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| R.P. Singh, M. Henry, J. Huerta-Espino, A. Mujeeb-Kazi, R.J. Peña, and M. Khairallah |
Introduction
Leaf
rust (caused by Puccinia triticina) and barley yellow dwarf
(BYD) (caused by barley yellow dwarf virus, BYDV) are important diseases
of wheat in several wheat growing regions. Genetic resistance offers the
most economical and environmentally safe control measure.
Sharma and Knott (1966) transferred a chromosome segment from Thinopyrum elongatum to chromosome 7DL of wheat (Fig. 1a). This segment carries leaf rust resistance gene Lr19, which has had limited use in wheat improvement due to its linkage with a gene that causes yellowness of wheat flour. In a recent study Singh et al. (1998) found that the presence of this alien segment increases wheat grain yield by about 10%.
Using tissue culture, Banks et al. (1995) transferred a chromosome segment carrying BYDV resistance from Th. intermedium to wheat and obtained eight lines, commonly referred to as TC lines. The Th. intermedium fragment carried the only known BYDV resistance gene named Bdv2. Among TC lines, TC14 carries the smallest translocation that replaces the terminal part of wheat chromosome 7DL (Fig. 1b).
The objective of our work was to recombine the two alien chromosome segments in a wheat background to identify recombinants that combine genes Lr19 and Bdv2, and lack the gene for yellowness of flour.
| Fig. 1. Fluorescent in situ hibridization (FISH) detail in partial mitotic cells of: | |||
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Wheat DNA was biontin labeled and Thinopyrum bessarabicum DNA was used for blocking. |
| a) Triticum aestivum cv. Oasis 86 with Lr19; | b) Triticum aestivum (TC14) with Bdv2; | c) Oasis 86//TC14/2*spear with Lr19 and Bdv2. | |
Materials and Methods
Two wheat varieties, 'Oasis 86' and 'Super Seri #2', carrying gene Lr19, were crossed with two other varieties, 'TC14/2*Spear' and 'TC14/2*Hartog' carrying Bdv2. Chromosome pairing was studied in meiosis of the F1 plants. By testing with an Lr19-avirulent race of P. triticina, 118 individual F2 plants derived F3 lines from each of the four crosses were evaluated for homozygosity for Lr19.
The Lr19 homozygous lines were evaluated for endosperm or flour yellowness by two methods: a) visual evaluation of endosperm yellowness by cutting the seed in half, and b) flour color determination using Minolta Color Meter, where "b" values were recorded. Acceptable "b" values are 8-12, while unacceptable light yellow to yellow "b" values are 15-20. Lines showing non-yellow endosperm and flour were advanced to the F5 generation by harvesting individual plants in the F4 generation that showed good agronomic features.
The F1 plants from the two crosses involving Oasis 86 described above were top-crossed with 'Yecora+Lr34', whereas the remaining two F1s were top-crossed with 'Seri.1B'. Yecora+Lr34 and Seri.1B are very similar to Oasis 86 and Super Seri#2 but do not carry any alien chromosome translocation. The top-crossed seedlings were first tested for resistance to PAV-Mex isolate of BYDV; plants with low virus titers in ELISA were retained, and then tested for the presence of Lr19-based resistance to leaf rust. Only those plants considered resistant to both diseases were grown and harvested. The leaf rust resistant F2 progenies of these plants were advanced to F3 and lines homozygous for gene Lr19 were identified for further work as described above for the F3 lines from simple crosses.
Cytological procedures for meiosis and fluorescent in situ hybridization (FISH) were similar to those of Mujeeb-Kazi et al. (1994) and Islam-Faridi and Mujeeb-Kazi (1995), respectively.
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| Fig. 2. Agarose gel showing differentiation betwwen lines homozygous for gwm37 (1), heterozygous (10) or not carrying the diagnostic marker (0) |
From 21 F4 lines (representing at least 21 recombination events), 235 individual F5 plants were selected that were homozygous for Lr19 and had white endosperm. An SSR marker, gwm37, mapping to 7DL and identified to be diagnostic for the Th. intermedium translocation (Ayala et al., 2001) was used to assess the presence or absence of the translocation. Because of its co-dominant nature, the marker allowed us to differentiate if the alien fragment was present in homozygous (1) or heterozygous (10) state, or whether it was absent (0) (Fig. 2). DNA extraction, PCR amplification, and separation of the amplified products on agarose gels were done as described by Ayala et al. (2001).
Five 7-day old seedlings of a total of 41 selected F5 lines were inoculated with 10 BYDV-PAV viruliferous aphids (Rhopalosiphum padi) for a 48-h inoculation period. After spraying with the insecticide Pirimor, plants were grown in the greenhouse for 30 days. Virus titers were assessed by double antibody sandwich ELISA (DAS ELISA) on the flag-1 leaf, 10, 20, and 30 days after inoculation. The test was repeated once. For each repetition a non-infected seedling was tested for each line as a control for ELISA.
Results and Discussion
Meiotic chromosome pairing in F1 plantsThe presence of 21 chromosome ring bivalents in at least some cells (Table 1) indicated that the two chromosomes with alien translocations paired at metaphase I, suggesting that recombinants could be expected.
Flour yellowness
The two crosses involving Super Seri#2 did
not give any Lr19 homozygous line with white flour. Of the 21 recombinants
identified (Table 2), 16 were from the simple cross Oasis
86//TC14/2*Spear, plus 3 more when the above cross was top crossed with
Yecora+Lr34. The remaining 2 white-floured recombinants were derived from
the cross Oasis 86//TC14/2*Hartog/3/Yecora+Lr34.
Of the 235 F5 lines (homozygous for Lr19 and white floured) tested, 121 did not carry gwm37, 28 were heterozygous for this marker, and 85 were homozygous. In total, 41 F5 lines were tested, 19 homozygous for the marker gwm37 and the remaining 22 lines, not carrying it. All lines that did not carry gwm37 were susceptible to BYDV (high virus titers in ELISA) (Table 3). Most lines where gwm37 was present were highly or moderately resistant to BYDV indicating the presence of the Bdv2 gene. However, in four cases, lines homozygous for gwm37 were susceptible (high titers). These results suggest that probably recombination also occurred between the molecular marker and the Bdv2 gene.
FISH preparations
Each F1 combination and three
recombined lines where genes Lr19, Bdv2 and gwm37 were present together
and possessed white flour were used for mitotic FISH preparations. The
translocations present in this germplasm were characteristic of Lr19 and
Bdv2 in the F1 heterozygote (Fig.
1c), and appeared to be of
similar length as in the TC14 lines in the advanced progeny with white
flour.
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Conclusions
- The Th. elongatum and Th. intermedium chromosome segments were recombined successfully.
- Recombined alien segments possessing genes Lr19, Bdv2, and white flour with or without the molecular marker gwm37 were identified.
- The recombined translocations could be useful for transferring the Bdv2 gene using leaf rust resistance as a marker, or vice-versa by using the gwm37 molecular marker.
- The status of the gene that enhances yield potential has yet to be determined.
References
Ayala, L., Henry, M. , González de León, D., van Ginkel, M., Mujeeb-Kazi, A., Keller, B., and Khairallah, M. 2001. A diagnostic molecular marker allowing the study of Thinopyrum intermedium derived resistance to BYDV in bread wheat segregating populations. Accepted for publication in Theor. Appl. Genet.
Banks, P.M., Larkin, P.J., Bariana, H.S., Lagudah, E.S., Appels, R., Waterhouse, P.M., Brettell, R.I.S., Chen, X., Xu, H.J., Xin, Z.Y., Qian, Y.T., Zhou, X.M., Cheng, Z.M., and Zhou, G.H. 1995. The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat. Genome 38:395-405.
Islam-Faridi, M.N., and Mujeeb-Kazi, A. 1995. Visualization of Secale cereale DNA in wheat germplasm by fluorescent in situ hybridization. Theor. Appl. Genet. 90:595-600.
Mujeeb-Kazi, A., Jahan, Q., and A. Vahidy. 1994. Application of a somatic and meiotic cytological technique to diverse plant genera and species in the Triticeae. Pak. J. Bot. 26:353-366.
Sharma, D., and D.R. Knott. 1966. The transfer of leaf-rust resistance from Agropyron to Triticum by irradiation. Can. J. Genet. Cytol. 8:137-143.
Singh, R.P., J. Huerta-Espino, S. Rajaram, and J. Crossa. 1998. Agronomic effects from chromosome translocations 7DL.7Ag and 1BL.1RS in spring wheat. Crop Sci. 38:27-33.
© CIMMYT April 2001
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