Rust resistance in Triticum cylindricum Ces. (4x ...

Rust resistance in miticum cylindricum Ces. (4x, CCDD) and its transfer into durum and hexaploid wheats

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D. Bai, G.J. Scoles, and D.R. Knott

Abstract: In order to counteract the effects of the mutant genes in races of leaf rust (Pucciniu recondita f.sp. tritici Rob. ex Desm.) and stem rust (P. graminis f.sp. tritici Eriks. & Henn.) in wheat, exploration of new resistance genes in wheat relatives is necessary. Three accessions of Triticum cylindricum Ces. (4x, CCDD), Acyl, Acy9, and A c y l l , were tested with 10 races each of leaf rust and stem rust. They were resistant to all races tested. Viable F , plants were produced from the crosses of the T. cylindricum accessions as males with susceptible MP and Chinese Spring p h l b hexaploid wheats ( T . aestivum, 6x, AABBDD), but not with susceptible Kubanka durum wheat (T. turgidum var. durum, 4x, AABB), even with embryo rescue. In these crosses the D genome of hexaploid wheat may play a critical role in eliminating the barriers for species isolation during hybrid seed development. The T. cylindricum rust resistance was expressed in the F, hybrids with hexaploid wheat. However, only the cross MPlAcyl was successfully backcrossed to another susceptible hexaploid wheat, LMPG-6. In the BC2F2of the cross MPlAcylIlLMPG-6/3lMP, monosomic or disomic addition lines with resistance to either leaf rust race 15 (infection types (IT) 1=, 1, or 1'; addition line 1) or stem rust race 15B-1 (IT 1 or 1'; addition line 2) were selected. Rust tests and examination of chromosome pairing of the F , hybrids and the progeny of the disomic addition lines confirmed that the genes for rust resistance were located on the added T. cylindricum C-genome chromosomes rather than on the D-genome chromosomes. The T. cylindricum chromosome in addition line 2 was determined to be chromosome 4C through the detection of RFLPs among the genomes using a set of homoeologous group-specific wheat cDNA probes. Addition line 1 was resistant to the 10 races of leaf rust and addition line 2 was resistant to the 10 races of stem rust, as was the T. cylindricum parent. The added C-genome chromosomes occasionally paired with hexaploid wheat chromosomes. Translocation lines with rust resistance (2n = 21 11) may be obtained in the self-pollinated progeny of the addition lines through spontaneous recombination of the C-genome chromosomes and wheat chromosomes. Such translocation lines with resistance against a wide spectrum of rust races should be potentially valuable in breeding wheat for rust resistance. Key words: wheat, Triticum cvlindricum, rust resistance, gene transfer, addition line, molecular cytogenetics. RCsumC : Afin de contrecarrer les effets de mutations chez les races des agents de la rouille de la feuille (Puccinia recondita f.sp. tritici Rob. ex Desm.) et de la rouille de la tige (P. graminis f.sp. tritici Eriks & Henn.) chez le blk, l'exploration de nouvelles sources de rksistances parmi les especes apparentkes au blk est nkcessaire. Trois lignkes de Triticum cylindricum Ces. (4x, CCDD), Acyl, Acy9 et A c y l l , ont kt6 infectkes avec 10 races chacune de rouille de la feuille et de rouille de la tige. Ces lignkes se sont avkrkes rksistances a toutes les races. Des plants F , viables ont kt6 obtenus suite au croisement des lignkes de T. cvlindricum (en tant que mile) avec des blks hexaploi'des (T. aestivum, 6x, AABBDD) sensibles, MP et Chinese Spring phlb, mais pas avec le blk dur sensible Kubanka (T. turgidum var. durum, 4x, AABB) et cela m6me avec le secours d'embryons. Dans ces croisements, le gknome D du blk hexaploi'de joue vraisemblablement un r61e important dans l'klimination des barrieres interspkcifiques lors du dkveloppement de la graine hybride. La resistance a la rouille de T. cylindricum s'exprimait dans les hybrides F l avec le blk hexaploi'de. Cependant, seule la F , MPlAcyl a pu 6tre rktrocroiske avec succes a un autre blk

I

Corresponding Editor: G. Fedak. Received April 13, 1994. Accepted September 6, 1994.

D. ~ a i ' ,G.J. Scoles, a n d D.R. Knott. University of Saskatchewan, Saskatoon, SK S7N OWO, Canada.

Genome, 38: 8-1 6 (1995). Printed in Canada 1 ImprimC au Canada

'

Present address: London Research Center, Agriculture Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada.


Bai et al

hexaploi'de sensible, LMPG-6. Dans la gCnCration BC2F2du croisement MPIAcy IIILMPG-6/3/MP, des IignCes d'addition monosomiques ou disomiques possCdant la rdsistance soit a la race 15 de la rouille de la feuille (types d'infection (IT) 1=, 1 ou 1+; lignCe d'addition 1 ) soit a la race 15B-1 de la rouille de la tige (IT 1 ou 1 +; IignCe d'addition 2) ont CtC sClectionnCes. Des tests de rksistance a la rouille et l'examen des appariements chromosomiques chez les hybrides F, et les progknitures des lignCes d'addition disomiques ont confirm6 que les genes de rCsistance sont situCs sur les chromosomes du gCnome C de T. cylindricurn plut6t que sur les chromosomes du complCment D. La ddtection de polymorphismes (RFLPs) a l'aide de sondes d' ADNc spdcifiques aux groupes homCologues a permis de dCterminer que la lignCe d'addition 2 porte le chromosome 4C de T. cylindricurn. La lignCe d'addition 1 Ctait rksistante aux 10 races de rouille de la feuille et la lignCe d'addition 2 aux 10 races de rouilles de la tige, tout comme 1'Ctait le parent T cylindricurn. Les chromosomes du gCnome C se sont parfois apparids avec des chromosomes du blC hexaploi'de. Ainsi, des lignCes de translocation rksistantes (2n = 21 11) pourraient Etre obtenues par autofkcondation des lignCes d'addition par suite de recombinaisons spontanCes entre les chromosomes du gCnome C et ceux du blC. De telles IignCes, resistantes faces a une large gamme de races de la rouille, seraient d'une grande valeur dans 1'amClioration gCnCtique du blC. Mots cle's : blC, Triticurn cylindricurn, rCsistance a la rouille, transfert de genes, IignCe d'addition, cytogCnCtique molCculaire. [Traduit par la rCdaction]

lntroduction

Materials and methods

Rust resistance genes from the relatives of wheat can be a valuable addition to the wheat genetic reservoir. When rust resistance is identified in a related species, the next step is to make hybrids with a susceptible wheat cultivar in order to transfer the resistance. The probability of success in transferring rust resistance depends on four factors: (i) the crossability of the species with wheat and the fertility of the hybrid, (ii) the ability of the chromosomes of the two species to pair and induce recombination, (iii) the expression of the alien resistance gene or genes in the wheat genetic background, and (iv) the genetic complexity of the alien rust resistance. Because the relatives of wheat are tremendously diverse in terms of both genomic complement and ploidy, transferring genes to wheat can range from being simple to being very difficult. As a consequence, the methods used depend on the genomic relationship between wheat and its relatives. Three accessions of Triticurn cylindricurn Ces. (4x, CCDD) maintained at the University of Saskatchewan were tested with leaf rust (Puccinia recondita f.sp. tritici Rob. ex Desm.) race 15 and stem rust (P. grarninis f.sp. tritici Eriks. & Henn.) race 15B-1 and showed consistent resistance to both rusts. T. cylindricurn shares the D genome with hexaploid wheat (7: aestivurn L., 6x, AABBDD). If the gene(s) for rust resistance is located on the D-genome chromosomes, it could be directly transferred into hexaploid wheat by simple backcross methods. Otherwise, backcrossing will lead to the production of rust-resistant C-genome chromosome addition lines. The objectives of this study were (i) to test the three 7: cylindricurn accessions with another nine races each of leaf rust and stem rust in order to understand the interaction of the rust-resistance genes with a wide spectrum of rust races, (ii) to transfer the rust-resistance genes into durum wheat ( T . turgidurn var. dururn L., 4x, AABB) and hexaploid wheat by hybridization and backcrossing, and (iii) to assess the homoeology with wheat chromosomes of the T. cylindricurn chromosomes carrying rust-resistance genes added to hexaploid wheat using homoeologous group-specific DNA probes.

The three rust-resistant accessions of T. cylindricurn, the susceptible durum wheat, and the three susceptible hexaploid wheats used in this study are listed in Table 1. The leaf rust races 1, 15, 58, 70, 70B, 75, 83, 100, 104, and 161 and the stem rust races 11, 15B- 1, 15B- lL, 29- 1, 48A, 56, 111, C 15, C 18, and C33 used in this study were originally obtained from Agriculture Canada Research Station, Winnipeg, but are maintained at the University of Saskatchewan. Fourteen homoeologous group-specific wheat cDNAs (Sharp et al. 1989) used as RFLP (restriction fragment length polymorphism) probes in identification of the homoeologous group of the T. cylindricurn chromosomes added to hexaploid wheat were kindly provided by Dr. M.D. Gale, Institute of Plant Science Research, Cambridge Laboratory, Norwich, U.K. The three accessions of T. cylindricurn were used as males in crosses with the susceptible wheat parents, and the F, plants were backcrossed twice to a susceptible hexaploid wheat, either MP or LMPG-6. In the crosses between durum wheat and 7: cylindricurn accessions, embryo culture was employed to rescue the F, hybrid embryos. The medium used for embryo rescue was N, (Chu 1978) plus 0.5 mg/L indole-3-butyric acid, 400 mg/L casein hydrolysate, 5% sucrose, and 8 g/L agar, pH 5.8 (adjusted with 0.2 NaOH) (N, plus medium). Embryos were cultured 15 days after pollination. All rust tests were done on seedlings grown in pots on a bench in a greenhouse, using the procedure described by Bai and Knott (1992). The seedlings were inoculated at the two-leaf stage with either leaf rust or stem rust. If the phenotype of each plant to both rusts was desired, each seedling was tested at the two-leaf stage with stem rust and 1 week later was tested at the three-leaf stage with leaf rust. After 12 to 14 days, when the pustules were fully developed, the infection types (ITS) were read on a scale from 0 to 4 using the system of Stakman et al. (1962). ITS 0, O;, and 1 were considered to be resistant, 2 to be moderately resistant, 3 to be moderately susceptible, and 4 to be susceptible. Symbols + and - were used to indicate


Genome, Vol. 38, 1995

that the pustules were larger or smaller, respectively, than typical for the IT. The F, plants and backcross progeny were tested only with leaf rust race 15 (LR 15) and stem rust race 15B-1 (SR 15B-I), while the i? cylindricum accessions, the wheat parents, and the selected i? cylindricum chromosome addition lines in hexaploid wheat were tested with 10 races each of leaf rust and stem rust. The mitotic chromosome numbers of the BC,F, plants were determined from squashes of root-tip cells using the methods described by Mujeeb-Kazi and Miranda (1985). Three root tips per plant and at least five cells per root tip were analyzed to determine chromosome numbers. Spikes from the F, plants, the parents, all resistant plants, and some susceptible plants (as controls) from the advanced generations (BC2F2, BC2F,, and BC,F,) were fixed in Carnoy's fixative (absolute alcohol - chloroform acetic acid, 6:3:1) for 48 h, and stored under refrigeration (4°C) in 70% alcohol until used. Anthers were stained in alcohol - acid carmine for 48 h and squashed in 45% acetic acid. Meiotic chromosome associations were analyzed at metaphase I in as many cells as could be counted. RFLPs among the hexaploid wheat and i? cylindricum genomes detected by homoeologous group-specific wheat cDNA probes were employed to assess the homoeology of the added i? cylindricum C-genome chromosomes carrying rust-resistance genes. The details of the protocols for genomic DNA extractions from the hexaploid wheats, the i? cylindricum parent, and the i? cylindricum C-genome chromosome addition lines in hexaploid wheat and for Southern blot analysis to detect RFLPs among them were previously reported by Bai et al. (1994).

Results Multiple race tests of wheat parents and three T. cylindricum accessions The three h'exaploid wheats, MP, LMPG-6, and Chinese Spring (CS) phlb, were susceptible to all 10 races each of leaf rust and stem rust, except that MP was resistant to stem rust race 111 and CS phlb was resistant to stem rust race 56 (Table 1). Kubanka durum wheat was susceptible to 8 races of stem rust. The three i? cylindricum accessions were resistant to all 10 races each of leaf rust and stem rust. Production of F, hybrids Three accessions of i? cylindricum, Acy 1, Acy9, and Acy 11, were crossed with both durum wheat and hexaploid wheats. The seed set in the crosses of durum wheat with T. cylindricum accessions was quite high, ranging from 23.75 to 43.33% (Table 2). However, the immature hybrid seeds appeared plump, the contents were watery, and no solid endosperm had formed by the 15th day after pollination. The hybrid embryos were small and could be detected only under a stereomicroscope; they then quickly degenerated. No F, plants from the crosses were obtained by culturing the 15-day-old hybrid embryos on N, plus medium. The seed set in the crosses of hexaploid wheats with the i? cylindricum accessions was less than that with durum, ranging from 7.14 to 27.27% (Table 2). The average seed set in the crosses involving CS p h l b (19.64%) was twice that in the cross of MPIAcy 1 (9.18%). Viable F, plants

Bai et al.


Table 2. Seed set in crosses of durum and hexaploid wheat with T. cylindricurn (4x, CCDD) and infection type (IT) to LR 15 and SR 15B-1 of the F, seedlings.

No. of F, seeds

Seed set

Cross

No. of florets pollinated

KubIAcy 1 KubIAcy9 KubIAcy 1 1 MPIAcy 1 CS phlblAcy 1 CS phlblAcy9 CS phlblAcy 1 1

72 60 80 98 14 22 76

23 26 19 9 1 6 15

3 1.94 43.33 23.75 9.18 7.14 27.27 19.74

(%)

IT to LR 15

IT to SR 15B-1

Embryo rescuea

-

-

-

-

-

-

1

2 1 0; 1-

U U U N N N N

xh 0; 2

"U, embryo culture unsuccessful; N, embryo culture not necessary. 'A range of infection types from resistant to susceptible scattered randomly on a single leaf; caused by a single isolate.

Table 3. Chromosome association in the F, hybrids between hexaploid wheat (6x, AABBDD) and T. cylindricurn (4x, CCDD).

I1 Cross

2n

Cells obs.

CS phlblAcyl

35

100

MPIAcy 1

35

75

I

Total

Ring

Rod

I11

IV

10.33 (8-17) 18.99 (14-3 1 )

10.67 (8-13) 7.06 (2-9)

6.67 (4-8) 1.75 (0-4)

4.00 (3-8) 5.31 (2-8)

0.67 (0-1) 0.63 (0-2)

0.33 (0-2) 0

NOTE:Numbers in parentheses are ranges. I, univalent; 11, bivalent; 111, trivalent; IV, quadrivalent.

Table 4. Seed set in backcrosses to hexaploid wheat of the F, hybrids between hexaploid wheat and T. cylindricurn.

Cross

No. of florets pollinated

MPIAcy I IILMPG-6 CS phl blAcy 1llMP CS phlblAcy91lMP CS phlblAcy 1 lllMP

444 310 180 84

No. of seeds

Seed set (%)

from these crosses were produced directly from germination of the mature hybrid seeds. The T. cylindricurn genes for resistance to LR 15 and SR 15B-1 were expressed in the F, hybrids (Table 2). Thus, both leaf rust and stem rust resistance from the three accessions of T. cylindricurn were dominant in the genetic background partially of hexaploid wheat and partially of T. cylindricurn. Meiotic associations of chromosomes were determined in F , plants from the crosses MPIAcy 1 and CS phlblAcy 1 (Table 3). On average more than seven bivalents per cell were observed in each cross.

Backcrossing of F, plants to wheat parents The term backcross is used here to refer to crossing an F , hybrid to a hexaploid wheat, but not necessarily to the original wheat parent. Although T. cylindricurn shares the D genome with hexaploid wheat, the seed set on backcrossing the hybrids to wheat was low, ranging from 0 to

about 3% (Table 4). No B C , F , seed was obtained from the F, hybrids involving CS phlb, although repeated backcrosses were made. The chromosome numbers in the 11 BC,F, plants from the cross MPlAcylIlLMPG-6 fell into four groups: 30-31 (28 + 2 or 3), 41-44 (42 - 1, + 0 or + 2), 4.7-48 (49 - 1 or - 2) and 53 (56 - 3) (Table 5). The functional gametes tended to have chromosome numbers near multiples of seven. Plants with higher chromosome numbers were more likely to be rust resistant.

Advanced backcross generations Of 11 BC,F, plants obtained from the cross MPlAcylIl LMPG-6, 5 with resistance to either SR 15B-1 or LR 15 were selected and crossed to MP. Of 27 BC2F, plants, 10 were resistant to SR 15B-1 but none were resistant to LR 15. Most of the BC2F, plants were self-fertile. Fourteen BC2F2families from susceptible F, plants were tested with SR 15B-1 and LR 15 and none of 173 plants was resistant


Table 5. Chromosome numbers of BC,F, plants from the cross MPIT. cylindricurn AcylIlLMPG-6 and infection types (IT) to LR 15 and SR15B-1.


Plant

IT with LR 15

IT with SR 15B- 1

Chromosome no. (2n)

"A range of infection types from resistant to susceptible scattered randomly on a single leaf; caused by a single isolate.

Table 6. Infection types (IT) to LR 15 and SR 15B- 1 and chromosome numbers of the BC2F2plants from the cross MPIT. cylindricurn AcylIlLMPG-613lMP.

Plant Rust susceptible 1 2 3 4 5 Stem rust resistant 6 7 8 9 10 11 12 13 14 15 Leaf rust resistant 16 17 18 19 20 21 22 23 24 25

IT with LR 15

Chromosome no. and meiotic associations

IT with SR 15B-1

(20 I1 + 1 I) (21 11) (19 I1 + 3 I) (21 11) (19 I1 + 1 IV

4 4 4 4 4

4 4 4 4 4

41 42 41 42 44

4 4 4 4 4 4 4 4 4 4

1

1 1 1 I 1 222 2

44 (22 11) 43 (21 I1 + 46 (20 I1 + 43 (21 I1 + 45(16II + 44 (20 I1 + 43 (21 I1 + 44 (21 I1 + 4 3 ( 2 1 I1 + 43 (21 I1 +

4 4 4 4 4 4 4 4 4 4

49 (23 I1 + 45 (20 I1 + 43 (21 I1 + 44 (22 11) 43 (20 I1 + 4 3 ( 2 1 I1 + 47 (19 I1 + 43 (21 I1 + 43 (19 I1 + 44 (20 I1 +

+

+

+

1= 11 1 1

1 2 2 2 2

+

+

to either rust. However, 6 BC2F2families from the F, plants resistant to stem rust segregated for resistance to both rusts. Although leaf rust resistance was dominant in F , plants, it did not appear in BC2F, plants but segregated in BC,F2. From 197 BC2F2plants, 18 resistant only to stem

+ 21)

1 I) 1 IV + 2 I) 1 I) 1 I11 + 21V 1 111 + 1 I) 1 I) 2 I) 1 I) 1 I)

3 I) 1 IV I I)

+

+ 21)

1 I)

1 111) 1 I) 1 IV + 1 I11 1 I) 1 IV + 1 I) 1 I11 + 1 I)

+ 2 I)

rust (IT 1, I f , or 2-) and 14 resistant only to leaf rust (IT, 1=, 1, 1 +,or 2) were selected. Chromosome numbers at meiosis were determined in 5 plants susceptible to both rusts, 10 resistant only to stem rust, and 10 resistant only to leaf rust (Table 6). The 5 susceptible plants had from

Bai et al


Fig. 1. Spikes and meiotic chromosome associations at metaphase I of disomic addition lines carrying T. cylindricurn C-genome chromosomes ( 2 n = 22 11) from cross MPIT. cylindricurn Acy ll1LMPG-613lNIP. (A) Leaf rust resistant addition line 1. (B) Stem rust resistant addition line 2.

41 to 44 chromosomes. The 20 resistant plants had chromosome numbers ranging from 43 to 49. Fifty-nine BC2F, plants of the progeny of a disomic addition line (2n = 22 11) with leaf rust resistance were

retested with LR 15 and examined cytologically. Three (5%) were susceptible and had 21 11, while the rest were resistant and had 21 I1 + 1 I (22%), 22 I1 (60%), or 22 I1 + 1 111 (13%). Sixty-five BC2F, plants of the progeny of a


Fig. 2. RFLP of nuclear DNAs from MP (lane A) and CS p h l b (lane B) bread wheats, T. cylindricum accession


Acyl (lane C), and the disomic addition line 2 with stem rust resistance (lanes D and E) restricted with HindIII. Probe 163, specific for homoeologous group 4, was used for hybridization. The triangles indicate the three DNA fragments each from wheat genomes A, B, and D, respectively. The addition lines have extra bands (arrow) corresponding to one of the T. cylindricum bands.

determined through the detection of RFLPs between wheat and the alien chromosome segments using a set of probes. The probes were hybridized to the HindIII and BarnHI restricted DNAs from the wheat parents, MP and CS phlb, 'I: cylindricurn accession Acy 1, and addition lines 1 and 2. In all hybridizations no polymorphism was detected between the two wheat parents, MP and CS phlb. However, polymorphism~were detected between wheat and 'I: cylindricurn in HindIII and BarnHI digests. Extra bands corresponding to one of the 'I: cylindricurn bands were detected in HindIIIor BarnHI-restricted DNA from addition line 2 against probes 144 and 163 (Fig. 2). Probes 144 and 163 were homoeologous group 4 specific (Sharp et al. 1989). Thus, the 'I: cylindricurn chromosome carrying a stem rust resistance gene(s) from Acyl in addition line 2 probably belongs to homoeologous group 4. However, the homoeology of the T. cylindricurn chromosome carrying leaf rust resistance in addition line 1 was not identified by this method.

Discussion

disomic addition line (2n = 22 11) with stem rust resistance were retested with SR 15B- 1 and examined cytologically. Five (8%) were susceptible and had 21 11, while the rest were resistant and had 21 I1 + 1 I (16%), 22 I1 (67%), or 21 I1 + 1 111 (9%). The cytological studies confirmed that the leaf rust and stem rust resistance genes were located on the added T. cylindricurn chromosomes. Thus, both monosomic (21 I1 + 1 I) and disomic (22 11) 'I: cylindricurn chromosome addition lines carrying either leaf rust or stem rust resistance were produced (Fig. 1). Addition line 1, with resistance to LR 15 from the cross MP/Acyl//LMPG-6//MP, was tested with another nine races of leaf rust (1, 58, 70, 70B, 75, 83, 100, 104, and 161) and was resistant to all nine (Table 1). Addition line 2, with resistance to SR 15B-1 from the cross MP/Acyl// LMPG-6/3/MP, was tested with another nine races of stem rust (11, 29-1, 15B-IL, 48A, 56, 111, C15, C18, and C33) and was resistant to all nine (Table I).

Assessment of the homoeology of the added T. cylindricum chromosomes using homoeologous group-specific DNA probes The 14 low copy number probes isolated from a wheat cDNA library by Sharp et al. (1989) can identify each of the 14 homoeologous chromosome arms of wheat and its relatives. Homoeology of alien chromosomes added to wheat can be

The seed set in the crosses of both durum and hexaploid wheat with T. cylindricurn was relatively high and the F, hybrid seeds were easily produced. The higher seed set in the crosses involving CS p h l b compared with those involving MP is probably a result of the Krl and Kr2 crossability genes carried by the former. Gill (1981) indicated that in crosses of wheat with its relatives, the mechanisms for species isolation expressed during hybrid seed development are endosperm abortion, hybrid embryo abortion, or both, and only the barrier caused by endosperm abortion can be overcome by embryo culture. Failure, in this study, to produce F, plants from plump but watery hybrid seeds from the crosses of Kubanka with all of the 'I: cylindricurn accessions, using embryo rescue, may be the result of both hybrid endosperm and embryo abortion. However, in the crosses of hexaploid wheat with the T. cylindricurn accessions, viable F , plants were produced directly from germination of the mature F, seeds. The gene or genes in the D genome that hexaploid wheat carries but durum wheat does not must play a critical role in overcoming the barriers for species isolation during hybrid seed development. Seven of the bivalents at metaphase I of meiosis in F, plants from the cross MPIAcyl must result from homologous pairing of the D-genome chromosomes contributed by the two parents. The increased pairing in the cross CS phlblAcy 1 (10.67 bivalents plus 1.OO multivalent, on average; Table 3) must be the result of homoeologous pairing among the A-, B-, and C-genome chromosomes promoted by the p h l b allele from CS. The seed set in backcrosses of the F, hybrids from the cross MPIAcyl to their wheat parents was much lower than in the initial crosses of the wheats with the T. cylindricurn accessions (Tables 2 and 4). Cytological study of the BC,F, plants showed that the functional gametes from the F, hybrids (5x, ABCDD) tended to have chromosome numbers near multiples of seven. Theoretically, the frequency of such gametes is very low, and consequently, the seed set on backcrossing the F, hybrids to wheat was low. Darvey (1978) suggested the use of the p h l b mutant for enhancing recombination frequencies in interspecific

Bai et al.


Fig. 3. Variation in meiotic chromosome associations at metaphase I of the monosomic and disomic T. cylindricurn C-genome chromosome addition lines with rust resistance. (A) 18 I1 + 1 I11 (triangle) + 1 IV (arrow) in a monosomic addition line with leaf rust resistance. (B) 20 I1 + 1 IV (arrow) in a disomic addition line with leaf rust resistance. (C) 19 I1 + 2 I11 (triangle) in a disomic addition line with stem rust resistance. (D) 1 I + 18 I1 + 1 I11 (triangle) + 1 IV (arrow) in a disomic addition line with stem rust resistance.

F l hybrids. In the present study, no BCIFl seed was set on any hybrids from crosses of the p h l b mutant with the T. cylindricurn accessions. Similar results were reported by Sharma and Gill (1986) and Charpentier et al. (1988). The increased homoeologous pairing in the crosses involving the p h l b mutant may cause higher sterility in the megagametophytes. However, Farooq et al. (1990) were able to obtain BCIF, seeds from the crosses CS p h l b / 7: ovaturn and CS p h l b / T. kotschyi by repeated backcrossing to wheat. Thus, many more pollinations should be made on the F , hybrids to try to produce BC,Fl seeds. In the advanced backcross generations (BC2F2,BC2F,, and BC2F,) from the cross NIP/Acyl//LMPG-6/3/MP, all plants resistant to leaf rust or stem rust had chromosome numbers of 21 I1 1 I or greater. Rust tests and cytology

+

on progeny of the selected disomic addition lines (2n = 22 11) indicated that the genes for leaf and stem rust resistance were located on the added T. cylindricurn chromosomes. The D-genome chromosomes in MP hexaploid wheat were completely paired with those in the T. cylindricurn accession Acyl (Table 3). If the rust resistance gene or genes in Acyl were located on the D genome, plants with 21 I1 of chromosomes and carrying rust resistance should have been selected from the advanced backcrosses. The fact that all resistant plants at the advanced backcross generations carried the extra 7: cylindricurn chromosomes indicates that the genes for resistance to both leaf rust and stem rust in accession Acyl were located on the C-genome rather than on the D-genome chromosomes. Thus, both monosomic and disomic T cylindricurn C-genome chromosome



addition lines in hexaploid wheat having either leaf rust resistance (addition line 1) or stem rust resistance (addition line 2) were produced. The T. cylindricurn C-genome chromosome in addition line 2 was determined to be of homoeologous group 4 by using a set of homoeologous group-specific wheat cDNA probes. Thus, chromosome 4C in accession Acyl carried the gene(s) for resistance to stem rust. However, the homoeologous group to which the T. cylindricurn C-genome chromosome in addition line 1 belonged was not determined. Both the data on rust resistance and the study using cDNA probes show that the genes for leaf and stem rust resistance are on different T. cylindricurn C-genome chromosomes. Addition line 1 was resistant to all 10 races of leaf rust and addition line 2 was resistant to all 10 races of stem rust, as was the T. cylindricurn parent. Thus, the wheat genetic background did not interfere with the expression of the T. cylindricurn genes for rust resistance. Such genes, which provide resistance against a wide spectrum of rust races, should be potentially valuable in breeding wheat for rust resistance. Occasionally, the added T. cylindricurn C-genome chromosomes paired with wheat chromosomes (Fig. 3). Thus, translocation lines with rust resistance can probably be directly produced by natural crossing-over. Rust-resistant plants with 21 pairs of chromosomes can be looked for in a large number of progeny of the monosomic or disomic addition lines. Meanwhile, the addition lines have been crossed with Prelude monosomic 5B to facilitate the transfer of the resistance genes to wheat chromosomes. Bai, D., and Knott, D.R. 1992. Suppression of rust resistance in hexaploid wheat (Triticurn aestivurn L.) by D-genome chromosomes. Genome, 35: 276-282. Bai, D., Scoles, G.J., and Knott, D.R. 1994. Transfer of leaf rust and stem rust resistance from Triticurn triaristaturn to durum and hexaploid wheat and its molecular cytogenetic identification. Genome, 37: 410-418.

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