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CELL BIOLOGY & MOLECULAR GENETICS

Determination of the Paternity of Wheat (Triticum aestivum L) x Jointed Goatgrass (Aegilops cylindrica Host) BC₁ Plants by Using Genomic In Situ Hybridization (GISH) Technique

^a Dep. of Plant, Soil and Entomological Sciences, Univ. of Idaho, Moscow, ID 83844-2339
^b USDA-ARS, P.O. Box 307, Aberdeen, ID 83210
^c Dep. of Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331

* Corresponding author (rzemetra{at}uidaho.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The release of herbicide resistant wheat (Triticum aestivum L.) raises concerns with gene flow between wheat and jointed goatgrass (Aegilops cylindrica Host). Hybrids between the two species and backcrosses with either species have been observed in the field. Gene flow is dependent on jointed goatgrass being the paternal parent of the BC₁ generation. Differences in the genomes of wheat (AABBDD) and jointed goatgrass (CCDD) could be used to determine the paternity of the BC₁ generation. Twenty BC₁ plants (10 of each paternal type) were used to determine if the number of C genome chromosomes based on genomic in situ hybridization (GISH) could be used to determine BC₁ paternity. Differences between the two BC₁ paternal types for number of C genome chromosomes indicates that C genome chromosome counts could be used to determine the paternity BC₁ plants providing a more accurate estimate of the potential for gene flow between the two species.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
JOINTED GOATGRASS IS AN increasingly troublesome weed in winter wheat fields in the western USA (Dewey, 1996). Jointed goatgrass and wheat share a common genome (D) with jointed goatgrass being a tetraploid with 28 chromosomes and a genomic constitution of CCDD and wheat being a hexaploid with 42 chromosomes and a genomic constitution of AABBDD (Kimber and Sears, 1987). Because of the close genetic relationship between these two species, there is currently no selective herbicide available commercially to control jointed goatgrass (Miller, 1995). A wheat cultivar with resistance to a herbicide that can kill jointed goatgrass would provide an effective means of control. This new strategy, however, could make the control of jointed goatgrass even more difficult if the herbicide resistance gene in wheat could move to jointed goatgrass in the field. The production of wheat x jointed goatgrass hybrids in wheat fields has long been noted; however, the hybrids were assumed to be sterile (Mayfield, 1927; Johnston and Parker, 1929; Priadcencu et al., 1967; Donald and Ogg, 1991). Recent studies showed that hybrids can be backcrossed by either wheat or jointed goatgrass to produce the BC₁ generation both in the greenhouse (Zemetra et al., 1998) and in the field (Snyder et al., 2000). These BC₁ plants can serve as parents for a second backcross to jointed goatgrass resulting in BC₂ plants with the potential for partial self-fertility (Zemetra et al., 1998). Thus the hybrids have the potential to serve as a bridge for gene movement across the two species. For a gene to move from wheat to jointed goatgrass, the hybrids must be continuously backcrossed to jointed goatgrass. If the hybrids were continuously backcrossed by wheat, then the genetic background of backcross progeny would become more similar to wheat. On the other hand, if the hybrids were continuously backcrossed by jointed goatgrass, the backcross progeny's genetic background would gradually be restored to that of jointed goatgrass, plus some genes retained from wheat, especially if the genes were on the D genome. If the genes from wheat included the herbicide resistance gene, then a herbicide resistant jointed goatgrass would occur. Therefore, it is of great importance to know whether hybrids are backcrossed to wheat or to jointed goatgrass. The higher the percentage of hybrids in the field that are backcrossed by jointed goatgrass, the greater the chance that the gene for herbicide resistance in wheat would move to jointed goatgrass. To determine the potential for gene flow in the field, a reliable technique to determine the paternity of field derived BC₁ plants becomes crucial.

Since the BC₁ generation varies in its chromosome constitution, the morphology of BC₁ plants varies from wheat-like to jointed goatgrass-like in leaf characteristics and overall appearance. However, the paternity of BC₁ plants cannot be identified by their leaf or spike characteristics (Snyder et al., 2000). Molecular markers have been used successfully to determine the paternity in adders (Tegelstrom and Hoggren, 1994), Arctic grizzly bears (Craighead et al., 1995), and bur oak (Dow and Ashley, 1998). However, because of the mixed genomic constitution of the BC₁ plants, molecular markers may not be useful in this case. A molecular marker unique to either jointed goatgrass or wheat could, in theory, exist in both types of BC₁ plants. Therefore, the detection of species-specific molecular markers in a BC₁ plant will not provide information about the paternity of that plant.

A promising way to determine the paternity of a BC₁ plant is to count its C genome chromosomes. Since the C genome is unique to jointed goatgrass, the hybrid (2n = 5x = 35 = ABCDD) has only one set of C genome chromosomes. If the hybrid is backcrossed to wheat, the resultant BC₁ plant will have between zero and seven C genome chromosomes. However if the hybrid is backcrossed to jointed goatgrass, the resultant BC₁ plant will have between seven and 14 C genome chromosomes. Therefore, by counting the number of C genome chromosomes, the paternity of BC₁ plants can be determined. A similar system using the number of A and B genome chromosomes could also be used but screening for a single genome such as C has been found to be easier when using techniques such as genomic in situ hybridization (Wang et al. 2000).

Genomic in situ hybridization (GISH) technique has been used to distinguish different genomes in allopolyploids and alien addition lines (Anamthawat-Jonssen et al., 1990; Mukai et al., 1993; Chen et al., 1995). Linc et al. (1999) successfully visualized the C genome chromosomes in jointed goatgrass by using GISH technique. Wang et al. (2000) successfully visualized seven C-genome chromosomes in wheat x jointed goatgrass hybrids by using the total genomic DNA of Aegilops markgrafii (Greuter) Hammer (a C-genome species) as a probe and the total genomic DNA of wheat as blocking DNA. Therefore GISH technique could be used to count the number of C genome chromosomes in BC₁ plants. The objective of this study was to test the hypothesis that a BC₁ plant whose paternal parent is wheat will have zero to seven C-genome chromosomes, while a BC₁ plant whose paternal parent is jointed goatgrass will have seven to 14 C-genome chromosomes. If this hypothesis is correct, then the paternity of a field derived BC₁ plant could be determined by counting its C genome chromosome number using the GISH technique.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plant Material
Wheat x jointed goatgrass hybrids were produced by artificial crossing using the soft white winter wheat cultivar Madsen (Allan et al., 1989) as female parent and a native collection of jointed goatgrass as male parent. Hybrids and their parental species were grown in the greenhouse. Hybrids were emasculated and pollinated by either wheat or jointed goatgrass pollen to produce BC₁ seeds of backcrosses to wheat or jointed goatgrass. All crosses were made by means of the approach method (Zemetra et al., 1998).

Chromosome Preparation
Ten BC₁ seeds of each parental type were sampled randomly. BC₁ seeds were germinated on water saturated filter paper in petri plates. When the primary roots reached 1 to 1.5 cm long, the root tips were collected and immediately pretreated in 1°C water for 24 h. Root tips were then fixed in Farmer's solution (95%, v/v, ethanol–glacial acetic acid, 3:1) for 2 d before use (Tsuchiya, 1971). After root tip collection, the seedlings were transplanted to peat pellets and grown in the greenhouse. Secondary roots were also collected from these seedlings, and pretreated in the same way as described above. Chromosome preparations were made with 2% (v/v) acetocarmine following the procedures in Tsuchiya (1971) for counting mitotic chromosome number and without staining for GISH. Chromosomes were observed with a light microscope with phase contrast (Nikon Model Labophot, Japan). For determining the mitotic chromosome number, two to four chromosome preparations were done for each BC₁ plant. Mitotic chromosome number was based on the chromosome number in a minimum of 10 mitotic metaphase cells with each chromosome preparation representing at least two of the metaphase cells used to determine the chromosome number.

C Genome Chromosome Visualization and Counting
To count number of the C genome chromosomes by GISH technique, it is necessary to probe the C-genome and block all the chromosomes from the other three genomes (A, B, and D). The total genomic DNA of Ae. markgrafii (genome CC) and wheat (genomes AABBDD) were extracted as described by Riede et al. (1996). The total genomic DNA of Ae. markgrafii was labeled with biotin-14-dATP (Gibco BRL) by nick translation as described in the product manual. The biotin labeled total genomic DNA of Ae. markgrafii was used to probe the C-genome chromosomes of jointed goatgrass in the BC₁ plants. The total genomic DNA of wheat was sheared by boiling in 0.4 M NaOH for 50 min as described by Cai et al. (1998) and used as blocking DNA to block all the A-, B-, and D-genome chromosomes in the BC₁ plants, including the D-genome chromosomes from jointed goatgrass. The GISH procedure for detecting the C-genome chromosomes in the BC₁ generation was the same as that described by Wang et al. (2000). Photographs were taken with a Zeiss Axiophot microscope (Zeiss, Germany) equipped with the PowerGene probe system (Perceptive Scientific Instruments, League City, TX).

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Total Number of Chromosomes in BC₁ Plants
The large number of univalents in the wheat x jointed goatgrass hybrids because of having single copies of the A, B, and C genomes resulted in great variation in the total number of chromosomes in BC₁ plants. Although the hybrid plant always had 35 chromosomes, its gametes could have a very wide range of chromosomes because of the irregular segregation of univalents. The chromosome number of BC₁ plants whose paternal parent was jointed goatgrass varied from 34 to 49, while that of BC₁ plants whose paternal parent was wheat varied from 40 to 56 (Table 1). Since the chromosome number of the two types of BC₁ plants overlapped, the paternity of field derived BC₁ plants could not be determined simply by counting the total number of chromosomes. Some BC₁ plants had a high number of chromosomes, which may have resulted from chromosome restitution. In the tribe Triticeae, meiotic restitution has long been reported in some intergeneric hybrids, including Triticum dicoccoides (Koern. ex Asch. & Graebner) Aarons. x Ae. squarrosa L. (Kihara and Lilienfeld, 1949); T. crassum Boiss x T. turgidum L. (Wagenaar, 1968a,b); T. aestivum x Hordeum vulgare L. (Islam and Shepherd, 1980); and T. turgidum x Ae. squarrosa (Xu and Joppa, 1995). In intergeneric hybrids with unpaired chromosomes or low meiotic pairing, the dyad may contain a complete set of chromosomes from the parental species and produce 2n gametes (Xu and Joppa, 1995). The BC₁ plant, BC₁-934, had 56 chromosomes (Table 1, Fig. 1C) . Since its paternal parent (wheat) provides 21 chromosomes, its maternal parent (hybrid) must have provided the remaining 35 chromosomes. That is to say, the hybrid produced a 2n female gamete that had as many chromosomes as its somatic (2n) cells because of meiotic chromosome restitution. The BC₁ plant, BC₁-934, had all 7C genome chromosomes from the hybrid (Fig. 1C), further indicating chromosome restitution.

View larger version (65K): [in this window] [in a new window]   Fig. 1. Visualization of C genome chromosomes in BC1 plants by GISH. A: Plant BC1-855 whose paternal parent was jointed goatgrass showed 12 C genome chromosomes. B: Plant BC1-932 whose paternal parent was wheat showed six C genome chromosomes. C: Plant BC1-934 whose paternal parent was wheat and most likely resulted from meiotic chromosome restitution showed seven C genome chromosomes.

In theory, if a BC₁ plant has seven C genome chromosomes, then its paternal parent could be either wheat or jointed goatgrass, causing ambiguity in paternity. In fact, for all the 20 BC₁ plants observed, the number of C genome chromosomes did not overlap for the two types of BC₁ plants. For the 10 BC₁ plants whose paternal parent was jointed goatgrass, the minimum number of C-genome chromosomes was nine, with 80% (8 of 10) having more than 11 C genome chromosomes (Table 1). Only when the female gamete from the hybrid does not have any C genome chromosomes would the number of C-genome chromosomes in a BC₁ produced with jointed goatgrass as the paternal parent be seven. Theoretically, the chance for a hybrid to produce a female gamete without any C genome chromosomes is very low, being (1/2)⁷ = 0.78%. The presence of at least 2 C genome chromosomes in either the wheat or jointed goatgrass backcrosses (Table 1) may indicate that the presence of at least some of the C genome chromosomes are required to produce a viable female gamete in the hybrid. Further work involving additional backcrosses and C chromosome specific markers would be necessary to confirm this observation. For BC₁ plants whose paternal parent was wheat, the number of C genome chromosomes ranged from four to seven. Although the chance of producing a gamete with seven C genome chromosomes is as low (0.78%), theoretically, as the chance of producing a gamete without any C genome chromosomes in hybrids, three of the wheat backcrosses were found to have seven C chromosomes Since the average number of C chromosomes transferred by the hybrid gamete in the wheat backcrosses was 5.9 and 5.2 in the jointed goatgrass backcrosses, it is reasonable to assume that a BC₁ plant with seven C genome chromosomes originated from a backcross to wheat. Therefore, the number of C genome chromosomes appears to provide a reliable criterion to differentiate the paternity of BC₁ plants and could be used on field-derived BC₁ plants to estimate better the potential for gene flow between herbicide resistant wheat and jointed goatgrass.

	ACKNOWLEDGMENTS

 
The Ae. markgrafii seeds were kindly provided by Dr. Harold Bockelman, USDA-ARS, Aberdeen, ID 83210, USA. This research was supported in part by grant from the USDA-CSREES National Jointed Goatgrass Initiative Program No. 97-34327-3965, and USDA-NRICGP No. 98-35315-6774. Contribution No. 00728 from the Idaho Agricultural Experiment Station, University of Idaho, Moscow, ID.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Contribution No. 00728 from the Idaho Agric. Exp. Stn., Univ. of Idaho.

Received for publication December 22, 2000.

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TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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