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a Dep. of Botany and Plant Sciences, University of California, Riverside, CA 92521
b USDA-ARS, 1301 N. Western Rd., Stillwater, OK 74075-2714
c Institute of Plant Breeding and Acclimatization, 05-870 Blonie, Poland
* Corresponding author (ajoel{at}ucrac1.ucr.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The levels of resistance of the four Russian triticales to the RWA population in the USA are much higher than those found in hexaploid wheats (Webster, 1990) and would be beneficial in developing new RWA-resistant wheat cultivars. This article describes attempts to transfer RWA resistance from two of the Russian triticales (PI 386148 and PI 386156) to wheat through chromosome engineering protocols.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To map the genetic location of the RWA resistance locus, plants with 4RLmon were crossed to a disomic addition of 4Rcer from ‘Blanco’ rye to a Brazilian wheat line BH1146, while plants with 4Rmon were crossed to a ditelosomic addition 4RLcer of ‘Imperial’ rye to ‘Chinese Spring’. The Blanco-BH1146 set of additions was produced by Lukaszewski (1988); Chinese Spring-Imperial 4RL addition was kindly provided by Dr. T.E. Miller of the John Innes Centre, Norwich, UK. Chromosome constitution of the resulting hybrids was verified by C-banding.
Plants with a rye chromosome associated with resistance to RWA were backcrossed several times to Pavon; the progenies from consecutive backcrosses were screened cytologically for the presence of the rye chromosomes or its misdivision products. To induce homoeologous recombination of rye chromosome with wheat, a plant with a centric wheat-rye translocation was crossed and backcrossed to the ph1b line of Pavon and translocation heterozygous-ph1b homozygous progeny were selected, grown and self-pollinated. The ph1b line of Pavon was produced by repeated backcrossing of the ph1b line of Chinese Spring to the monosomic 5B of Pavon, with ten backcrosses completed. Progeny from the selected ph1b plants were screened by C-banding and recombinant wheat-rye chromosomes were identified, backcrossed twice to Pavon, self-pollinated, and the resulting progenies were tested for resistance to RWA.
The final test of RWA resistance was performed in a greenhouse at the USDA-ARS Plant Science and Water Conservation Laboratory, Stillwater, OK during October 2000. Three to four seeds of each entry were planted in potting media in cone containers held in racks. PI 386148 and Pavon were included as resistant and susceptible controls. Total number of plants tested per entry ranged from seven to 48 depending on the availability of seed. Seedlings were infested five days after planting and damage ratings were recorded for each plant 25 d after infestation. Damage ratings were recorded by a scale of 1 to 9 (1 = no damage, 9 = dead plant) (Webster et al., 1991). In general, a 1 to 3 rating is considered resistant, 4 to 6 moderately resistant (or moderately susceptible), and 7 to 9 susceptible.
Concurrent studies conducted at the ARS laboratory in Stillwater, OK attempted to transfer RWA resistance gene(s) from the Russian triticale PI 386156 to wheat by irradiation. Beginning in January 1992, individual spikes of PI 386156 were X-rayed at the mature pollen stage and used as males to pollinate Pavon, with a subsequent backcross to Pavon. Pavon/PI 386156 BC1F1's were tested for RWA resistance and resistant selections were crossed to ‘Karl 92’. Additional RWA tests and selection resulted in the line 97RWA936-5-1 (Karl 92//Pavon *2/PI 386156) which was examined cytologically.
All C-banding was done according to Lukaszewski and Xu (1995). Wheat-rye translocation chromosomes were analyzed with fluorescent in situ hybridization with total genomic DNA (FISH) by the protocol of Schwarzacher et al. (1989) with minor modifications as outlined in Zwierzykowski et al. (1998) except that total genomic DNA of S. cereale was used as a probe, sheared wheat DNA was used as a block, and the probe to block ratio was 1:25.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PI 386148 x Pavon F1 hybrid had very little male and female fertility. Among 20 viable BC1 progeny screened cytologically, 63 complete and three telocentric rye chromosomes, and two centric wheat-rye translocations were present. From among these progeny, five plants with minor or no RWA symptoms were backcrossed to Pavon. They had two to four rye chromosomes present, with 4Rm present in each plant. Among their progeny, 74 viable seedlings were screened by C-banding and found to contain a total of 81 complete rye chromosomes and 10 newly formed rye telocentrics, and seven centric translocations, either wheat-rye or rye-rye.
In all tests for resistance to RWA performed at UC Riverside, Pavon was susceptible and did not survive the two-week tests while triticale PI 386148 usually emerged from the tests without any symptoms of RWA feeding. Among BC1 progeny, the levels of RWA resistance ranged from immunity typical of the triticale parent to moderate resistance. While the standard 1-to-9 scale was used to assign resistance levels to individual plants, given the imprecision of the tests, the scores were never treated as absolute indicators of resistance. With these qualifications, the BC1 plants were scored 1 to 4 and plants with the scores of 1 to 3 were used in backcrosses. Among BC2, the RWA resistance scores ranged from 2 to 9; all plants with 4Rm showed improved RWA resistance relative to Pavon, with evident variation. Observations of BC2 made it clear that the RWA resistance in PI 386148 was controlled by at least two independent factors with one of them associated with the presence of a rye chromosome tentatively identified as 4Rmon. As both BC2 plants with 4RLmon had improved resistance relative to Pavon, one of the loci must have been located on 4RLmon. The other locus (or loci) was not associated with any of the remaining six rye chromosomes. Moderate resistance in two BC2 plants without any detectable rye chromatin suggested that it was located in the wheat genomes. No further studies of its (their) location were performed. Once 4Rmon was identified as the rye chromosome with RWA resistance locus, no additional tests of resistance were performed until on the finished lines.
From BC2 on 4Rmon demonstrated an affinity to the homoeologous group-7 of wheat as it segregated with 7D; all 42-chromosome backcross plants with 4Rmon were disomic 4D and monosomic 7D (20'' + 7D' + 4Rmon'). The presence of 4Rmon in Pavon was associated in Riverside CA with early seed abortion resulting in severely shriveled seed at maturity and poor germination. Reciprocal crosses in BC3 demonstrated that this syndrome was absent when 4Rmon was introduced into the ovule via pollen. Consequently, from BC3, backcrosses were also made using double monosomics 20'' + 7D' + 4Rmon' as male. Male transmission rate of complete 4Rmon in BC3 and BC5 was 21% among 67 progeny screened; female transmission in BC3 - BC5 was 26.5% among 147 progeny tested.
An attempt to map the genetic position of the RWA resistance locus on 4RLmon relative to the centromere and the telomere was unsuccessful. Screening of a sample of backcross progeny from a plant with 4RLmon + 4Rcer indicated nonMendelian segregation of the two chromosomes and the absence of cytologically identifiable recombinants. Analysis of metaphase I showed absence of pairing of the two chromosomes in a sample of 53 meiocytes scored.
Among the BC4 progeny screened, a plant was identified with a centric translocation of 4RLmon to a wheat chromosome arm identified as 7DS (Fig. 1) . This identification was based on the C-banding pattern of the chromosomes of Pavon, the original C-banded karyotype of Chinese Spring (Lukaszewski and Gustafson, 1983), and the standardized C-banded karyotype of wheat (Gill et al., 1991). Since it involves the rye arm with the RWA resistance locus in an apparently compensating configuration (as evidenced by its transmission through the pollen), the subsequent effort concentrated on this translocation. The early seed abortion effect was absent in plants with 4RLmon as well as in the translocation line pointing to 4RSmon as the responsible arm.
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To further reduce the amount of rye chromatin present, homoeologous recombination of 4RLmon with wheat was induced by a removal of the Ph1 locus. For this purpose, translocation 7DS.4RLmon was combined in BC5-BC6 with the ph1b mutation and plants with chromosome constitution 19'' + 7DS.4RLmon + 7D + 5Bph1b'' were selected and grown. Among 3563 progeny screened by C-banding, four chromosomes were identified that appeared to be wheat-rye recombinants. Three of those had 7DS.4RLmon translocations that were missing the terminal C-band on 4RLmon. These chromosomes were presumed to have acquired terminal segments on the long arm from unidentified wheat chromosome(s). Two of these three chromosomes were recovered in backcrosses to Pavon and labeled 7DS.4RLmon-1 and -2. In situ DNA hybridization demonstrated that both were in fact recombined wheat-rye translocations (Fig. 1) with relatively long terminal wheat segments on the long arms. The fourth suspected wheat-rye recombinant chromosome recovered in the original screening was wheat chromosome 6A with a telomeric C-band on the short arm. However, in situ DNA hybridization with total genomic DNA failed to confirm the presence of a rye segment in that chromosome.
On the basis of the three observed recombinant chromosomes (two of which were confirmed by FISH) among 3563 selfed progeny screened, and with the assumption that all progeny were disomic for the critical chromosomes, the recombination frequency of 4RLmon in wheat would appear to have been 0.04% on a per-chromosome basis. As this is based on one class of recombined chromosomes, the actual recombination frequency might have been about 0.08% or one recombinant chromosome per 
600 progeny screened.
The progenies of plants segregating for the recombinant chromosomes 7DS.4RLmon-1 and -2 were exposed to RWA in standardized tests and showed clear segregation for resistance. This indicates that a RWA resistance locus is located in the proximal segments of 4RLmon present in both recombinant chromosomes. All progeny with the suspected recombinant 6A were uniformly susceptible.
Following the second backcross of the two recombined chromosomes to Pavon (after their recovery), selfed progenies of heterozygotes were screened by C-banding. Among 112 progeny with 7DS.4RLmon–1, no homozygotes were found and only about one half of the progeny were heterozygous for the translocation, suggesting the absence of, or very low, male transmission. On the other hand, two translocation homozygotes were found among 21 karyotyped progeny of heterozygotes with 7DS.4RLmon–2. These two homozygotes were grown to maturity and found to be extreme dwarfs with seed set reduced to about 30% of normal. The harvested seed had low germination rate and the progeny had very low vigor. To date only one of the grown plants survived to maturity and produced progeny. Its chromosome constitution is not known. Low transmission rate of one recombinant and very poor vigor of the homozygotes of the other one suggest that unlike the centric translocation 7DS.4RLmon, recombinant chromosomes 7DS.4RLmon–1 and –2 are incapable of normal compensation for the missing arm of 7D. Observations of meiosis in a homozygote of 7DS.4RLmon–2 showed regular presence of a quadrivalent; in a heterozygote of 7DS.4RLmon–1 up to two quadrivalents were present (Fig. 2) . When both quadrivalents were present, one involved the 7DS.4RLmon–1 translocation and the other involved wheat chromosomes only.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification of 4Rmon as the rye chromosome carrying a RWA resistance locus in this study was based on its general similarity, including its arm ratio and its heterochromatin pattern, to chromosome 4R of S. cereale and the published karyotype of S. montanum (Bennett et al., 1978). This is not a precise way of identifying chromosomes, especially that the C-banded karyotype of S. montanum of Bennett et al. (1978) also was created on the basis of the chromosomes' "relative similarity with respect to C-banding patterns and arm ratios to the chromosomes of cultivated rye" (Bennett et al., 1978) and not on any genetic criteria. On the other hand, during the construction of the C-banded karyotype of S. cereale, chromosomes 4R and 7R were assigned to homoeologous groups 4 and 7 of wheat at random (N.L. Darvey, personal communication, 1998) and were originally listed as 4/7R and 7/4R (Darvey and Gustafson, 1975). Following a nomenclature conference, this was simplified to 4R and 7R despite a conflict with some genetic data (Sybenga, 1983). Chromosome 4Rcer spontaneously substitutes into wheat homoeologous group 7 and not 4, while chromosome 7Rcer shows little ability to compensate for either group 4 or 7 chromosomes of wheat (Koller and Zeller, 1976). A similar pattern was observed in triticale-wheat hybrids (Lukaszewski et al., 1982). This alone suggests that a reversal of the 4Rcer and 7Rcer designations would more closely reflect the genetic relationships of these two chromosomes to wheat.
Cytologically, S. montanum differs from S. cereale by three translocations (Koller and Zeller, 1976). Devos et al. (1993) established homoeology of individual chromosome segments of S. cereale to chromosomes of wheat and concluded that the 4-7 translocation present in the Triticeae also was probably present in S. montantum. If the chromosome designations have been used consistently, one arm of chromosome 4Rmon as identified here should be colinear with 4BS and 4DS of wheat while the other arm, except for a short proximal region, should be homoeologous to the short arms of group-7 chromosomes of wheat. It should differ from 4Rcer by the absence of a terminal segment homoeologous to wheat 6S on the arm otherwise homoeologous to 7S. Because of this, chromosome 4Rmon may be structurally more closely related to a wheat homoeologue, either of groups 4 or 7, than 4Rcer. Still, the 7DS.4RLmon translocation should be noncompensating.
Severe grain shriveling associated with the presence of 4Rmon in early backcrosses to Pavon was similar to that observed in several additions of 4Rcer to wheat grown under similar conditions. Both in 4Rmon and 4Rcer, the effect was associated with the short arm. This suggests that 4Rmon and 4Rcer are in fact homoeologous. Recovery of the centric translocation 7DS.4RLmon in the backcross as male, its normal transmission through pollen in subsequent backcrosses, normal seed set of the translocation homozygote in Pavon, and no significant yield reduction relative to Pavon and sister sib-lines with standard chromosome constitution in a replicated field trial (P.S. Baenziger, personal communication, 2001) indicate that it is a compensating translocation. It involves an arm identified as 7DS both on the karyotype of the Chinese Spring wheat (Lukaszewski and Gustafson, 1983) and on the standard karyotype of wheat (Gill et al., 1991). However, recently discovered problems with the telocentrics of 7D in Chinese Spring indicate that the original identification of 7DS and 7DL might have been incorrect. Data of Friebe et al. (1996a) suggest that perhaps the nomenclature of 7DS and 7DL needs to be reversed. If this is the case, the centric translocation produced in this study would be 7DL.4RLmon and to be compensating, the rye arm involved would have to be homoeologous to the short arms of group-7 chromosomes of wheat, as shown by Devos et al. (1993). However, until the questions of chromosome nomenclature are clarified, the identity of chromosome arms in the centric translocation recovered in this study will remain unclear.
Recombination frequency of the rye arm in the translocation 7DS.4RLmon with wheat homoeologues was very low. Only three recombinants were observed among 3563 progeny screened, of which one could not be recovered among the progeny. The identity of the terminal wheat segments on the long arms of the two recovered recombinants is not known. Clear differences in the behavior of the two recombinant chromosomes suggest that two different wheat chromosomes may be involved. Lack of male transmission of 7DS.4RLmon–1 and plant morphology, low fertility and poor germination of seed from the 7DS.4RLmon–2 homozygote indicate that neither one is a compensating combination. It ought to be noted here that Chinese Spring nullisomics and tetrasomics 7D are morphologically similar to the euploids and they are quite female and male fertile. Therefore, compensation problems of the recombined chromosomes are unlikely to result from dosage effects of some parts of group-7 homoeologues. Absence of ring bivalents 7DS.4RLmon–1 + 7D and the involvement of 7DS.4RLmon–1 in a quadrivalent with three wheat chromosomes shows that the terminal wheat segment on the long arm of the recombinant is not from 7D. Loss of the compensating ability following induced homoeologous recombination of both recombinants, and aberrant morphology and poor vigor of the progeny of the 7DS.4RLmon–2 homozygote seem to exclude other group-7 chromosomes as the source of the terminal wheat segment in the recombinants. This is surprising in the light of the speculations above on the affinity of 4RLmon to the 7S chromosomes of wheat.
The ph1b-induced homoeologous recombination does not only affect the manipulated alien chromosome, but the wheat homoeologues as well. Taking into account the relative affinities of alien chromosomes to wheat and wheat homoeologues themselves, there is little doubt that each round of the ph1b-induced homoeologous pairing does considerable damage to the wheat genome and such damage may be responsible, in part, for the aberrant behavior of the lines with recombinant chromosomes. However, two backcrosses to Pavon were made to remove most of such damage, and segregation of residual heterozygosity for structural aberrations would be expected among the homozygotes of 7DS.4RLmon–2 and their progeny. Collateral damage to wheat chromosomes caused by the absence of Ph1 does not explain the clear loss of the compensating ability of 7DS.4RLmon–1 relative to the centric translocation 7DS.4RLmon. Regardless, by the virtue of forming quadrivalents, the two recombinant wheat-rye translocations would be a source of meiotic instability and, therefore, are unsuitable for practical use.
Irradiation has been used with fair success to transfer alien chromosome segments into wheat (see Friebe et al., 1996b) and may be the only available approach when the pairing affinity of the donor and recipient chromosomes is low. This was clearly the case in this study. As a rule, translocations recovered following irradiation are products of random breakage and fusion, hence are noncompensating, and their practical application may be limited. Additionally, at times the recovered translocations could have just as well been produced by more conventional approaches, such as the 1RS.1AL centric translocation in ‘Amigo’ (Sebesta and Wood, 1978). In this experiment, irradiation followed by several cycles of screening and selection resulted in an apparent addition of a complete rye chromosome to wheat. Disomic alien additions to wheat tend to lose the extra chromosomes over the generations which explains continuous segregation of RWA resistance in 97RWA936-5-1. The centric translocation present in this line, tentatively identified as 5DS.5RLmon, might have been a result of a centric misdivision-fusion of univalents in early generations of hexaploid triticale x wheat hybrids rather than a consequence of irradiation.
Despite numerous questions raised by this experiment as to the identity of the chromosomes involved and the best approach to introgress the desired loci into wheat, it is clear that with low recombination frequency and structural differences between the donor and recipient chromosomes, transfers of small segments of alien chromatin by induced homoeologous recombination may be impractical. For a reasonably high precision of transfer such as the manipulation of 1RS in wheat (Lukaszewski, 2000), at least 50–60 primary wheat-rye recombinant chromosomes would be required. At the frequencies observed here, even at the worst case scenario that only one half were actually recovered because of inadequate screening, at least 30000 to 40000 progeny would have to be screened; quite a difficult task with the current technology, whether cytological or otherwise. Ironically, the least involving approach, that of routine screening of the progenies of double monosomics 20'' + 7D' + 4Rmon' in backcrosses, produced a transfer (centric translocation) which seems to have the least negative impact on wheat even though it involves an entire arm of a rye chromosome. On the other hand, the X-ray approach to affect a small transfer, while perhaps offering a better chance of success for a chromosome as structurally different from wheat as 4Rmon, would require more sophisticated screening to avoid fixation of a complete chromosome, especially in an inherently unstable addition to a full wheat complement. In this particular instance, the moderate level of protection against RWA offered by 4Rmon does not seem to justify the effort that would be required for engineering of an agronomically acceptable transfer of a small segment of rye chromatin.
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ACKNOWLEDGMENTS |
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Received for publication January 16, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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