Published in Crop Sci. 44:2063-2067 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Origin of the Blue-Aleurone Gene in Sebesta Blue Wheat Genetic Stocks and a Protocol for Its Use in Apomixis Screening
L. A. Morrisona,
R. J. Metzgerb,* and
A. J. Lukaszewskic
a Herbarium, Dep. of Botany and Plant Pathology, Oregon State Univ., Corvallis, OR 97331-2910
b Dep. of Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331-3002
c Dep. of Botany and Plant Sciences, University of California, Riverside, CA 92521
* Corresponding author (bob.metzger{at}oregonstate.edu)
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ABSTRACT
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Tracing the pedigree of the blue aleurone locus in the Sebesta Blue wheat (Triticum aestivum L.) genetic stocks has proven difficult. There is limited information on the original intergeneric crosses that moved the blue-aleurone trait from Thinopyrum ponticum (Podp.) Z.W. Liu & R.-C. Wang into wheat. Blue Baart, an unregistered genetic stock developed at the University of California-Davis, was the direct parental source for the blue-aleurone trait in Sebesta Blue. Its wheat pedigree is unclear because of the introgressing, bulk population technique by which it was developed from the original intergeneric wheat x Thinopyrum cross. In situ probing with total genomic DNA in the spring and winter Sebesta Blue lines showed a 4EL Thinopyrum translocation and numerous unidentified chromosomal rearrangements in the two winter lines. A protocol is presented for use of the Sebesta Blue genetic stocks for apomixis studies.
Abbreviations: PBB, Pugsley's Male Sterile/Blue Baart • SB, Sebesta Blue
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INTRODUCTION
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AS POINTED OUT by Zeven (1991), development of synthetic blue-seeded wheats derived from intergeneric crosses with Thinopyrum Á. Löve (= Agropyron Gaertn.) and Secale L. species has a long history in North America, Europe, and Asia. Research in the USA had its roots in the breeding work of two USDA agronomists, William Sando at the Beltsville, MD, facility and Coit Suneson at the Agricultural Experiment Station at the University of California-Davis. The blue aleurone locus carried by the Sebesta Blue (SB) genetic stocks registered in this issue (Metzger and Sebesta, 2004) was obtained from Blue Baart, an experimental line developed by Suneson from one of Sando's intergeneric hybrid crosses between a hexaploid wheat (T. aestivum) and a Thinopyrum species. An exact lineage cannot be traced for either of the wheat or Thinopyrum parents of Blue Baart because of limited information available in the literature. Further, many of the key players in the early, blue-seeded breeding work are now deceased or no longer professionally active. Also complicating reconstruction of the pedigree is the fact that research material often passed from breeder to breeder without documentation.
SB development at Oregon State University (OSU) was originally begun to produce blue-seeded marker lines exhibiting xenia for research on apomixis. The SB genetic stocks rank among several blue-seeded wheats that have been used successfully as marker lines in a variety of applications (Zeven, 1991; Hucl and Matus-Cádiz, 2001) where a visible seed marker proves useful. Preregistered forms of SB have already been released for research dealing with hybrid wheat development and pollen flow studies.
Given a common heritage shared with other blue-seeded lines developed from Blue Baart (Qualset et al., 1983), reconstruction of SB's wheat and Thinopyrum lineage is of historical interest and also instructive of the long and involved effort that led to current blue-seeded marker lines. The following account reconstructs as much as can be ascertained about the pedigree of SB. It also reports preliminary data on the genetics of the SB blue-aleurone trait and provides a proposed protocol for SB use in apomixis research. For consistency with taxonomic usage in the older literature, the traditional concept of Agropyron as understood by Suneson and his colleagues will be followed in the discussion of the Blue Baart pedigree.
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Blue Baart Pedigree
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In North America, hybridization work to transfer Agropyron genes for disease resistance, perennial habit, and forage traits into wheat originally started in the early part of the 20th century (Smith, 1942; Armstrong, 1945). With the discovery of the blue-aleurone phenotype as a secondary trait of the Agropyron genetic material (Suneson and Pope, 1946), development of blue-seeded wheat marker lines became a separate research focus (Knott, 1958; Suneson, 1962).
The two Agropyron species typically used in this work were A. elongatum (Host) Beauv. and A. intermedium (Host) Beauv. (= A. tricophorum K. Richter). Dramatic changes in generic concepts have recently produced a confusing taxonomy for Agropyron. Thus, these two species have been variously classified in the genera Elytrigia Desv., Lophopyrum Á. Löve, and Thinopyrum Á. Löve. The varying species names for A. elongatum according to ploidy level introduces yet another complexity into the taxonomy. Currently, most taxonomists treat A. elongatum and A. intermedium as members of Thinopyrum, i.e., Th. elongatum (Host) D.R. Dewey (2n = 14), Th. ponticum (Podp.) Z.W. Liu & R.R.-C. Wang (2n = 70), and Th. intermedium (Host) Barkworth & D.R. Dewey (2n = 42).
Suneson began developing blue-seeded lines on obtaining Triticum–Agropyron hybrid material from Sando in 1938 (Love and Suneson, 1945). In literature reports on Suneson's early blue-seeded lines, neither the original wheat cultivar nor the Agropyron species parents were identified. According to Hurd (1959), the Agropyron parent was A. elongatum. Dr. Warren Pope (personal communication, 2002), one of Suneson's coworkers, has confirmed this Agropyron parentage, clarifying that Sando used the decaploid form of A. elongatum (= Th. ponticum). According to Hurd (1959), Suneson was providing breeders with blue-seeded wheat material developed from 10 generations of backcrossing to the white-seeded, spring wheat cultivar Baart as early as 1949.
The wheat backcross parentage for Blue Baart proves more complex than reported by Hurd. As noted in later research reports (Suneson et al., 1963a, 1963b), Blue Baart was produced in Suneson's Wheat Composite Cross I (CCI) breeding program. Because of the CCI natural outcrossing and bulking method, in which a diverse mixture of wheat cultivars played a part, the wheat pedigree for Blue Baart cannot be traced. There is another interesting historical note on the wheat parentage of Blue Baart. During the World War II period, Suneson injected an element of symbolic patriotism into his breeding program by crossing the white-seeded Baart with both red- and blue-seeded wheat lines to produce a red, white, and blue wheat (C.O. Qualset, personal communication, 2002). Two other blue-aleurone wheats, ‘Blue Onas’ and ‘Pugsley’s Male Sterile/Blue Baart' (PBB), were developed in the CCI program. Blue Baart and Blue Onas were disomic substitution lines. PBB was a ditelocentric addition line that was derived from a male-sterile, white-seeded wheat selection provided to Suneson in 1959 by the Australian breeder A.T. Pugsley (Suneson, 1962; Bolton, 1968).
As reported by Metzger and Sebesta (2004), development of SB was stepwise. After obtaining seed of Blue Baart from Suneson in 1958, Metzger began a crossing program initially to create a regional blue-seeded substitution line. One of the more successful parental stocks was Norco, an unreleased soft white winter line identified as WA4995, which had been developed by Dr. Clarence Peterson, the USDA wheat breeder at Washington State University at Pullman, WA. From this preliminary work, a male-fertile, blue-seeded substitution line from the Blue Baart x Norco cross was selected for the next step to induce an Agropyron–wheat translocation. Dr. Emil Sebesta, the USDA cytogeneticist at Oklahoma State University, crossed ‘Pavon 76’ with irradiated pollen of the Blue Baart x Norco line. Two stable translocation lines, designated 78 x Ci12 and 78 x Ci6 and each with consistent 3:1 blue to white seed ratios, were identified from Sebesta's irradiated lines. The two winter SB genetic stocks (SB-1, -2) were developed by a two-step series of crosses, with 78 x Ci12 first crossed to a white-seeded form of ‘Chinese Spring’ (CS-Tsts3D) and then to the soft white winter cultivar Gene (winter SB pedigree: Gene/3/78xCi12/CS-Tsts3D//Gene). The spring SB genetic stock (SB-3) was developed by crossing 78 x Ci6 to the hard white spring cultivar Sonalika (spring SB pedigree: 78xCi6/2*Sonalika). Extensive generation testing preceded selection of the winter and spring SB genetic stocks for registration (Metzger and Sebesta, 2004).
PBB was also included in the early stages of the OSU program with seed obtained from Dr. Floyd Bolton, who had used this line in a doctoral research study of blue aleurone and purple pericarp inheritance (Bolton, 1968). Although PBB does not figure in the pedigree of the SB genetic stocks, it has indirectly played a role in complicating the story for SB's Agropyron lineage. In the OSU blue-seeded breeding program, PBB x soft white winter wheat crosses were also made to develop a set of experimental addition lines. The line developed from the cross PBB x Norco was named Blue Norco. In the 1970s, Metzger began distributing Blue Norco seed to North American breeders. Whelan (1989), who obtained Blue Norco from one of these breeders, erroneously cited A. intermedium (= A. tricophorum) as the parental source of the blue-aleurone trait due to a mistake in reporting the Agropyron parentage to him.
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Blue-Aleurone Genetics
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In Th. ponticum, the blue aleurone locus, designated Ba(b), is located on the long arm of chromosome 4E (Dubcovsky et al., 1996). It is incompletely dominant, a trait readily observed in the natural segregation of red and blue seed in this species. In wheat (Triticum L. and Aegilops L.), blue aleurone is naturally present in only one species, the wild diploid Tr. boeoticum Boiss., where it also shows a form of incomplete dominance. The T. boeoticum locus, designated Ba(a), is located on the long arm of chromosome 4A (Dubcovsky et al., 1996), and is expressed either as a solid or mottled blue with variable intensity found in the first- and second-formed seeds of each spikelet. Apparently, the analogous Ba(a) and Ba(b) loci function differently in hexaploid wheats. SB lines consistently exhibit a strong degree of xenia [Ba(b) locus] with the blue color ranging from dark (three doses) to light (one dose). On the other hand, lines derived from a cross Norco//Saragolla(4x)/T. boeoticum [Ba(a) locus] do not exhibit xenia. Segregation ratios illustrate these differences between Ba(a) and Ba(b) behavior. For crosses in reciprocal combinations of advanced SB-1 and SB-2 lines, segregants consistently give a 3-blue [Ba(b)]: 1-white seeded ratio (Table 1). On the other hand, segregants from the Saragolla/T. boeoticum reciprocal crosses give inconsistent blue [Ba(a)] to white ratios ranging between 1:3 and 1:1 (Table 1).
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Table 1. Average percentage of blue-seeded segregation on F1 progeny of reciprocal blue x white crosses derived from experimental lines developed from T. boeoticum [Ba(a) locus] and Th. ponticum [Ba(b) locus].
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Sequential C-banding/FISH (fluorescence in situ hybridization) was done on lines SB-1, -2, and -3. A single pair of Th. ponticum–wheat translocation chromosomes is present in SB-3, a 42-chromosome line. Based on the C-banding pattern, the wheat chromosome in this translocation is 4B. As the Th. ponticum chromosome responsible for blue aleurone is known to be 4EL (Dubcovsky et al., 1996), it is assumed that the Th. ponticum fragment in SB-3 is from 4EL and, therefore, the translocation is 4BS.4BL.4EL (Fig. 1)
. Although segregation ratios suggest a straightforward Thinopryum–Triticum translocation, sequential C-banding/FISH analysis of SB-1 and SB-2 has revealed severely rearranged karyotypes (Fig. 2)
. Both have 44 chromosomes and carry two pairs of Th. ponticum–wheat translocated chromosomes. The Th. ponticum segment in one of these translocations appears identical by size and hybridization pattern to the T. ponticum segment in SB-3. Absence of any identifying marks on the wheat segment precludes its identification. The other Th. ponticum segment is very small, present in a miniature chromosome that may be a fragment of 2D. In addition, both lines have several translocations within the wheat genome among which is an acrocentic 4BS.
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Fig. 1. Sequential C-banding (left) and FISH (right) of SB-3. Chromosomes labeled T are translocations 4BS.4BL.4EL. Note the hybridization pattern on 4EL.
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Fig. 2. Sequential C-banding (left) and FISH (right) of SB-1. Chromosomes labeled T1 and T2 are two translocations of Th. ponticum segments into unidentified wheat chromosomes. Note that the hybridization pattern on T1 is the same as on 4BS.4BL.4EL in SB3.
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Apomixis Study Protocol
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Although expected at a rare rate, apomictic forms of wheat may exist. If apomictic wheat lines could be identified, they would become important in hybrid wheat production, offering a more cost-effective production method than is currently possible with male-sterile breeding systems (Flament et al., 2001). The strong xenia effect exhibited by the blue aleurone trait of the SB lines provides a visible marker method for detecting the form of apomixis known as pseudogamy. In pseudogamic apomixis (Grant, 1981), an apomictic seed is produced as a consequence of two tandem events: (i) fertilization of the endosperm and (ii) meiotic restitution by the somatic development of an entirely maternal embryo. Meiotic restitution is already known to occur in the wheats, functioning as a mechanism to reverse hybrid sterility (Shamina et al., 1999). It is possible that it could serve yet another role in pseudogamy, thus leading to the occasional production of an apomictic wheat seed. The SB blue-seeded, aleurone-marker trait could provide the means for detecting such an event. Should apomixis occur naturally in wheat as it does in other polyploid members of the Tribe Triticeae, identifying apomictic wheat forms would enable researchers to exploit a genetic system that is already in place. Otherwise, development of apomictic wheats using either introgressive hybridization or transgenic techniques faces significant challenges (Flament et al., 2001).
As envisioned by Metzger, the SB apomictic screening program would encompass two successive cycles (Fig. 3)
. In Step 1 of the first X cycle, an extensive sampling of white- or red-seeded wheats would be interplanted with SB. At the appropriate growth stage, these non-blue wheats would be treated with a gametocide to induce male sterility and to encourage pollination by pollen from the untreated SB lines. For Step 2, only hybrid blue F1x seed produced on the non-blue wheats would be planted. If apomictic wheats are present, two classes of plants should be observed: (i) Class 1, F1x plants segregating for F2x blue and non-blue seed, thereby indicating normal F1x seed formation resulting from fertilization of both endosperm and egg; and (ii) Class 2, F1x plants producing only white (or red) F2x seed, thereby suggesting an F1x apomictic event.
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Fig. 3. Two cycles of screening to identify apomictic plants of wheat (white-seeded form shown here). Possible apomictic lines (Class 2, F2x generation) are selected in the 1st Cycle and confirmed in the 2nd Cycle (Class 4, F2y generation)—i.e., production of only white seed verifies somatic embryo development. Segregating plants for white and blue seed (Classes 1 and 3) verify fertilization by the male SB parent.
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For a firm verification of apomixis, Steps 1 and 2 would be repeated in a second y cycle except that only white (or only red) Class 2-F2x seed produced in Cycle 1 would be interplanted with SB lines (Fig. 3). True apomicts identified in the first Class 2-Fx cycle should also produce white (or red) Class 2-F2y seed in the second cycle. Clearly, success of this screening program depends on the validity of the assumption that apomictic forms exist in hexaploid wheat.
Received for publication September 25, 2003.
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REFERENCES
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ABSTRACT
INTRODUCTION
