Crop Science 41:1062-1065 (2001)
© 2001 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Breeding Behavior of the Cytogenetically Engineered Wheat-Rye Translocation Chromosomes 1RS.1BL
Adam J. Lukaszewski*
Dep. of Botany and Plant Sciences, Univ. of California, Riverside CA 92507
* Corresponding author (ajoel{at}ucrac1.ucr.edu)
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ABSTRACT
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The rye (Secale cereale L.) chromosome arm in the wheat-rye centric translocation 1RS.1BL in wheat (Triticum aestivum L.) had been engineered by induced homoeologous recombination to remove the associated breadmaking quality defect. From a range of primary wheat-rye recombinant chromosomes, two classes of multipoint translocated chromosomes were assembled, MA and Te, that contain Gli-B1 and Glu-B3 loci of wheat and are missing the Sec-1 locus of rye. Their disassembly rates by recombination with normal 1BS and 1RS and the segregation ratios from heterozygotes were studied in populations involving the original, unmodified translocation 1RS.1BL and chromosomes 1B from several wheat cultivars. Overall, the four-point translocation chromosomes MA recombined with 1BS with 4.4% frequency; their intercalary rye segment recombined with 1RS with 3.2% frequency. The overall recombination frequency of the three-point translocation chromosome Te1 with 1BS was 18.1% but only 5.4% resulted in the disassembly of the chromosome. The remaining 12.7% recombination was in the terminal wheat segment and in most cases it introduced new alleles at the wheat storage protein loci. The male transmission rate of all engineered chromosomes was similar to that of the original 1RS.1BL translocation. In competition with 1B, it was reduced to about 22 to 37%. As a result of the disassembly and reduced transmission, the probability of selection of homozygotes for unaltered engineered chromosomes among the progenies of heterozygotes with 1B was about 15 to 16%.
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INTRODUCTION
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TO REMEDY the breadmaking quality defect of the centric wheat-rye translocation 1RS.1BL in wheat, its rye arm 1RS has been recombined with wheat arm 1BS by means of the ph1b mutation and from a range of the primary (single breakpoint) recombinants, series of secondary and tertiary recombinant chromosomes were assembled (Lukaszewski, 2000). Of these, the short (rye) arms in chromosomes MA1 and MA2 were double, four-breakpoint translocations; in chromosomes Te1 and Te2, they were double, three-breakpoint translocations. The rye chromosome arms in the MA and Te chromosomes have two wheat segments: the distal segment introduces wheat storage protein loci Gli-B1 and Glu-B2; the proximal wheat segment removes a rye segment with the Sec-1 locus. The two wheat segments are separated by an intercalary rye segment containing four disease resistance loci, Pm8, Lr26, Yr9, and Sr31. Chromosomes Te differ from chromosomes MA by the absence of the terminal rye segment, originally from the primary wheat-rye recombinant chromosome 1B+40. Because these engineered chromosomes are multipoint translocations, they may disassemble by recombination in hybrids with wheats carrying either the original 1RS chromosome arm or those with normal 1BS arms. This article summarizes the observations on the behavior of the engineered chromosomes MA and Te in hybrids with normal and 1RS.1BL wheats.
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MATERIALS AND METHODS
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To study the frequency of recombination of various segments in the engineered chromosomes MA and Te, plants of Pavon wheat with chromosomes MA1, MA2, or Te1 were crossed to normal Pavon and to lines of Pavon with substitutions of chromosomes 1B from cv. Begra (abbreviated 1BBE), Wheaton (1BWH), and from two breeding lines W3702 (1BPI) and NE93554 (1BNE). Pavon MA1 was also crossed to Pavon 1RS.1BL, where the 1RS arm was from the original Aurora/Kavkaz translocation and 1BL was from Pavon (Lukaszewski, 1997). The 1B substitution lines were developed by repeated backcrosses to the ditelocentric 1BL (Dt1BL) line of Pavon and selection for the complete chromosome 1B. At the time the crosses were made, 1BWH and 1BPI had six backcrosses completed, 1BBE five and 1BNE three. Additionally, recombination frequencies and the F2 segregation ratios of chromosome Te1 were also studied in hybrids with line NE93554.
The chromosome constitution of all F1 hybrids was confirmed cytologically and they were backcrossed as male to Pavon Dt1BL and had most of their heads bagged for controlled self-pollination. The resulting progenies were individually screened by C-banding to determine the structure of the translocated chromosomes and/or by the acid polyacrylamide gel electrophoresis (A-PAGE) as described before (Lukaszewski, 2000) to determine the status of the Gli-B1 and/or Sec-1 storage protein loci. Whenever necessary, samples of the cytologically identified recombinant chromosomes were challenged by the TJD 84-32-122C race of a leaf rust (Puccinia recondita Rob. ex Desm.) to determine the status of the Lr26 locus. This race was selected for virulence on Pavon and avirulence on Pavon 1RS.1BL and was kindly provided for the original engineering effort by Dr. D.V. McVey, USDA-ARS, St. Paul, MN. Lr26 is located on the intercalary rye segment that separates the distal and proximal wheat segments in the engineered chromosomes. Depending on the configuration of the recombined chromosomes, the presence or absence of the locus indicated which of the two intercalary wheat segments was involved in recombination. The differences in the observed recombination and transmission rates were tested by 
2.
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RESULTS
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The wheat segments in chromosomes MA1 and MA2 recombined with normal 1BS with practically identical frequencies of 4.38 and 4.36% (Table 1), respectively. Of the 21 and 15 recombinants of MA1 and MA2 recovered, respectively, 11 of MA1 and 13 of MA2 were tested for the status of Lr26. This test indicated that of the overall average recombination frequency of 4.37%, 1.82% recombination was in the distal wheat segment and 2.55% in the proximal wheat segment. Crossover frequency of the intercalary rye segment in chromosomes MA1 in hybrids with the original translocation 1RS.1BL, as determined by the frequency of the backcross progeny kernels double positive or double null for Gli-B1 and Sec-1, was 3.24% (Table 1). The rye segment in question is identical in all engineered chromosomes MA and Te and tests of additional combinations were deemed unnecessary.
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Table 1. Recombination frequencies of engineered chromosomes MA and Te in hybrids with normal chromosomes 1B and 1RS.1BL.
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Male transmission of chromosomes MA1 and MA2 from heterozygotes with normal 1B was consistently reduced and ranged from 22.7% to about 37.5% (Table 2). The exception was the hybrid with chromosome 1BPI where the transmission rate did not deviate from random (48.6% in a sample of 109 progeny tested). While this sample was relatively small, the transmission rate was significantly higher than that from any remaining hybrids and all hybrids combined (2 = 4.40 P > 0.05%). Normal transmission rates of primary recombinants T-9, T-21, and T-26 from hybrids with 1BPI have been consistently observed in a different experiment involving over 1200 backcross progeny (data not shown). Male transmission of MA1 from hybrids involving the original translocation 1RS.1BL was 50.6% in a sample of 401 backcross progeny tested. Female transmission rate of chromosomes MA was always close to 50% in all crosses and backcrosses made.
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Table 2. Male transmission rates of the engineered chromosomes MA and Te from hybrids with 1B or 1RS.1BL chromosomes.
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As a result of the lower-than-normal male transmission rate (average of 33.6%, excluding 1BPI), the segregation ratios observed among the progeny of self-pollinated heterozygotes with 1B (except 1BPI) significantly deviated from 1:2:1 (Table 3). Recombination further reduced the frequencies of the structurally unaltered homozygotes to 15%, or 60% of the expected based on normal transmission rate. However, one class of recombinants produced by crossover in the distal wheat segment has the same value as the Te chromosomes. Consequently, the frequencies of useful homozygotes and MA/Te-like heterozygotes would be slightly higher. Segregation ratios from heterozygotes with 1RS.1BL were not tested but normal (50%) male and female transmission rates and low crossover frequency of the intercalary rye segment suggest that it would not substantially deviate from 1:2:1.
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Table 3. Segregation ratios of the engineered chromosomes MA and Te from heterozygotes with normal 1B and 1RS.1BL.
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Chromosomes Te differ from chromosomes MA by the absence of the terminal rye segment. The distal wheat segment originated from the primary recombinant T-9 and is identical in Te1 and Te2. These two chromosomes differ only by the position of the proximal translocation breakpoint in the proximal wheat segment. Of the two chromosomes, only Te1 was tested here. The overall crossover frequency of the wheat segments in the short arm of this chromosome was tested cytologically by polymorphism for the telomeric C-band 3.4 on 1BS (Gill et al., 1991) offered by 1BBE and 1BNE. It was 18.1% in a sample of 860 backcross progeny screened. These crossovers were apportioned to the terminal and the proximal wheat segments on the basis of the presence of the intercalary rye segment as detected by the leaf rust test for the presence of Lr26. Of the 156 recombinant chromosomes recovered, 100 were tested and 70 chromosomes were found to have recombined in the distal wheat segment and 30 in the proximal wheat segment. Using the proportion of 7:3, it was calculated that of the overall recombination frequency of 18.1%, about 12.7% recombination was in the distal and 5.4% in the proximal wheat segments.
The overall male transmission rate of Te1 was 35.4% in a sample of 860 progeny screened. As a result of reduced male transmission and 5.4% crossover frequency in the proximal wheat segment, only 15.5% of the progeny of heterozygotes can be expected to be homozygous for the structurally unaltered Te1. This is 62% of the expected frequency based on normal segregation and the absence of recombination.
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DISCUSSION
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In hybrids combining chromosomes MA with normal chromosomes 1B, single crossovers in any of the two intercalary wheat segments can produce four classes of chromosomes as shown in Fig. 1. Double crossovers in both wheat segments simultaneously cannot be ruled out but their probability seems low taking into account strong positive chiasma interference in the B-genome of wheat (Curtis and Lukaszewski, 1992).
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Fig. 1. The structure and recombination patterns of the engineered translocation chromosomes MA and Te. Shaded boxes indicate rye chromatin; blank boxes indicate wheat chromatin. Elongated x's indicate the positions of crossovers. A: recombination of the short arm (S) of chromosomes MA with 1BS; B: recombination of MA(S) with 1RS; C: recombination of Te(S) with 1BS; and D: recombination of Te(S) with 1RS. All chromosomes have normal 1BL arms; drawings not to genetic or physical scale.
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Crossovers in the distal wheat segment separate the terminal rye segment from the rest of the arm. The genetic length of that rye segment was estimated 0.4 centimorgans (Lukaszewski, 2000) and its loss may have no discernible effect on the agronomic characteristics of the translocation lines. Of the two chromosomes produced in such crossover event, one is identical to chromosomes Te and may be useful in breeding, the other is identical to the primary recombinant 1B + 40 (Lukaszewski, 2000). In this study, the distal wheat segment in MA chromosomes recombined with 1BS with a combined frequency of 1.8%. It appears, therefore, that recombination in the distal wheat segment of MA chromosomes produces a chromosome without breeding value with an approximate frequency of about 0.9%.
Recombination in the proximal wheat segments of MA chromosomes separates the intercalary rye segment with the disease resistance loci Lr26, Sr31, Yr9, and Pm8, the distal wheat segment with the Glu-B3/Gli-B1 wheat storage protein loci, and the terminal rye segment from the rest (and the bulk) of the arm. In this study, these segments recombined with an average frequency of 2.55%. The proximal wheat segments in MA1 and MA2 chromosomes differ in length. Genetic mapping of the translocation breakpoints in the original primary recombinants from which MA1 and MA2 were produced suggested that 1B + 44 (used in the construction of MA2) was more proximal than 1B + 38 (used in the construction of MA1) but the difference was not statistically significant (Lukaszewski, 2000). This study confirms that there may be very little difference in the positions of the 1B + 38 and 1B + 44 breakpoints and, consequently, very little difference in the lengths of the proximal wheat segments in MA1 and MA2.
In hybrids with the 1RS.1BL wheats, recombination in the proximal or the terminal rye segments does not affect the structure of the engineered chromosome. Recombination within the interstitial rye segment produces two chromosomes that cytologically are indistinguishable from either the 1RS.1BL translocation or the MA chromosomes but genetically, one chromosome has all three Gli-B1, Glu-B3, and Sec-1 loci while the other one is null at all three loci. Recombination in the intercalary rye segment of MA chromosomes was observed with 3.2% frequency.
Relative to MA, chromosomes Te have simplified structure in that they do not have the terminal rye segment. They are three-point, double translocations. This structural difference clearly affected their overall recombination frequency: it was 18.1% relative to 4.4% for MA chromosomes. Recombination in the terminal wheat segment may change its allelic composition, including that at the Gli-B1 and Glu-B3 loci, but does not change the structure of the chromosome. Recombination in the proximal wheat segment has the same effects as exchanges in the proximal wheat segments of MA chromosomes. Such exchanges in the proximal wheat segment of Te1 took place with 5.4% frequency that is not statistically different from 4.4% recombination in the corresponding wheat segments of MA chromosomes. Recombination of the intercalary rye segment in Te was not studied but there is no reason to believe that it would be higher than that of the identical segment in MA1. In fact, it is possible that the structural heterozygosity at the telomere would reduce pairing efficiency and, therefore, recombination of that rye segment with 1RS.
This study tested recombination of MA1 with 1RS of the original 1RS.1BL translocation. However, both engineered chromosomes MA and Te are also capable of pairing and recombination with 1RS arms in either 1RS.1AL or 1RS.1DL centric translocations. The frequencies of such events probably would be comparable to those observed here for MA1 and none of the recombination products would be of immediate breeding value.
Based on the tests performed here, chromosomes MA appear marginally more stable, especially in hybrids with 1B-wheats, and as such may be much easier to manage in breeding. A small difference in chromosome structure dramatically increased recombination frequency of Te relative to MA. However, most of that increase was in the terminal wheat segment of Te and in most cases it introduced new allelic variation at the storage protein loci.
Overall, the disassembly rates of the engineered chromosomes MA and Te were low, ranging from 4.4 to 5.4%. Taking into account that some of the recombination products would be useful in breeding, the disassembly rates producing chromosomes useless in breeding were even lower. Unfortunately, the recovery rate of the engineered chromosomes was further reduced by low male transmission rates. In the original description of the 1RS.1BL translocation, Mettin et al. (1973) reported complete compensation of the translocated chromosome for wheat chromosome 1B. However, numerous later reports clearly documented lower male transmission rate of the 1RS.1BL translocations from heterozygotes with chromosome 1B (Rayburn and Mornhinweg, 1988; Schelgel and Meinel, 1994; Bullrich et al., 1998; Kozub et al., 2000). Reduced male transmission rate was evident in every step of engineering of the translocation (Lukaszewski, 1995, 1997, 2000) and in every aspect of this study. It is not entirely clear if it is a result of poor compensation ability of the 1RS chromatin for 1BS or if a discrete segregation distortion locus is present on 1RS. There were indications that such a locus might have been located on the 1RS arm in the vicinity of the Sec-1 locus (Lukaszewski, 2000) but no detailed mapping was performed. Nevertheless, this study shows that similar to the original translocation 1RS.1BL, engineered chromosomes MA and Te also suffer from reduced male transmission from heterozygotes with 1B. No such reduction is evident from heterozygotes with normal 1RS.1BL translocation. On the other hand, statistically significant differences in the male transmission rates from heterozygotes of MA1 with chromosomes 1B from several different wheats suggested that the compensating ability of chromosomes might be dependent, at least to a degree, on their allelic composition. In this study, chromosome MA1 fully compensated for 1BPI but not for 1BPA or 1BWH. In earlier observations, primary recombinants T-9 and T-21 also fully compensated for the same 1BPI but not for 1BPA. Kozub et al. (2000) observed a relationship between the transmission rate of the 1RS.1BL translocation and the presence of certain alleles at the storage protein loci. This may well have indicated different compensating ability of different chromosomes marked by different alleles.
Coupled with the disassembly of the engineered chromosomes, low male transmission rate lowers the probability of recovery of the desired translocation homozygotes among the progenies of heterozygotes. Excluding the MA1 + 1BPI combination, the overall male transmission of the engineered chromosomes was 33.7%. Factoring in the disassembly rates, 4.4% for MA and 5.4% for Te1, the probability of recovery of unaltered MA homozygotes from heterozygotes with 1B was 15%; from heterozygotes with 1RS.1BL it was 23.4%. The probability of recovery of Te1 homozygotes was 15.5%. Reduced male transmission rate never hampered extensive utilization in breeding of the original translocation 1RS.1BL, as evidenced by hundreds of cultivars containing it (Braun et al., 1998). It is to be hoped that the low disassembly rates of the engineered chromosomes may not negatively affect their adoption in breeding.
Received for publication July 27, 2000.
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REFERENCES
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ABSTRACT
INTRODUCTION
