ITEMS FROM IRAN

DRYLAND AGRICULTURAL RESEARCH INSTITUTE
Sararood, Kermanshah, P.O. Box 67145-1164, Iran.

 

M. Aghaee-Sarbarzeh and H.S. Dhaliwal and Harjit-Singh (Department of Biotechnology, Punjab Agricultural University, Ludhiana 141 004, India.)

Suppression of rust resistance genes from distantly related species in Triticum durum­Aegilops amphiploids. [p. 78-81]

Summary. To transfer resistance gene(s) from less closely related wild species, Ae. caudata and Ae. umbellulata amphiploids were developed with susceptible T. durum cultivars and subsequently backcrossed to the hexaploid wheat Chinese Spring. Under field conditions, the gene(s) conditioning resistance to stripe and leaf rust from the C and U genomes were suppressed by the A/B genomes of T. durum in the amphiploids. Differential reactions in some of the amphiploids at the seedling stage to individual pathotypes indicated the selective specificity of the suppression system. Recovery of resistant plants in the F2, F3, and backcross generations of all amphiploids with Chinese Spring indicated the absence of suppressor genes in Chinese Spring for these resistance genes.

Introduction. Related wild and progenitor species of wheat represent a large reservoir of useful variability that can be exploited for wheat improvement. Wide hybridization has contributed significantly to germ plasm enhancement of bread wheat. Many agronomically important traits, including resistance to diseases and pests and abiotic stresses, have been transferred from related species and genera into wheat (Knott and Dvorak 1976, Sharma and Gill 1983, Gale and Miller 1987, Jiang et al. 1994, Friebe et al. 1996). Alien resistance genes are useful only when they express in the cultivated background. Genetic suppression of disease resistance of related species by the D genome has been reported frequently (Kerber 1983, Bai and Knott 1992, Dhaliwal et al. 1993, Innes and Kerber 1994, Ma et al. 1997).

Studies at the Punjab Agricultural University (PAU), Ludhiana, showed that among less closely related wild species, the diploid Aegilops species with C, U, and M genomes are excellent sources of resistance to leaf and stripe rust (Dhaliwal et al. 1993, Singh and Dhaliwal 2000). Therefore, a study was initiated to transfer the rust resistance gene(s) from these species into hexaploid wheat. The present paper reports the suppression of resistance gene(s) of C and U genome of Ae. caudata and Ae. umbellulata, respectively, by gene(s) on the A/B genomes of wheat.

Material and methods. The plant materials used in this investigation are listed in Table 1. To transfer stripe rust and leaf rust resistance from diploid Aegilops species with C or U genomes to hexaploid wheat, amphiploids were developed between these species and susceptible T. durum cultivars as pollen parents. To synthesize amphiploids, the coleoptile of 4-5-day-old F1 seedlings obtained from these crosses were treated with 0.25 % colchicine in 2 % DMSO solution for 4 hours (Gill et al. 1988). These amphiploids were used as a bridge to transfer leaf and stripe rust-resistance genes into cultivated hexaploid wheat. To induce homoeologous chromosome pairing, the amphiploids were first crossed with a line of Chinese Spring carrying the PhI gene from Ae. speltoides (Chen et al. 1994, Aghaee Sarbarzeh et al. 2000) and further backcrossed to Chinese Spring.

The parents, F1s, amphiploids, and the derivatives of crosses with Chinese Spring were scored for field reaction to leaf rust and stripe rust following modified Cobb's scale (Peterson et al. 1948). In addition, these materials were evaluated at the seedling stage against individual pathotypes of leaf rust and stripe rust using the standard procedure (Nayar et al. 1997).

Results and discussion. Accession 13749 of Ae. umbellulata was resistant under field conditions and at the seedling stage to pathotype N of stripe rust and pathotypes 77-4, 77-5, 104B, and 104-2 of leaf rust (Table 2). However, the durum wheats Bijaga Yellow and Malvi Local were susceptible to both the rusts under the field conditions. Bijaga Yellow was resistant to races 77-4 and 77-5 of leaf rust and race N of stripe rust, but susceptible to races 104B and 104-2 of leaf rust, whereas the durum wheat Malvi Local showed susceptibility to all the pathotypes of rusts, except 104B of leaf rust at the seedling stage (Table 2).

The amphiploid of Bijaga Yellow with 13749 was susceptible under the field conditions and at seedling stage to two races of leaf rust 77-5 and 104-2, and race N of stripe rust (Table 2). The resistance of the amphiploid to race 77-4 may be from either of the parents or a combination of resistance genes from both of them. However, resistance to race 104B, which was due to gene(s) from Ae. umbellulata, could express in the amphiploid. As Chinese Spring also was susceptible to race 104B, this race can therefore be used to discriminate between segregating population for rust resistance of Ae. umbellulata. The amphiploid of Malvi Local with Ae. umbellulata also was susceptible to both the rusts under the field conditions and at the seedling stage to race 77-5 of leaf rust and race N of stripe rust, but resistant to races 77-4 and 104-2, to which Malvi Local was susceptible. The two races, 77-4 and 104-2, can be used for screening purposes at seedling stage among the derivatives.

Susceptibility of the amphiploids and their F1 hybrid with CS(PhI) indicated the presence of suppressor gene(s) on A/B genome of the durum wheat cultivars for the resistance genes of Ae. umbellulata Acc. 13749. The consistent resistance of the donor species, Ae. umbellulata (13749), to all races and differential reaction of the amphiploid to different pathotype of leaf rust at the seedling stage (Table 2) may indicate that at least two different genes for rust resistance were present in the U genome of Ae. umbellulata accession 13749. For instance, in the amphiploid 'Bijaga Yellow-Ae. umbellulata 13749', one of the genes was effective against race 104B, which could express in the amphiploid, and the other was effective against races 104-2 and 77-5, but it was suppressed in the amphiploid because of gene(s) on the A and/or B genomes of durum wheat. This may be attributed to selective specificity of the suppression system. Differential specificity of suppressor genes has already been reported. Nelson et al. (1997) reported a locus designated as SuLr23 from Ae. tauschii, which suppressed Lr23 of durum wheat in an amphiploid of these two species. However, the gene SuLr23 could not suppress other Lr genes in the F1 hybrid of the amphiploid and hexaploid wheat. Susceptibility of the amphiploid 'Bijaga Yellow-Ae. umbellulata' to the race N of stripe rust and 77-5 of leaf rust, to which both the parents were resistant, may be attributed to gene interaction in the amphiploids.

The Ae. caudata accession 3556 showed consistent resistance to prevalent races of leaf rust and stripe rust under the field conditions (Dhaliwal et al 1993) and to pathotypes 77A-1, 77-1, 77-2, 77-4, and 77-5 of leaf rust at seedling stage (Table 2). Whereas, T. durum cv. A206 and WH868 were susceptible to these rusts under the field conditions but resistant to races 77-5 and 104B at seedling stage. The amphiploids of these two cultivars with Ae. caudata, and their F1 hybrid with CS(PhI) were also susceptible under the field condition as well as to individual pathotypes of leaf rust to which the durum wheat were susceptible.

Susceptibility of the amphiploids and their F1 hybrid with CS(PhI) under the field conditions as well as to individual pathotypes of leaf rusts at seedling stage, clearly indicated suppression of resistance gene(s) from the resistant Aegilops species by gene(s) on A/B genome of durum wheat. Suppression of rust resistance genes by the A or B genome of wheat has already been reported (Kerber 1983, Ma et al. 1997). Innes and Kerber (1994) reported suppression of resistance to leaf rust in an amphiploid of a susceptible durum wheat and a resistant Ae. tauschii.

Recovery of resistant plants from the F2 and subsequent backcross generations (BC1 and BC2) with CS in crosses involving Ae. caudata, suggests the absence of suppression gene(s) on A/B genomes of CS, so that in these generations resistance gene(s) could segregate from the suppression gene(s) of durum wheat. It also indicates the absence of suppressor genes in the D genome of CS for resistance gene(s) of Ae. caudata.

The T. durum cultivar WH890 also was susceptible under the field conditions and to most of the pathotypes of leaf rust and pathotype P of stripe rust (Table 2). However, in contrast to the previous amphiploids, the resistance gene(s) from U genome of Ae. umbellulata 3732 could express in the amphiploid WH890­Ae. umbellulata 3732 under field conditions, whereas at the seedling stage, the amphiploid was susceptible to all the individual pathotypes of leaf rust, except race 77-3. As the donor species, Ae. umbellulata 3732 was resistant to all the races of leaf rust (Table 2), but the amphiploid was resistant only to race 77-3. We concluded that the donor species carried at least two different genes. One of the genes expressed throughout the life of the plant, which was also effective against race 77-3, and the other gene(s) is an APR gene(s), which expresses only at the adult plant stage. The possibility of suppression of this gene(s) at seedling stage also can not be ruled out. Recovery of plants in later generations (e.g., F2 and backcross generations) that were resistant to races to which durum wheat, the amphiploid, and the hexaploid wheat were susceptible (Table 2) further confirm the suppression of some of the resistance gene(s) of Ae. umbellulata by the A/B genome of T. durum parents and that Chinese Spring did not carry any suppressor for resistance gene(s) of Ae. umbellulata.

Although suppression of alien resistance gene(s) by gene(s) present on the genomes of cultivated species is a barrier in alien gene transfer, but this obstacle can be avoided by selecting wheat stocks or cultivars lacking the suppressing genes.

References.

• Aghaee-Sarbarzeh M, Harjeet-Singh, and Dhaliwal HS. 2000. PhI gene derived from Aegilops speltoides induces homoeologous chromosome pairing in wide crosses of Triticum aestivum. J Hered (In press).
• Bai DP and Knott DR. 1992. Suppression of rust resistance in bread wheat (Triticum aestivum) by D-genome chromosomes. Genome 35:276-82.
• Dhaliwal HS, Singh H, Singh KS, and Randhawa HS. 1993. Evaluation and cataloguing of wheat germplasm for disease resistance and quality. In: Biodiversity and wheat improvement (Damania AB ed). John Wiley and Sons Pub, New York, NY. pp. 123-140.
• Friebe B, Jiang J, Raupp WJ, McIntosh RA, and Gill BS. 1996. Characterization of wheat-alien translocations conferring resistance to disease and pest: current status. Euphytica 91:59-87.
• Gale MD and Miller TE. 1987. The introduction of alien genetic variation into wheat. In: Wheat breeding: its scientific basis (Lupton FGH ed). Chapman and Hall. pp. 173-210.
• Gill KS, Dhaliwal HS, and Multani DS. 1988. Synthesis and evaluation of T. durum-T. monococcum amphiploids. Theor Appl Genet 76(6):912-16.
• Harji-Singh and Dhaliwal HS. 2000. Interspecific genetic diversity for resistance to wheat rusts in wild Triticum and Aegilops species. Wheat Inf Serv 90(In press).
• Innes RL and Kerber ER. 1994. Resistance to wheat leaf rust and stem rust in Triticum tauschii and inheritance in hexaploid wheat of resistance transferred from T. tauschii. Genome 37(5): 813-22.
• Jiang J, Friebe B, and Gill BS . 1994. Recent advances in alien gene transfer in wheat. Euphytica 73:199-212.
• Kerber ER. 1983. Suppression of rust resistance in amphiploids of Triticum. In: Proc 6th Internat Wheat Genet Symp (Sakamoto S ed). Plant Germ-Plasm Insitute, Kyoto, Japan. pp. 813-814
• Knott DR and Dvorak J. 1976. Alien germplasm as a source of resistance to disease. Ann Rev Phytopathol 14:211-235.
• Ma H, Singh RP, and Mujeeb-Kazi A. 1997. Resistance to stripe rust in durum wheat, A-genome diploids, and their amphiploids. Euphytica 94(3):279-86.
• Nayar SK, Prashar M, and Bhardwaj SC. 1997. Manual of current techniques in wheat rusts. Research Bulletin No. 2. Regional Station, DWR, Flowerdale, Shimla, India.
• Nelson JC, Singh RP, Antrique JE, and Sorrells ME. 1997. Mapping genes conferring and suppressing leaf rust resistance in wheat. Crop Sci 37:1928-23.
• Peterson RF, Campbell AB, and Hannah AE. 1948. A diagrammatic scale for estimating of rust intensity of leaves and stem of cereals. Can J Res 26:469-500.
• Sharma HC and Gill BS. 1983. Current status of wide hybridization in wheat. Euphytica 32:17-31.
  

Induced transfer of rest resistance from Aegilops species into wheat and characterization of the derivatives using microsatellite markers. [p. 82-87]

M. Aghaee-Sarbarzeh and H.S. Dhaliwal (Department of Biotechnology, Punjab Agricultural University, Ludhiana 141 004, India).

Summary. The nonprogenitor species Ae. umbellulata and Ae. caudata are very good sources of resistance to stripe and leaf rust. To transfer rust resistance from these species to bread wheat, synthetic amphiploids were developed from crosses with susceptible T. durum cultivars. The amphiploids were crossed with T. aestivum cv. Chinese Spring with the PhI gene of Ae. speltoides to induce homoeologous chromosome pairing. Resistant plants with nearly normal chromosome number and partial to high fertility were recovered from these crosses, some of which were characterized with a set of wheat microsatellite (STMS) markers. The results showed that in some of the resistant derivatives chromosomal exchanges between the alien chromosomes and their homoeologous wheat chromosome carrying gene(s) for resistance had taken place.

Introduction. Among the wheat foliar diseases stripe and leaf rust are the most damaging diseases of wheat (Knott 1989, McIntosh et al. 1995). Although a number of resistance genes have been transferred into cultivated wheat during the last 3-4 decades, most of them have become ineffective because of the emergence of new rust pathotypes thereby necessitating continuous search for new sources of resistance.

Species related to wheat, including both distantly related and progenitor species, represent a large reservoir of useful variability including rust resistance that can be exploited in wheat improvement (Jiang et al. 1994, Friebe et al. 1996). A large number of transfers carrying useful alien genes have been produced, but very few have been exploited commercially. Most of the introduced alien segment from wild relatives into wheat either do not compensate well for the loss of wheat chromatin or contain undesirable genes causing depression in yield and performance of the plant (Knott 1993, Jiang et al. 1994). Undesirable effects can be avoided by transferring the desired gene(s) with least amount of unwanted alien chromatin.

Alien introgression has been greatly facilitated, first, by induced homoeologous chromosome pairing for recombination between wheat and alien chromosomes, and second, by the identification of the alien chromatin in the recipient progenies by molecular cytogenetic techniques (Heslop-Harrison et al. 1990, Mukai and Gill 1991). Recent studies have shown that the PhI gene(s) from Ae. speltoides in the background of T. aestivum (Chen et al. 1994, Jiang et al. 1994) is useful in inducing pairing between homoeologous chromosomes in intergeneric crosses of Triticeae (Aghaee-Sarbarzeh et al. 2000) facilitating precise transfer of alien genes with least linkage drag. Evaluation of wild Triticum and Aegilops species at the Punjab Agricultural University has revealed that nonprogenitor Aegilops species with the C, U, and M genomes are good sources of resistance to stripe and leaf rust (Dhaliwal et al. 1993, Harjit-Singh et al. 1998). The aim of this study was to transfer rust resistance from these species into wheat with the use of the PhI gene system.

Material and methods. The plant materials used in this investigation are listed in Table 3. Amphiploids were synthesized following the procedure used by Gill et al (1988) after crossing susceptible T. durum cultivars as females with stripe and leaf rust-resistant accessions of Ae. umbellulata (Acc. 3732 and 13749) and Ae. caudata (Acc. 3556). These amphiploids were used as the bridge for the transfer of resistance genes into cultivated bread wheat.

To induce homoeologous pairing, the amphiploids were first crossed with CS(PhI) and further backcrossed to either Chinese Spring or T. aestivum cv. WL711. Seed of CS(PhI) were obtained from Dr. Bikram S. Gill, Kansas State University, Manhattan, USA. The parents, F1s, amphiploids, and the derivatives of crosses with CS(PhI) were scored for field and seedling reaction to stripe and leaf rust following the standard procedures.

To identify possible chromosome/chromosome arm involved in translocation with alien chromosome in the resistant derivatives, a set of 40 microsatellite markers (one marker/chromosome arm) was selected based on the linkage map developed by Röder et al. (1998) (Table 4). PCR was performed and the products were separated on 3 % high-resolution agarose gels. Ethidium bromide-stained gels were visualized under UV light and photographed using a UVP Gel Documentation System Model GDS 7600.

Table 4. Wheat microsatellite markers used for characterization of some of the resistant derivatives of crosses of amphiploids/CS(PhI).

Genome	STMS markers (gwm #)	Total
A	666, 136, 312, 359, 155, 369, 397, 601, 617, 304, 570, 334, 63, 60	14
B	140, 582, 388, 257, 340, 285, 251, 368, 335, 234, 219, 508, 577, 537	14
D	642, 33, 382, 261, 645, 114, 624, 583, 190, 469, 428, 350	12

Results and discussion. Aegilops caudata Acc. 3556 and Ae. umbellulata Accs. 3732 and 13749 exhibited consistent resistance to stripe and leaf rust under field conditions for many years (Table 5). They also were resistant to individual pathotypes of stripe and leaf rust at the seedling stage (Dhaliwal et al. 1993).

Based on disease reaction under field and controlled conditions, resistant derivatives were selected and studied cytologically (Table 5 and Table 6). The selected plants were partially to nearly fertile with 40 to 44 chromosomes. Although, some plants with 21 bivalents also were obtained (e.g., plant no. 16, W1087-6, Table 6) additional backcrossing is needed to restore chromosome balance, increase the fertility level, and remove unwanted alien chromatin.

The results of the molecular characterization of some of the derivatives of 'CS(PhI)/amphiploid (T. durum cv. WH890-Ae. umbellulata Acc. 3732) crosses indicated that 23 ( 57.5 %) out of 40 STMS markers examined amplified in Ae. umbellulata Acc. 3732.

The amplification patterns of STMS markers revealed several cases in which the wheat chromosome arms were missing or replaced by its homoeologous segments from Ae. umbellulata (Figs. 1a-1d). The STMS markers used for chromosome 1A (gwm666 for the long arm and gwm136 for the short arm) showed that the gwm666 band was missing in plant no. 12, whereas, the wheat specific band of gwm136 was present in this plant but absent in plant no. 16 (Figs. 1a and 1b). This suggests that in plant no. 12 the long arm and in plant no. 16 the short arm of chromosome 1A were involved in chromosomal exchanges.

Presence of both the donor and recipient parent specific bands synthesized by gwm136 in plants no. 7, 18, and 19 (Fig. 1b) may indicate addition of an alien chromosome carrying this marker, probably 1U, to the chromosome complement of these plants. In plant no. 19, the chromosome number of 2n = 44 (Table 6) further shows the addition of a pair chromosomal structural changes were also observed in chromosome 1BS of plant no 15 (Fig. 1c), and 2DS of plants no. 19 (Fig. 1d).

These chromosomal translocations must have taken place due to the effect of the PhI gene. Chen et al (1994) and Aghaee-Sarbarzeh et al (2000) provided cytological evidences of the effectiveness of the PhI gene in inducing homoeologous chromosome pairing between wheat chromosomes and alien chromosomes from distantly related species of wheat. The molecular results of the present study further provide the molecular evidences of chromosomal exchanges between wheat chromosomes and those of its distantly related species, Ae. umbellulata, induced by the PhI gene of Ae. speltoides.

Synthetic amphiploids of T. durum and Aegilops species have been found to be very effective for transferring resistance to wheat diseases from nonprogenitor C- and U-genome species compared to direct crosses of these species with wheat. Induced homoeologous chromosome pairing in crosses of synthetic amphiploids with CS(PhI) and molecular characterization of the derivatives with the wheat microsatellites would be a good strategy for transfer of desired variability from related species, such as Ae. umbellulata and Ae. caudata, into wheat genome.

Although the resistance of the derivatives under the field conditions and at the seedling stage indicate the presence of alien resistance gene(s), because of several chromosomal structural changes monitored by cytological (Table 5) and molecular studies (Table 7 and Fig. 1), it was difficult to associate any of the alien chromosome/fragment to resistance genes. Further backcrossing along with selection for resistance will be needed to reduce the unwanted alien chromatin. In this process, the STMS markers identified for each chromosome are very useful to follow the alien chromatin and select the derivatives with least linkage drag. Selecting the resistant derivatives, which still show the amplification pattern similar to that of recipient parent, indicate the presence of least alien chromatin in the derivatives.
Although, chromosome banding techniques, such as C-banding and in situ hybridization (Le et al. 1989) have been widely used for identification of wheat chromosomes involved in translocation, translocation breakpoints, and the sizes of transferred alien segments (Friebe et al 1996), the results of this study, however, indicates the wheat microsatellites can be efficiently used as a supplementary tool for easy and early identification of the wheat chromosomes involved in translocations.

References.

• Aghaee-Sarbarzeh M, Harjit-Singh, and Dhaliwal HS. 2000. PhI gene derived from Aegilops speltoides induces homoeologous chromosome pairing in wide crosses of Triticum aestivum. J Hered 91:417-421.
• Chen PD, Tsujimoto H, and Gill BS. 1994. Transfer of PhI gene promoting homoeologous pairing from Triticum speltoides into common wheat and their utilization in alien genetic introgression. Theor Appl Genet 88:97-101.
• Dhaliwal HS, Harjit-Singh, Gill KS, and Randhawa HS. 1993. Evaluation and cataloguing of wheat germplasm for disease resistance and quality. In: Biodiversity and wheat improvement (Damania AB ed). John Wiley & Sons Pub. pp 123-140.
• Friebe B, Jiang J, Raupp WJ, McIntosh RA, and Gill BS. 1996. Characterization of wheat-alien translocation conferring resistance to disease and pest: current status. Euphytica 91:59-87.
• Gill KS, Dhaliwal HS, and Multani DS. 1988. Synthesis and evaluation of T. durum-T. monococcum amphiploids. Theor Appl Genet 76:912-916.
  Harjit-Singh, Grewal TS, Dhaliwal HS, Pannu PPS, and Bagga PS. 1998. Sources of leaf rust and stripe rust resistance in wild relatives of wheat. Crop Improv 25:26-33.
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