Deletion Mapping of a Nematode Resistance Gene on Rye Chromosome 6R in Wheat

D mutants of wheat chromosomes (Endo, Four deletion mutants of rye chromosome 6R were identified in 1995; Endo and Gill, 1996) have proven to be progeny of wheat (Triticum aestivum L.) lines of ph1bph1b genotype valuable tools for mapping molecular markers and morand monosomic for chromosome 6R. The rye chromosome carried a phological characters to physical segments of chromoresistance gene against the cereal cyst nematode (CCN) (Heterodera somes. By allocating markers to these types of chromoavenae Woll.) and this chromosome originated in triticale line T-701 somes (Werner et al., 1992; Hohmann et al., 1994, 1995; ( Triticosecale Witt.). The deletion mutants were selected on the Mickelson-Young et al., 1995; Gill et al., 1996a,b) the basis of dissociation of three isozyme loci on the long arm of the validity of maps based on conventional recombination rye (Secale cereale L.) chromosome. Three plants and their progeny studies can be confirmed. In addition, deletion-based showed expression of the rye genes -Amy-R1 and Got-R2 but lacked maps have the advantage of not requiring complete the gene PgdR2. The other plant and its progeny showed expression of the -Amy-R1 gene while the genes Got-R2 and PgdR2 were synapsis and recombination of pairing chromosomes absent. The four deletion chromosomes displayed long-arm terminal which are essential for conventional mapping analyses. deficiencies of different sizes which enabled mapping of the rye isoStructural differences between pairing chromosomes zyme genes and the CCN resistance gene. The distal to proximal order can prevent full synapsis and distort segregation ratios of the 6R isozyme loci was found to be: PgdR2, Got-R2, and then (Burnham, 1962) thus hindering recombination analysis. -Amy-R1. Bioassay tests demonstrated that the CCN resistance gene Cytogeneticists find both deletion and recombination (CreR ) was located on an interstitial section of the long arm of 6R maps useful first, for selection of suitable probes for use adjacent to Got-R2. with homoeologous chromosomes from related species and second, to assist in planning strategies for the effiI.S. Dundas, Dep. of Plant Science, Waite Campus, Adelaide Univ., cient transfer of useful genes from alien chromosomes PMB 1, Glen Osmond, S.A., 5064, Australia; D.E. Frappell, Dep. of to wheat. Zoology, La Trobe Univ., Bundoora, Vic. 3093, Australia; D.M. Crack, Devos et al. (1993) presented a recombination-based Dep. of Molecular Biosciences, Adelaide Univ., North Terrace, Adelaide. S.A. 5000, Australia; and J.M. Fisher (retired), Applied and map of the rye chromosomes showing the distribution Molecular Ecology, Waite Campus, Adelaide Univ., PMB 1, Glen of molecular markers and a suggested history of the Osmond, S.A. 5064, Australia. This research was undertaken in the structural rearrangements involving those chromoformer Dep. of Agronomy, Adelaide Univ., Australia, and was initially somes. The rye map showed numerous translocations led by the late Professor Colin Driscoll and more recently supervised by Dr. Ken W. Shepherd (now retired). This research was funded involving most of the rye chromosomes (with the excepby the Wheat Research Committee of South Australia (now Grains tion of 1R) as compared with their wheat homoeoResearch and Development Corporation of Australia). Received 7 logues. The complex structure of the rye genome sugDec. 2000. *Corresponding author ([email protected]). gests that gene transfer from these chromosomes to wheat by homoeologous recombination may be difficult. Published in Crop Sci. 41:1771–1778 (2001). 1772 CROP SCIENCE, VOL. 41, NOVEMBER–DECEMBER 2001 With the exception of rye chromosome 1R (Endo et al., Isozyme Screening and Maintenance of Stocks 1994), there are no other known sets of chromosome Detection of changes to the structure of rye chromosome deletion lines available for rye which could be used to 6R was based on the dissociation of rye isozyme markers elucidate the structures of those chromosomes further. Amy-R1, Got-R2, and PgdR2, previously traced to the long Several years before the study of Devos et al. (1993) arm of chromosome 6R by Ainsworth et al. (1987), Tang and was published, a project was initiated to transfer a CCN Hart (1975), and Salinas and Benito (1983), respectively. To determine isozyme phenotypes, seeds were surface steriresistance gene on rye chromosome 6R to a wheat chrolized in a 12% (v/v) sodium hypochlorite solution for 2 min, mosome. Fisher (1982) had previously described a line rinsed thoroughly with water, and germinated on moist filter of triticale (T-701) showing high levels of resistance to paper at about 25 C. After 4 d, extracts were taken from the CCN. Asiedu et al. (1990) found that the resistance endosperm of each seedling and analyzed for the isozymes of character was controlled by a single dominant gene lo-amylase (E.C. 3.2.1.1) and 6-phosphogluconate dehydrogecated on rye chromosome arm 6RL of triticale T-701. nase (6-PGD) (E.C. 1.1.1.44). Two days later, extracts were Homoeologous recombination was chosen as the pretaken from green leaf tissue and tested for glutamate oxaloferred method of achieving transfer of the CCN gene acetate transaminase (GOT) (E.C. 2.6.1.1). The isozymes of to wheat because a similar technique had been used -amylase were resolved on flat bed IEF gels following the successfully by Riley et al. (1968), Sears (1973), and technique of Ainsworth et al. (1987) and the gels scored for Joshi and Singh (1979) to incorporate alien disease rea band controlled by the -Amy-R1 locus. The isozymes of 6-PGD and GOT were separated by discontinuous polyacrylsistance genes into wheat and because Koebner and amide slab system of Rao and Rao (1980) and Hart (1975), Shepherd (1985, 1986) and Koebner et al. (1986) had respectively, and unique bands controlled by PgdR2 and Gotreported the production of recombinants between a rye R2 loci identified. chromosome and wheat in the absence of the wheat Seedlings suspected of containing 6R-wheat recombinants Ph1 pairing gene. were transplanted to the glasshouse and crossed with wheat However, the absence of confirmed recombination beSchomburgk as the male parent. Progeny tests on either selfed tween the 6R chromosome segment and homoeologous or backcrossed F1 seedlings from suspected recombinants were wheat chromosomes of group 6 in a ph1bph1b backconducted to confirm the isozyme patterns of the parent plant. ground was reported earlier (Dundas et al., 1992). Several deletion mutants involving rye chromosome 6R were Chromosome Studies found (Dundas et al., 1992) and this paper describes their Actively growing root tips were collected from 4-d-old seedisolation and detailed structure. These deletion stocks lings and pretreated for 24 h in iced water. Root tips were may prove useful for further physical mapping of that fixed in freshly prepared 1:3 acetic ethanol and stored until chromosome and even provide a strategy for future gene required at 10 C. Squash preparations were made after softtransfer from chromosome 6R to a wheat chromosome. ening the root tips in 45% (v/v) acetic acid for 10 min at room temperature. Dried slides were C-banded by the method of MATERIALS AND METHODS Koebner and Shepherd (1985). Before examination, slides were made permanent by mounting the coverslips on imGenetic Stocks mersion oil. Dried metaphase I chromosome spreads were Crosses were made by Dr. Robert Asiedu (formerly of C-banded following the method for mitotic chromosomes of Waite Agricultural Research Institute, Adelaide) between tritKoebner and Shepherd (1985) with the exception that denaicale T-701 and wheat to produce a disomic chromosome subturing of slides was performed for 15 min in a 5% (v/v) stitution line 6R(6D). Dr. Asiedu later crossed this substituBa(OH)2 solution at room temperature (about 25 C). tion line with Sears’ ph1b mutant (Sears, 1977) in a ‘Chinese Spring’ background (Asiedu et al., 1990). F2 progeny of those Nematode Bioassays plants were screened for the presence of the isozymes proThe assay method of Fisher (1982) was conducted in three duced by the genes Got-D2 and Got-R2 located on the long stages and control plants of Schomburgk and Chinese Spring arms of 6D (Hart, 1975) and 6R (Tang and Hart, 1975), respec(susceptible) and substitution 6R(6D) (resistant) were intively, to ensure that both of these chromosomes were present. cluded in each bioassay. Families with confirmed dissociations The monosomic status of chromosome 6R in the F2 plants was of 6RL markers or control plants of 6RL addition stock were confirmed by C-banding of mitotic squash preparations. screened for the presence or absence of isozymes of -AmyF2 plants homozygous for the ph1b gene were selected on R1. After several weeks, leaf samples were taken for dethe basis of two criteria, first, for the increased frequency of termination of the presence of Got-R2 isozymes. Wheat plants univalents and rod bivalents at meiosis (Koebner and Shepcontaining the short arm telocentric 6RS chromosome were herd, 1985) and second, for the presence of homoeologous selected using the dot-blot method of Rogowsky et al. (1991) pairing after test crossing with the related species Aegilops and employing the rye-specific probe pAW161 (Guidet et variabilis Eig (Asiedu et al., 1990). From 10 F2 plants homozyal. 1991). Statistical tests for significant differences between gous for the ph1b gene, 2787 progeny seeds were produced. means for the genotypes in the bioassay tests were conducted. In addition, 999 seeds representing progeny of 12 F3 plants monosomic for 6R chromosome, which had been derived from five different F2 families, were produced. RESULTS Seed stocks of ‘Schomburgk’ wheat used in crossing activities with suspected recombinants were kindly provided by Dr. A. Isozyme Dissociations Rathjen at the Waite Campus, Adelaide University. The addiA total of 3786 wheat seedlings from plants which tion line containing the telocentric 6RL chromosome was prohad undergone either one or two meiotic cycles in a duced by Dr. R. Asiedu in family B83-9C-136-3-3-12 while the 6RS addition line was isolated in family B84-9C-2-1-7b-791-5-7. homozygous ph1b background were screened for dissoDUNDAS ET AL.: MAPPING OF NEMATODE RESISTANCE ON RYE CHROMOSOME 6R 1773 ciation between three rye isozymes located on the long band 6RS4 between the telomere and the interstitial band 6R53 also appears to have been lost. The evidence arm of chromosome 6R. About 291 seedlings showing apparent dissociation of the three marker isozymes were for the deletion of a segment of the short arm is two-fold. First, the interstitial C-band on the short arm appears to isolated from the above stocks and 172 of these plants were progeny tested, the remainder either failing to have been repositioned immediately proximal to the telomere (Fig. 1g and g ). Second, the reduced length survive to maturity or being sterile. Several plants were found with confirmed dissociations of isozyme markers. of the short arm of this chromosome is reflected in the higher than normal values for the positions of the long Three of the confirmed dissociation plants and their progeny possessed the rye isozyme genes -Amy-R1 arm C-bands in Table 1. The remaining section of the long arm appears to be unchanged. and Got-R2 but lacked the gene PgdR2. The fourth plant and its progeny had the -Amy-R1 gene but lacked the other two isozymes. Cytological examination showed Type 6RL21 [del(6R)Ster→L21:] that all four dissociation lines had retained the shortThis chromosome was found in plant 791 of the same arm telomeric knob of 6R chromosome but had lost family as Deletion Type 6RL22 (above). The distal segterminal segments of the long arm. ment of the long arm carrying the telomeric and subterminal C-bands as well as most of band 6RL21 has been Morphology of Chromosome 6R lost (Fig. 1c, h and h ). The positions of the remaining and Deletion Chromosomes C-bands on the long and short arms correspond closely to those of the normal 6R chromosome (Table 1). Figures 1 and 2 show the normal C-banded staining pattern of rye chromosome 6R from triticale T-701. The long arm of 6R displays a faintly staining telomeric Type 6RL1807 [del(6R)Ster→L1807:] band, two more intensely staining bands proximal to This chromosome was found in plant 54 of family the telomere and three less deeply staining pairs of dots B84-9C-2-2-3-1411 after two meiotic cycles with on the interstitial segment (Fig. 1a, d and d , e and e ). ph1bph1b. The segment carrying the telomeric and two The short arm displays an intensely staining telomeric most distal C-bands on the long arm is deleted from this knob which is separated from the remainder of the arm chromosome leaving the long and short arms of nearly by a secondary constriction. A pair of small interstitial the same length (Fig. 1i and i , Table 1). The breakpoint dots occur about halfway between the centromere and on this chromosome occurs through band 6RL18 at a the distal tip of the short arm (Fig. 1a, d and d , f and f ). point such that approximately 70% of the proximal porThe C-bands of chromosome 6R have been classified tion of this band is retained. The positions of the reaccording to a system based on the International System maining C-bands on the long and short arms correspond for Human Chromosome Nomenclature (ISCN, 1978). closely to those of a normal 6R chromosome (Table 1). The short arm of the present 6R has been assigned to a single region with bands numbered consecutively from Type 6RL1801 [del(6R)Ster→L1801] the centromere towards the telomere. The long arm of 6R chromosome has been divided into two regions, with This chromosome was found in progeny of plant 95 the most intensely staining C-band on that arm as the of the same family as Deletion Type 6RL1807 (above). landmark. Bands of these two regions are numbered The segment carrying the telomeric and two distal consecutively from proximal to distal. Measurements C-bands on the long arm is deleted from this chromowere taken of distances of C-bands from the centromere some reducing the length of that arm to less than that on several 6R chromosomes and are expressed as ratios of the short arm (Fig. 1j and j , Table 1). The breakpoint to the length of the short arm of the chromosome unon this chromosome occurs through band 6RL18 at a der study (Table 1). For example, the distance from the point such that approximately 10% of the proximal porcentromere to the long-arm telomere of chromosome tion of this band is retained. The positions of the re6R (band 6RL25) is about 1.48 times the average length maining C-bands on the long and short arms correspond of the short arm of that chromosome. closely to those of a normal 6R chromosome (Table 1). Each deletion chromosome has been classified according to the band in which the breakpoint occurred Meiotic Behavior and Transmission (Figure 2, Table 1). Metaphase I spreads of pollen mother cells from all of the deletion lines (now of Ph genotype) were examType 6RL22 [del(6R)Ster→::S3→L22] ined to determine if any wheat chromatin had been This chromosome was found in progeny of plant 657 incorporated into the deletion chromosomes as indiof family B84-9C-2-1-7b after one meiotic cycle with cated by pairing between the deletion chromosome and ph1bph1b genotype. The distal segment of the long arm wheat chromosomes. Forty-five cells of deletion line carrying the telomeric and subtelomeric C-bands is de6RL22, 61 cells of deletion line 6RL21, 93 cells of deleleted from this chromosome (Fig. 1b, g and g ). The tion line 6RL1807 (Fig. 1k), and 41 cells of line 6RL1801 breakpoint occurs just distal to band 6RL21 (Fig. 2) so were checked. In all meiocytes, selected because the that a small amount of band 6RL22 is visible. As well deletion chromosome carrying the intensely staining 6R short arm telomere could be seen clearly, that rye chroas the long-arm deletion, a segment of the short-arm 1774 CROP SCIENCE, VOL. 41, NOVEMBER–DECEMBER 2001 Fig. 1. Photomicrographs and drawings of C-banded preparations of a normal 6R chromosome from triticale T-701 and four deletion mutants in a wheat background. (a) disomic substitution line of normal chromosome 6R(6D) showing 2n 42 chromosomes, (b) monosomic addition line of deletion chromosome 6RL22 showing 2n 43 chromosomes, (c) disomic substitution line of deletion chromosome 6RL21 (6D) showing 2n 42 chromosomes, (d) and (d ) normal 6R chromosome showing prominent short arm telomere and interstitial C-bands on the long arm, (e) and (e ) telocentric 6RL chromosome, (f) and (f ) telocentric 6RS chromosome, (g) and (g ) deletion chromosome 6RL22, (h) and (h ) deletion chromosome 6RL21, (i) and (i ) deletion chromosome 6RL1807, (j) and (j ) deletion chromosome 6RL1801 and (k) metaphase I spread with monosomic addition line of deletion chromosome 6RL1807 showing 21 wheat bivalents and the deletion chromosome as a univalent. Note that Fig. (a) to (c) show whole cell spreads with intact mitotic wheat chromosomes indicating that the 6R deletion mutants did not arise from chromosome breakage during slide preparation. In Fig. (a) to (c) and (k), arrowheads denote 6R chromatin and in Fig. (d ) to (j ), the small arrows indicate the approximate positions of the centromeres. Bars represent 10 m with Fig. (d) to (j) being of the same magnification. of 6 seedlings of one family, deletion line 6RL21—14% mosome occurred as a univalent and was never observed of 21 seedlings of two families, deletion line 6RL1807— to pair with any wheat chromosome (Fig. 1k). 28% of 32 seedlings of one family, and deletion line The transmission rates of the normal 6R and dele6RL1801—33% of 16 seedlings of one family. tion mutants of 6R were low. Female transmission, estimated after crosses of ph1bph1b plants monosomic Nematode Bioassays for the deletion chromosome with wheat Schomburgk, were as follows: whole 6R chromosome—26% of 474 Control plants with the whole 6R chromosome had few or no nematode females per plant while susceptible seedlings over 18 families, deletion line 6RL22—17% DUNDAS ET AL.: MAPPING OF NEMATODE RESISTANCE ON RYE CHROMOSOME 6R 1775 Fig. 2. Idiogram of rye chromosome 6R of triticale T-701 showing the locations of isozyme loci -Amy-R1, Got-R2, and PgdR2 and a nematode resistance gene CreR (lower) with respect to C-bands and band nomenclature (upper) Vertical arrowheads denote approximate sites of breakpoints on the long arm resulting in four different deletion mutants of chromosome 6R. Arm lengths and positions of C-bands were determined from measurements given in Table 1. control plants of Schomburgk and Chinese Spring had where wheat-6R recombinant chromosomes have actually been isolated. significantly higher numbers of nematode females present (Table 2). Plants containing the telocentric 6RL Differences in the pairing behavior and structural arrangement of rye chromosomes 1R and 6R could acwere resistant to the nematode as compared with sib plants without the 6RL chromosome (P 0.009) while count for the differing rates of recovery of recombinants. First, Naranjo (1982) reported much higher rates of those carrying the 6RS telocentric chromosome were susceptible as compared to sib plants without that chropairing of 1R compared with other rye chromosomes with wheat chromosomes in plants without the normal mosome (P 0.186). Plants with the deletion chromosome types 6RL22, 6RL21 and 6RL1807 showed signifiPh1 pairing gene. Second, Riley and Kimber (1966) reported no pairing between the long arm of chromocantly low levels of nematode female development as compared to sib plants from the same families lacking some II of rye, since designated as 6R (Gupta, 1971), in a nulli-5B wheat missing the Ph1 pairing gene. Third, that rye segment (P 0.044, 0.029, 0.042, respectively). However, plants with the deletion chromosome type molecular marker studies (Benito et al., 1991; Devos et al., 1993) and chromosome pairing studies (Naranjo 6RL1801 showed high numbers of nematode females comparable to the susceptible controls (P 0.06) (Taand Fernández-Rueda, 1991, 1996; Cuadrado et al., 1997) show that the distal regions of 6R chromosome consist ble 2). of 7R and 3R chromosome segments whereas 1R chromosome shows closer colinearity with wheat group 1 DISCUSSION chromosomes. As recombination in cereal chromosomes has been reported to occur at higher levels at the Screening for Wheat-Rye Recombination distal regions of the chromosomes (Lukaszewski and The present study set out to isolate rare recombinants Curtis, 1993), homoeologous crossing-over occurring between rye chromosome 6R and wheat chromosomes between chromosome 6R in the present study and wheat in the attempt to transfer a gene for nematode resistance chromosomes would most likely have involved group 7 from the long arm of 6R into wheat by means of Sears’ or group 3 chromatin and rarely involved chromosome ph1b mutant. The Chinese Spring ph1b mutant acts by sections carrying the group 6 markers in this study. A allowing wheat chromosomes to pair and recombine strategy to circumvent this difficulty is discussed later. with those of related species (Sears, 1977). No wheatrye recombinant chromosomes were found, unlike in Origin of 6R Mutants the earlier pairing studies involving rye chromosome 1R (Koebner and Shepherd, 1985, 1986; Koebner et al., Apparent spontaneous breakage of cereal chromo1986). In the absence of the wheat Ph1 pairing gene, somes has been previously reported. Rye chromosomes either through nullisomy for 5B or the presence of the showing loss of telomeric C-bands have been described by Gustafson et al. (1983) and Dille and Gustafson ph1b gene, increased meiotic associations have been observed between wheat and rye chromosomes but still (1990), and the deletion of a larger segment of the long arm of 6R by Friebe and Larter (1988). In wheat, the at a low level (Upadhya and Swaminathan, 1963; Lacadena, 1967; Dhaliwal et al., 1977; Hutchinson et al., 1983; occurrence of deletion chromosomes has been associated with the presence of alien chromosomes of AegiSchnaider and Priilinn, 1984; Jouve and Giorgi, 1986; Wu et al., 1989; Naranjo and Fernández-Rueda, 1991; lops or Triticum spp. (Finch et al., 1984; Tsujimoto and Tsunewaki, 1985; Kota and Dvorak, 1988; Endo, 1988, Cuadrado et al., 1997). Given that the homoeologous relationship between rye chromosome 6R and chromo1995; Tsujimoto and Noda, 1988; Endo and Gill, 1996) although the cause is unknown. somes 6A, 6B, and 6D of wheat is well established (Gupta, 1971), the question arises as to why no wheatThe long-term stability of the present 6R deletion chromosomes is unknown at this stage. While transmisrye recombinants were recovered in the present study. Despite the reports of pairing between rye chromosome sion of the deletion chromosomes to progeny is not a cause for concern, previous studies with Drosophila 6R and wheat (above), we can find no published papers 1776 CROP SCIENCE, VOL. 41, NOVEMBER–DECEMBER 2001 Table 2. Number of white female nematodes on the roots of wheat sib lines with and without telocentric 6RL and 6RS chromosomes, deletion mutants of rye chromosome 6R and on control susceptible (S) and resistant (R) lines after inoculation with H. avenae. Number plants Average number Wheat line tested females per plant