Published online 16 January 2008
Published in Crop Sci 48:199-202 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Variable Production of Tetraploid and Hexaploid Progeny Lines from Spring Wheat by Durum Wheat Crosses
S. P. Lanning,
N. K. Blake,
J. D. Sherman and
L. E. Talbert*
Plant Sciences and Plant Pathology Dep., Montana State Univ., Bozeman, MT 59717
* Corresponding author (usslt{at}montana.edu).
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ABSTRACT
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Hexaploid spring wheat (Triticum aestivum ssp. aestivum) and tetraploid durum wheat (Triticum turgidum ssp. durum) are both widely grown in the Great Plains of North America. Transfer of genes between classes may be beneficial to improvement efforts. However, ploidy level differences lead to variable degrees of sterility in crosses between the two classes. Our aim was to quantify variation among a set of spring wheat genotypes for production of viable euploid progeny after crosses with durum wheat. Hexaploid spring wheat parents varied for their ability to produce viable F5 progeny, with attrition occurring at all stages of the inbreeding process. Polymerase chain reaction primers specific for the seven D chromosomes indicated that most lines derived from spring wheat by durum wheat crosses were euploid, with a preponderance of tetraploid progeny lines for most parents. Our results showed variability among hexaploid genotypes for their ability to produce viable progeny and suggested that ‘Choteau’ hard red spring wheat may be a good bridge variety for interploidy crosses.
Abbreviations: PCR, polymerase chain reaction • SNP, single-nucleotide polymorphism
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ACKNOWLEDGMENTS
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Work supported in part by USDA/CREES-NRI-CAP award 2006-55606-16629.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication June 16, 2007.
Variable Production of Tetraploid and Hexaploid Progeny Lines from Spring Wheat by Durum Wheat Crosses
S. P. Lanning,
N. K. Blake,
J. D. Sherman and
L. E. Talbert*
Plant Sciences and Plant Pathology Dep., Montana State Univ., Bozeman, MT 59717
* Corresponding author (usslt{at}montana.edu).
Hexaploid spring wheat (Triticum aestivum ssp. aestivum) and tetraploid durum wheat (Triticum turgidum ssp. durum) are both widely grown in the Great Plains of North America. Transfer of genes between classes may be beneficial to improvement efforts. However, ploidy level differences lead to variable degrees of sterility in crosses between the two classes. Our aim was to quantify variation among a set of spring wheat genotypes for production of viable euploid progeny after crosses with durum wheat. Hexaploid spring wheat parents varied for their ability to produce viable F5 progeny, with attrition occurring at all stages of the inbreeding process. Polymerase chain reaction primers specific for the seven D chromosomes indicated that most lines derived from spring wheat by durum wheat crosses were euploid, with a preponderance of tetraploid progeny lines for most parents. Our results showed variability among hexaploid genotypes for their ability to produce viable progeny and suggested that ‘Choteau’ hard red spring wheat may be a good bridge variety for interploidy crosses.
Abbreviations: PCR, polymerase chain reaction • SNP, single-nucleotide polymorphism
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INTRODUCTION
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HEXAPLOID HARD RED SPRING WHEAT (Triticum aestivum ssp. aestivum; genome:AABBDD; 2n = 6x = 42) and tetraploid durum wheat (Triticum turgidum ssp. durum; genome:AABB; 2n = 4x = 28) are cultivated in the same area of the northern Great Plains. Successful varieties within these classes of wheat share several properties, including adaptability to short, dry growing seasons, resistance to endemic diseases and insects, and high grain protein concentration. High grain yield is a primary objective for breeders of both crops. Given that breeding objectives are similar between durum wheat and hexaploid wheat, there is the possibility that unique alleles may be present in each beneficial to the other class. Durum wheat and hard red spring wheat grown in the northern Great Plains represent an example of different gene pools selected for a common set of characteristics.
A requirement for successful exchange of favorable alleles is that viable euploid progeny lines be produced. Love (1940) analyzed chromosome constitution of lines derived from crosses between T. aestivum and T. turgidum ssp. durum. Four different hexaploid parents were used. Selection for the T. aestivum morphology through generations of inbreeding resulted in 40% of the F6 lines having 42 chromosomes (the T. aestivum number), while the remainder were aneuploids or 28 chromosome types. The frequency of hexaploid progeny varied from 3.5 to 85% depending on the cross. Surviving progeny lines tended to be euploid due to lack of viability of aneuploid gametes (Love, 1940). Several valuable genes have been transferred between tetraploid wheat and common wheat, both from wild emmer (T. turgidum ssp. dicoccoides) and durum. These include genes for rust resistance (reviewed in McIntosh et al., 1995), powdery mildew resistance (McIntosh et al., 1998), and high grain protein (Mesfin et al., 1999).
Synthetic hexaploid wheat lines developed from crosses between tetraploid wheat and D genome donor Aegilops tauschii may also be used to transfer genes from tetraploid to hexaploid wheat. However, the presence of the D genome from Ae. tauschii results in numerous agronomic deficiencies in the synthetic wheat (Cox et al., 1995a,b), and ensures that several generations of prebreeding are required before commercial varieties can be developed. Direct interploidy crosses among adapted lines may provide favorable genetic recombination without the need for extensive backcrossing to an adapted parent.
Anecdotal evidence has suggested that hexaploid parents vary for their ability to produce viable progeny in crosses between hexaploid spring wheat and tetraploid durum wheat (e.g., Lanning et al., 2003a) and for the proportion of hexaploid progeny lines produced after generations of inbreeding. For this report, we wanted to quantify this variation and identify a hard red spring wheat variety that may serve as an efficient bridge to generate sufficient progeny to identify genes from durum wheat that positively impact important quantitative traits.
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MATERIALS AND METHODS
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Ten hexaploid wheat genotypes were crossed with each of three durum wheat cultivars. Hexaploid cultivars adapted to dryland conditions in the northern Great Plains included Choteau (Lanning et al., 2004), Outlook (Lanning et al., 2003b), McNeal (Lanning et al., 1994), MTHW9420 (Lanning et al., 2001), and MT9565 developed by Montana State University, Hank developed by Westbred LLC, and Len (CItr17790) and Ernest (PI592761) developed by North Dakota State University. Bobwhite, developed by the International Maize and Wheat Improvement Center, and Chinese Spring were chosen based on their historical use in wheat breeding studies. Durum wheat cultivars adapted to the northern Great Plains included Monroe (Cantrell et al., 1986) and Mountrail (Elias and Miller, 2000), developed by North Dakota State University, and AC Avonlea (Clarke et al., 1999), developed by Agriculture and Agri-Food Canada.
Heads from each of the 10 hard red spring genotypes were emasculated and pollinated by each of the three durum wheat genotypes. At least three heads were pollinated for each hexaploid–tetraploid combination. The number of florets was counted for each emasculated head. The percentage of florets producing seed was determined by dividing the number of F1 seed produced by the number of florets. All F1 seed was planted in pots in the greenhouse. Percentage germination of F1 seed, percentage F1 plants producing heads, and the number of F2 seed per F1 head was determined. F2 seed was planted in the greenhouse with 216 seed per flat. Up to three flats were planted per cross depending on available F2 seed. A single F3 seed per fertile F2 head was planted in flats in the greenhouse to produce F4 families. The F4 families were planted at the Post Research Farm near Bozeman, MT. Data were analyzed by ANOVA using PROC GLM in SAS software version 9.1 (SAS Institute, 2003). Each of the three durum male parents crossed to the 10 hexaploid female genotypes comprised a replication. Least significant difference was computed to allow comparison of individual means. Attrition of genotypes (hexaploid parents) and replications (durum parents) after the F1 due to lack of viable seed production prevented use of ANOVA in later generations of inbreeding. Chi-square test of contingency (Steel et al., 1997) was used to test whether hexaploid parental lines Choteau and MT9565 produced similar frequencies of hexaploid progeny lines. Statistical comparisons among the other hexaploid parents were not conducted due to the small sample size resulting from attrition during the process of line derivation.
A single F5 head was harvested from each row for each of the crosses that had survived the inbreeding process. One F5 seed per head was germinated for a set of 182 independently derived F4 plants, and DNA was isolated from the seedling. To determine ploidy level, the DNA was assayed with polymerase chain reaction (PCR) markers specific to each of the seven D genome chromosomes to verify presence or absence of the chromosome. Microsatellite primer sets used for the analysis were (chromosome marked by the primer set is given parenthetically): GWM337 (1D), GWM261 (2D), GWM161 (3D), GWM645 (3D), GWM194 (4D), GWM182 (5D), GWM292 (5D), GWM469 (6D), and GWM111 (7D). Verification of D genome chromosome specificity was accomplished using nullisomic-tetrasomic lines of Chinese Spring (Sears, 1954). It was necessary to use a second microsatellite primer set for chromosomes 3D and 5D (listed above) with some crosses because of poor amplification with the initial primer set. For a similar reason, a chromosome 6D primer set from the wheat single-nucleotide polymorphism (SNP) project, BE442574_cpF1 and BE442574D_R1, was substituted for primer set GWM469. The chromosome 7D wheat SNP primer set, BG313770D_F2 and BG313770_cpR1, was used to further assay the progeny lines as GWM111 gave ambiguous results (see "Results and Discussion"). Microsatellite primer sequences are available at GrainGenes 2.0 (http://wheat.pw.usda.gov/GG2/index.shtml). Wheat SNP primer sequences are available at the Wheat SNP database (http://wheat.pw.usda.gov/SNP/new/index.shtml). A PCR using the microsatellite primers was performed according the methods of Roder et al. (1998). Polymerase chain reactions with the wheat SNP primer sets included 50 to 100 ng DNA, 1x GoTaq Flexi Buffer (Promega Corp., Madison, WI), 0.2 mM of each dNTP, 1.5 mM MgCl, 1 pmol of each primer, 1 unit GoTaq DNA polymerase (Promega Corp.) in 25 µL total volume. Reaction conditions were 94°C, 5 min; 10 cycles touchdown PCR: 94°C, 20 s, 63 to 58°C, 20 s (decrease 0.5°C each cycle), 72°C, 2 min; 35 cycles: 94°C, 20 s to 58°C, 20 s (decrease 0.5°C each cycle), 72°C, 2 min; final extension: 72°C, 5 min. Products from microsatellite primer amplification were visualized on 12% polyacrylamide gels and those amplified with the wheat SNP primers were run on 1% agarose gels. Both types of gels were stained with ethidium bromide.
Mitotic chromosome counts in root tips were performed on a subset of hexaploid and tetraploid progeny lines to validate ploidy determination by PCR.
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RESULTS AND DISCUSSION
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The 10 hexaploid varieties differed for their ability to produce seed and for survivability of progeny following generations of inbreeding (Table 1
). The amount of crossed F1 seed produced was significantly lower for Outlook, Len, MTHW9420, Ernest, and Bobwhite than for the other hexaploid parents. Poor survival of F1 plants occurred for McNeal, MTHW9420, and Bobwhite, with no F1 progeny surviving in crosses with Outlook. Seed set per F1 head varied among hexaploid parents, with no viable seed produced in the crosses with McNeal (Table 1). Lack of viability and fertility in the F1 generation resulted in a variable number of F2 seed produced among crosses (Table 1). Survival of progeny through two generations of single seed descent also varied among genotypes, with no plants surviving for MTHW9420 or Bobwhite. Choteau and MT9565 were the most productive parents in regards to production of viable F4 progeny.
Our next objective was to determine the ploidy level of the lines produced from the hexaploid x tetraploid crosses (Table 2
). This was accomplished by analyzing 182 F5 plants, each tracing back to a single F2 seed, with PCR markers specific to each of the seven D genome chromosomes. The 182 plants included a plant from all viable F5 families from crosses with Choteau, MT9565, and a sample of progeny lines from the other crosses. Presence of a D-genome band indicated that the chromosome was present, and if all seven D-genome chromosome specific bands were present, the line was considered hexaploid. If no D-genome chromosome-specific bands were produced, the line was considered tetraploid. Aneuploids were indicated if some but not all of the chromosome-specific primer sets amplified a band. In the case of chromosome 7D, primer set GWM111 amplified bands in several progeny lines putatively identified as tetraploid by results from the other six chromosomes. This marker has been mapped to other chromosomes in some populations (Liu et al., 2005; Messmer et al., 1999); thus, it is likely that amplification was occurring on a second chromosome. The chromosome 7D wheat SNP primer set gave amplification results that matched those seen with the other D- genome chromosomes, and these results were used for classification.
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Table 2. Number of tetraploid, hexaploid, and aneuploid progeny indicated by polymerase chain reaction analysis of F5 progeny lines from hexaploid by tetraploid crosses.
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Our results indicated that 96 tetraploid lines, 64 hexaploid lines, and 19 aneuploids were produced by inbreeding hybrids between hard red spring wheat and durum wheat to the F5 generation (Table 2). A subset of eight tetraploid and eight hexaploid lines, as indicted by PCR, was also analyzed by counting chromosomes in root tips. All chromosome counts were consistent with the ploidy level indicated by PCR results (data not shown). Since only 10% of the progeny in this study were aneuploids, assaying with one or two D genome primers should be sufficient to determine ploidy of progeny from hexaploid by durum crosses. There was a preponderance of tetraploid progeny lines from most of the crosses (Table 2). The two hexaploid parents that produced the most progeny lines were Choteau and MT9565. A 2 x 2 Chi-square test of contingency showed that a higher frequency of hexaploid progeny lines came from the Choteau crosses than the MT9565 crosses (P = 0.0008). However this is impacted by heterogeneous results of Choteau crosses with the durum genotypes, evidenced by more frequent hexaploid progeny from the Choteau/AC Avonlea cross as compared to Mountrail or Monroe crosses. In fact, a 2 x 3 Chi-square test of contingency showed that the three durum parents also differed in the frequency of hexaploids produced (P = 0.02) with AC Avonlea crosses producing the most. Crosses with Chinese Spring showed the highest frequency of hexaploid progeny, although these crosses were not as productive as those with Choteau or MT9565. High attrition in the inbreeding process of Chinese Spring progeny was likely due to relative poor agronomic qualities of Chinese Spring (such as late flowering), which impeded seed production in the greenhouse, rather than to sterility.
To verify that recombination between the parental genomes occurred during derivation of the lines, we assayed 28 hexaploid progeny lines and 29 tetraploid progeny lines of Choteau with four codominant microsatellite markers, including GWM550 (chromosome 1B), BARC212 (chromosome 2A), GWM340 (chromosome 3B), and BARC170 (chromosome 4A). All progeny lines contained a mixture of alleles from the hexaploid and tetraploid parents (data not shown).
In summary, our results show that hexaploid hard red spring wheat varieties vary for their propensity to produce viable progeny in crosses with durum wheat. The variety Choteau and experimental line MT9565 each produced a relatively high number of viable F5 progeny lines from crosses with tetraploid durum wheat. Additionally, Choteau produced a higher frequency of hexaploid lines, suggesting that this variety may serve as an efficient bridge for transferring genes between durum wheat and hard red spring wheat.
Work supported in part by USDA/CREES-NRI-CAP award 2006-55606-16629.
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication June 16, 2007.
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ABSTRACT
INTRODUCTION
