-074

Jubault et al.

191

Fig. 1. Karyotype of the P chromosomes in the wheat –

A. cristatum addition lines visualized using the genomic in situ

hybridization technique. The rDNA site on 5P was detected us-

ing the rDNA probe pTa71 (arrowed).

Table 1. Chiasmata frequency per pollen

mother cell in the hybrids CS – Ae. variabilis

and (wheat – A. cristatum addition lines) –

Ae. variabilis .

Ae. variabilis

hybrids

Mean

Standard

deviation

1.70

0.21

4.56

0.86

2PS

2.14

0.22

2PL

2.85

0.28

4.10

0.57

3.92

0.77

4PS

3.44

0.24

3.38

0.28

5PL

3.30

0.23

4.77

0.32

6PS

3.53

0.69

( iv ) The perennial F 1 hybrid T. aestivum CS – A. cristatum ,

from which the addition lines were derived.

probes that detect each homoeologous chromosome arm in

wheat (Chen, Q. et al. 1992, 1994). This work did not pro-

vide any evidence of structural rearrangements that differen-

tiate the P genome from the A, B, and D genomes of wheat.

Different strategies were designed to produce wheat–alien

chromosome translocations (Jiang et al. 1994). Among them,

induced homoeologous pairing is the method of choice if the

gene synteny is conserved. It can be achieved either by de-

leting the Ph1 gene or by suppressing its inhibitory activity

on chromosome pairing by adding the chromosome of cer-

tain strains of a few species, including A. cristatum ,to

wheat. The present study aimed to assess the level of ho-

moeologous pairing between wheat and A. cristatum chro-

mosomes by using the Ph suppressor genetic system in

A. cristatum and the ph1b mutation, which is, in fact, a dele-

tion of a segment with the Ph1 gene (Sears 1977).

Cytogenetical methods

Mitotic and meiotic chromosome preparations were made

using standard techniques.

In situ hybridization

Genomic DNA of Agropyron cristatum , labelled by nick

translation with biotin-14-dATP (Invitrogen, Carlsbad, Calif.),

was used as a GISH probe. pTa71, containing a 9-kb Eco RI

fragment, including the 18S–5.8S–26S rDNA gene and a

spacer isolated from Triticum , was used as a FISH probe. It

was labelled by random priming with digoxigenin-11-dUTP

(Roche Diagnostics, Mannheim, Germany). Total genomic

DNA isolated from CS was autoclaved to yield fragments of

100–500 bp and used as blocking DNA.

Chromosome preparations were incubated in 100 ng

Rnase A/

Materials and methods

L and 0.05% pepsin in 10 mmol HCl/L, fixed

with 1% paraformaldehyde, dehydrated (70%, 90%, and

100%) and air dried. The hybridization mixture consisted of

50% deionized formamide, 10% dextran sulfate, 2× SSC,

1% SDS, labelled DNA (100 ng per slide), and blocking

DNA. Chromosome preparations and predenatured (92 °C for

6 min) probes were denatured at 85 °C for 10 min. In situ hy-

bridization was carried out overnight in a moist chamber at

37 °C. After hybridization, slides were washed for 5 min in

50% formamide in 2× SSC at 42 °C, followed by several

washes in 4× SSC – Tween. Biotinylated probes were im-

munodetected by fluorescein isothiocyanate-conjugated avidin

antibodies (Vector Laboratories). Digoxigenin-labelled probes

were detected with antidigoxigenin antibody conjugated with

rhodamine (Roche Diagnostics). The chromosomes were

mounted and conterstained in Vectashield (Vector Labora-

tories, Burlingame, Calif.) containing 2.5

Material

Materials used in these experiments were as follows:

( i ) Wheat – A. cristatum addition lines for chromosomes

1P–6P and telocentric chromosomes 2PS, 2PL, 4PS,

5PL, and 6PS. Disomic addition lines were achieved by

self-pollination, except those for chromosomes 2P and

4P. As a result of poor transmission of the 2P and 4P

added chromosomes through pollen in the monosomic

addition lines, addition lines for these 2 chromosomes

were produced by haplodiploidisation with maize pol-

len (David et al. 1999). However, the 2P addition line is

sterile and is maintained in the monosomic state, and

the 4P addition line has a reduced fertility.

( ii ) Hybrids between the addition lines (except 2P) and

Aegilops variabilis, and between T. aestivum CS and

Ae. variabilis .

( iii ) ph1b homozygous wheat – A. cristatum monotelo-

centric addition plants for 2PL, 5PL, and 6PS derived

from the cross between the corresponding ditelocentric

addition lines and T. aestivum ‘Courtot’ ph1b .

,6-diamidino-2-

phenylindole/mL. Fluorescence images were captured using a

CoolSnap HQ camera (Photometrics, Huntington Beach, Ca-

lif.) on an Axioplan 2 microscope (Zeiss, Oberkochen, Ger-

many) and analysed using MetaVue™ (Universal Imaging

Corporation™, Downingtown, Pa.).

′

192

Genome Vol. 49, 2006

Fig. 2. Meiosis in the pollen mother cells of ( a ) the hybrid between the 4PS ditelosomic addition line and Ae. variabilis , showing a

meiotic configuration of 1t + 23I+6II;( b ) the monotelosomic addition line 2PL ph1b, showing a meiotic configuration of1t+

2I+17II+2III. The telocentric chromosomes 4PS and 2PL are indicated by the arrows.

Data analysis

The homoeologous pairing level in the hybrids between

the addition lines and Ae. variabilis has been compared with

the homoeologous pairing level of the reference hybrid

T. aestivum CS – Ae. variabilis by a one-way analysis of

variance (ANOVA, Fisher–Snedecor’s test) and a compari-

son among means using the Dunnet’s test. Analysis was

done with SAS software (SAS Institute Inc., Cary, N.C.).

A. cristatum Ph suppressor system. Chromosome pairing, if

any, could only occur between homoeologous chromosomes.

Chromosome configurations in each hybrid were observed,

and the pairing level for each of them was compared with

that of the control hybrid T. aestivum CS – Ae. variabilis .

For each genotype, the chiasmata frequency per PMC (50

cells) was calculated on 3 plants (Table 1).

The one-way ANOVA showed a significant difference on

the homoeologous pairing level among the 11 genotypes

tested ( P < 0.01%). Comparison of means using Dunnett’s

test (

Results and discussion

= 5%) showed that all the genotypes, except those

with 2PS and 2PL chromosomes, displayed a significantly

higher level of homoeologous pairing than the control. Con-

sequently, all the P chromosomes tested, except for 2PS and

2PL chromosomes, seem to promote homoeologous pairing.

The A. cristatum Ph suppressor system appears polygenic.

However, even if each of chromosomes 1P, 3P, 4P, 5P and

6P have a promoting effect on homoeologous pairing, i.e., a

suppressing effect on the Ph genetic system, this effect has

the same magnitude as that of the Ph2 gene on wheat chro-

mosome 3DS (Driscoll 1972), but is much weaker than the

effect of the ph1b deletion. Indeed, Sears (1977) observed

18.2 chiasmata per cell in the hybrid T. aestivum ph1b –

Ae. variabilis. Here, the highest mean number of chiasmata

per cell is 4.9 (hybrid between the 6P disomic addition line

and Ae. variabilis ), further evidence that the A. cristatum

system involves only minor genes. So far, Ph inhibition has

only been genetically studied in Triticum speltoides . In that

species, 2 systems are involved in the promotion of homoeo-

logous pairing, with 1 system being composed of 2 major

genes segregating independantly of each other, and the other

being composed of several minor genes modifying the ef-

fects of the major genes (Chen and Dvorak 1984). Chen,

P.D. et al. (1994) transferred the 2 major genes ( Ph I genes)

to T. aestivum . Although the homoeologous pairing level in

F 1 interspecific hybrids with the Ph I line was not as high as

with the ph1b mutant, it was concluded that the Ph I

Integrity of P chromosomes added in the addition lines

A prerequisite for the evaluation of pairing between P and

ABD chromosomes was to check the P added chromosomes

for any wheat chromatin retained in the course of extraction

of the addition lines. Each of the P chromosomes was visu-

alized using genomic in situ hybridization with the total

genomic DNA of A. cristatum as a probe. No wheat trans-

locations were detected on the P chromosomes (Fig. 1). The

hybridization of the probe pTa71, which detects the sites of

rDNA, allowed the development of a physical marker for the

5P chromosome.

Chromosomal location of the A. cristatum genetic

system Ph suppressor and its potential to induce

P–ABD homoeologous pairing

We planned to assess the level of allosyndetic pairing in

the F 1 hybrid T. aestivum – A. cristatum , in which meiosis

was studied by Chen et al. (1989). For an unknown reason,

degeneration of pollen mother cells prior to meiosis was ob-

served, and consequently, the frequency of pairing between

the P chromosomes and those of wheat could not be studied.

Meiotic behaviour of the hybrids (2 n =36or2 n =35+t)

among the 5 disomic addition lines (1P, 3P, 4P, 5P, and 6P)

and the 5 ditelocentric addition lines (2PS, 2PL, 4PS, 5PL,

and 6PS) with Ae. variabilis (2 n =4 x = 28, UUSS) should

enable the identification of chromosome(s) carrying the

lines

Jubault et al.

193

could be effective in the transfer of alien genetic variation

(Chen, P.D. et al. 1994; Aghaee-Sarbarzeh et al. 2000).

In the hybrids between the ditelosomic addition lines and

Ae. variabilis , the P telocentric chromosomes are easily

identified on meiotic preparations. Despite the promoting ef-

fect on homoeologous pairing of the telocentric chromo-

somes 4PS, 5PL, and 6PS, these have never been observed

paired among 450 cells screened (Fig. 2 a ). Genes involved

in the A. cristatum system are not efficient enough when

used separately to induce homoeologous pairing between

A. cristatum and wheat genomes. Nevertheless, as the 7P ad-

dition line was not extracted, we cannot exclude the occur-

rence on the 7P chromosome of one or few genes having a

major suppressor effect on Ph1 .

Asay, H.K., and Johnson, D.A. 1990. Genetic variances for forage

yield in crested wheatgrass at six levels of irrigation. Crop Sci.

30 : 79–82.

Chen, K.C., and Dvorak, J. 1984. The inheritance of genetic varia-

tion in Triticum speltoides affecting heterogenetic chromosome

pairing in hybrids with Triticum aestivum . Can. J. Genet. Cytol.

26 : 279–287.

Chen, P.D., Tsujimoto, H., and Gill, B.S. 1994. Transfer of Ph I

genes promoting homoeologous pairing from Triticum spel-

toides to common wheat. Theor. Appl. Genet. 88 : 97–101.

Chen, Q., Jahier, J., and Cauderon, Y. 1989. Production and

cytogenetical studies of hybrids between Triticum aestivum L.

Thell., and Agropyron cristatum (L.) Gaertn. C. R. Acad. Sci.

Paris, 308 : 425–430.

Chen, Q., Jahier, J., and Cauderon, Y. 1991. Enhanced meiotic

chromosome pairing in intergeneric hybrids between Triticum

aestivum and diploid Inner Mongolian Agropyron . Genome, 35 :

98–102.

Chen, Q., Jahier, J., and Cauderon, Y. 1992. Production and cyto-

genetic analysis of BC 1 ,BC 2 , and BC 3 progenies of an in-

tergeneric hybrid between Triticum aestivum (L.) Thell., and

tetraploid Agropyron cristatum (L.) Gaertn. Theor. Appl. Genet.

84 : 698–703.

Chen, Q., Lu, Y.L., Jahier, J., and Bernard, M. 1994. Identification

of wheat– Agropyron cristatum monosomic addition lines by

RFLP analysis using a set of assigned DNA probes. Theor.

Appl. Genet. 89 : 70–75.

David, J.L., Dusautoir, J.C., Raynaud, C., and Roumet, P. 1999.

Heritable variation in the ability to produce haploid embryos via

pollination with maize and embryo rescue in durum wheat. Ge-

nome, 42 : 338–342.

Devos, K.M., Atkinson, M.D., Chinoy, C.N., Francis, H.A., Harcourt,

R.L., Koebner, R.M.D., Liu, C.J., Masojc, P., Xie, D.X., and Gale,

M.D. 1993. Chromosomal rearrangements in the rye genome rela-

tive to that of wheat. Theor. Appl. Genet. 85 : 673–680.

Dewey, D.R. 1984. The genomic system of classification as a guide

to intergeneric hybridization with the perennial Triticeae . Gene

manipulation in plant improvement. Edited by J.P. Gustafson.

Plenum Publishing Corp., New York. pp. 209–279.

Driscoll, C.J. 1972. Genetic suppression of homoeologous chromo-

some pairing in hexaploid wheat. Can. J. Genet. Cytol. 14 : 39–42.

Jiang, J., Friebe, B., and Gill, B.S. 1994. Recent advances in alien

gene transfer in wheat. Euphytica, 73 : 199–212.

Limin, A.E., and Fowler, D.B. 1987. Cold hardiness of forage

grasses grown on the Canadian prairies. Can. J. Plant Sci. 67 :

1111–1115.

Lukaszewski, A.J. 2000. Manipulation of the 1RS.1BL trans-

location in wheat by induced homoeologous recombination.

Crop Sci. 40 : 216–225.

Qu, L.J., Foote, T.N., Roberts, M.A., and Money, T.A. 1998. A

simple PCR-based method for scoring the ph1b deletion in

wheat. Theor. Appl. Genet. 96 : 371–375.

Sears, E.R. 1977. An induced mutant with homoeologous pairing

in common wheat. Can. J. Genet. Cytol. 19 : 585–593.

Sears, E.R. 1993. Use of radiation transfer alien chromosome seg-

ments to wheat. Crop Sci. 33 : 897–901.

Sharma, H.C., Gill, B.S., and Uyemoto, J.K. 1984. High levels of

resistance in Agropyron species to barley yellow dwarf and

wheat streak mosaic viruses. Phytopathol. Z. 119 : 143–147.

Attempts of homoeologous pairing induction using the

ph1b mutation

The ph1b mutation was introduced into monotelosomic

addition lines for the telocentric chromosomes 2PL, 5PL,

and 6PS. Homozygosity for ph1b was checked using the

PCR test described by Qu et al. (1998). Despite the absence

of the Ph1 gene, the added telocentric chromosomes 2PL

and 6PS were not observed paired among 331 cells screened

(Fig. 2 b ). Only the telocentric chromosome 5PL was found

paired, with a pairing frequency of 0.018 among 108 cells

screened.

Finally, allosyndetic associations between the A. cristatum

and wheat genomes, if any, are rare, even in the absence of

the Ph1 gene. Our results confirm the observations of Chen

et al. (1989) on the meiotic behaviour in different F 1 hybrids

between wheat and A. cristatum . These authors argued that

the high level of chromosome pairing in the F 1 hybrids is

likely to be due to homologous pairing within Agropyron

chromosomes and homoeologous pairing between wheat

chromosomes. Allosyndetic associations between wheat and

A. cristatum , if any, are rare in the F 1 hybrids. Under the hy-

pothesis that the colinearity of the maps of wheat and

A. cristatum is real, then the absence of allosyndetic pairing

would be due to differences in the nature and importance of

repeated sequences in the 2 species. This limitation for pair-

ing has already been found, e.g., with chromosome 1R of

rye, which is completely colinear to chromosomes 1A, 1B,

and 1D (Devos et al. 1993), and which compensates well for

the loss of 1B chromatin, has a very low recombination rate

with chromosome 1B (Lukaszewski 2000).

Consequently, the induction of homoeologous pairing be-

tween A. cristatum and wheat chromosomes does not appear

to be a suitable method for fully exploiting A. cristatum in

wheat breeding. The development of translocation lines us-

ing ionizing radiation (Sears 1993) could be a better way to

exploit the A. cristatum genome in wheat improvement.

References

Aghaee-Sarbarzeh, M., Harjit-Singh, M., and Dhaliwal, H.S. 2000.

Ph I Gene derived from Aegilops speltoides induces

homoeologous chromosome pairing in wide crosses of Triticum

aestivum. J. Hered. 91 : 417–421.

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: