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Jointed goatgrass is an increasingly troublesome by tyndale


									 Determination of the Paternity of Wheat (Triticum aestivum L) Jointed Goatgrass
              (Aegilops cylindrica Host) BC1 Plants by Using Genomic
                      In Situ Hybridization (GISH) Technique
 Zhining Wang, Robert S. Zemetra,* Jennifer Hansen, An Hang, Carol A. Mallory-Smith, and Charlotte Burton

                          ABSTRACT                                            al., 1998). Thus the hybrids have the potential to serve
   The release of herbicide resistant wheat (Triticum aestivum L.)            as a bridge for gene movement across the two species.
raises concerns with gene flow between wheat and jointed goatgrass            For a gene to move from wheat to jointed goatgrass, the
(Aegilops cylindrica Host). Hybrids between the two species and               hybrids must be continuously backcrossed to jointed goat-
backcrosses with either species have been observed in the field. Gene         grass. If the hybrids were continuously backcrossed by
flow is dependent on jointed goatgrass being the paternal parent of           wheat, then the genetic background of backcross prog-
the BC1 generation. Differences in the genomes of wheat (AABBDD)              eny would become more similar to wheat. On the other
and jointed goatgrass (CCDD) could be used to determine the pater-
                                                                              hand, if the hybrids were continuously backcrossed by
nity of the BC1 generation. Twenty BC1 plants (10 of each paternal
type) were used to determine if the number of C genome chromosomes
                                                                              jointed goatgrass, the backcross progeny’s genetic back-
based on genomic in situ hybridization (GISH) could be used to                ground would gradually be restored to that of jointed
determine BC1 paternity. Differences between the two BC1 paternal             goatgrass, plus some genes retained from wheat, espe-
types for number of C genome chromosomes indicates that C genome              cially if the genes were on the D genome. If the genes
chromosome counts could be used to determine the paternity BC1                from wheat included the herbicide resistance gene, then
plants providing a more accurate estimate of the potential for gene           a herbicide resistant jointed goatgrass would occur. There-
flow between the two species.                                                 fore, 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

J   ointed goatgrass is an increasingly troublesome
    weed in winter wheat fields in the western USA
(Dewey, 1996). Jointed goatgrass and wheat share a
                                                                              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
common genome (D) with jointed goatgrass being a                              for gene flow in the field, a reliable technique to deter-
tetraploid with 28 chromosomes and a genomic constitu-                        mine the paternity of field derived BC1 plants be-
tion of CCDD and wheat being a hexaploid with 42                              comes crucial.
chromosomes and a genomic constitution of AABBDD                                 Since the BC1 generation varies in its chromosome
(Kimber and Sears, 1987). Because of the close genetic                        constitution, the morphology of BC1 plants varies from
relationship between these two species, there is cur-                         wheat-like to jointed goatgrass-like in leaf characteris-
rently no selective herbicide available commercially to                       tics and overall appearance. However, the paternity of
control jointed goatgrass (Miller, 1995). A wheat culti-                      BC1 plants cannot be identified by their leaf or spike
var with resistance to a herbicide that can kill jointed                      characteristics (Snyder et al., 2000). Molecular markers
goatgrass would provide an effective means of control.                        have been used successfully to determine the paternity
This new strategy, however, could make the control of                         in adders (Tegelstrom and Hoggren, 1994), Arctic griz-
jointed goatgrass even more difficult if the herbicide                        zly bears (Craighead et al., 1995), and bur oak (Dow and
resistance gene in wheat could move to jointed goatgrass                      Ashley, 1998). However, because of the mixed genomic
in the field. The production of wheat jointed goatgrass                       constitution of the BC1 plants, molecular markers may
hybrids in wheat fields has long been noted; however,
                                                                              not be useful in this case. A molecular marker unique
the hybrids were assumed to be sterile (Mayfield, 1927;
                                                                              to either jointed goatgrass or wheat could, in theory,
Johnston and Parker, 1929; Priadcencu et al., 1967; Don-
                                                                              exist in both types of BC1 plants. Therefore, the detec-
ald and Ogg, 1991). Recent studies showed that hybrids
                                                                              tion of species-specific molecular markers in a BC1 plant
can be backcrossed by either wheat or jointed goatgrass
                                                                              will not provide information about the paternity of
to produce the BC1 generation both in the greenhouse
(Zemetra et al., 1998) and in the field (Snyder et al.,                       that plant.
2000). These BC1 plants can serve as parents for a second                        A promising way to determine the paternity of a BC1
backcross to jointed goatgrass resulting in BC2 plants                        plant is to count its C genome chromosomes. Since the
with the potential for partial self-fertility (Zemetra et                     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,
Zhining Wang, Robert S. Zemetra, and Jennifer Hansen, Dep. of                 the resultant BC1 plant will have between zero and seven
Plant, Soil and Entomological Sciences, Univ. of Idaho, Moscow, ID
83844-2339; An Hang and Charlotte Burton, USDA-ARS, P.O. Box                  C genome chromosomes. However if the hybrid is back-
307, Aberdeen, ID 83210; Carol A. Mallory-Smith, Dep. of Crop and             crossed to jointed goatgrass, the resultant BC1 plant will
Soil Science, Oregon State Univ., Corvallis, OR 97331. Contribution           have between seven and 14 C genome chromosomes.
No. 00728 from the Idaho Agric. Exp. Stn., Univ. of Idaho. Received           Therefore, by counting the number of C genome chro-
22 Dec. 2000. *Corresponding author (
                                                                              mosomes, the paternity of BC1 plants can be deter-
Published in Crop Sci. 42:939–943 (2002).                                     mined. A similar system using the number of A and B
940                                            CROP SCIENCE, VOL. 42, MAY–JUNE 2002

genome chromosomes could also be used but screening                these seedlings, and pretreated in the same way as described
for a single genome such as C has been found to be                 above. Chromosome preparations were made with 2% (v/v)
easier when using techniques such as genomic in situ               acetocarmine following the procedures in Tsuchiya (1971) for
hybridization (Wang et al. 2000).                                  counting mitotic chromosome number and without staining
                                                                   for GISH. Chromosomes were observed with a light micro-
   Genomic in situ hybridization (GISH) technique has              scope with phase contrast (Nikon Model Labophot, Japan).
been used to distinguish different genomes in allopoly-            For determining the mitotic chromosome number, two to four
ploids and alien addition lines (Anamthawat-Jonssen                chromosome preparations were done for each BC1 plant. Mi-
et al., 1990; Mukai et al., 1993; Chen et al., 1995). Linc         totic chromosome number was based on the chromosome
et al. (1999) successfully visualized the C genome                 number in a minimum of 10 mitotic metaphase cells with
chromosomes in jointed goatgrass by using GISH tech-               each chromosome preparation representing at least two of the
nique. Wang et al. (2000) successfully visualized seven            metaphase cells used to determine the chromosome number.
C-genome chromosomes in wheat            jointed goatgrass
hybrids by using the total genomic DNA of Aegilops                  C Genome Chromosome Visualization and Counting
markgrafii (Greuter) Hammer (a C-genome species) as
                                                                      To count number of the C genome chromosomes by GISH
a probe and the total genomic DNA of wheat as blocking             technique, it is necessary to probe the C-genome and block
DNA. Therefore GISH technique could be used to                     all the chromosomes from the other three genomes (A, B,
count the number of C genome chromosomes in BC1                    and D). The total genomic DNA of Ae. markgrafii (genome
plants. The objective of this study was to test the hypoth-        CC) and wheat (genomes AABBDD) were extracted as de-
esis that a BC1 plant whose paternal parent is wheat               scribed by Riede et al. (1996). The total genomic DNA of Ae.
will have zero to seven C-genome chromosomes, while                markgrafii was labeled with biotin-14-dATP (Gibco BRL) by
a BC1 plant whose paternal parent is jointed goatgrass             nick translation as described in the product manual. The biotin
will have seven to 14 C-genome chromosomes. If this                labeled total genomic DNA of Ae. markgrafii was used to
hypothesis is correct, then the paternity of a field derived       probe the C-genome chromosomes of jointed goatgrass in the
                                                                   BC1 plants. The total genomic DNA of wheat was sheared by
BC1 plant could be determined by counting its C genome             boiling in 0.4 M NaOH for 50 min as described by Cai et al.
chromosome number using the GISH technique.                        (1998) and used as blocking DNA to block all the A-, B-,
                                                                   and D-genome chromosomes in the BC1 plants, including the
            MATERIALS AND METHODS                                  D-genome chromosomes from jointed goatgrass. The GISH
                                                                   procedure for detecting the C-genome chromosomes in the
                       Plant Material                              BC1 generation was the same as that described by Wang et
                                                                   al. (2000). Photographs were taken with a Zeiss Axiophot
   Wheat jointed goatgrass hybrids were produced by artifi-        microscope (Zeiss, Germany) equipped with the PowerGene
cial crossing using the soft white winter wheat cultivar Madsen    probe system (Perceptive Scientific Instruments, League City,
(Allan et al., 1989) as female parent and a native collection      TX).
of jointed goatgrass as male parent. Hybrids and their parental
species were grown in the greenhouse. Hybrids were emascu-
lated and pollinated by either wheat or jointed goatgrass pol-             RESULTS AND DISCUSSION
len to produce BC1 seeds of backcrosses to wheat or jointed
goatgrass. All crosses were made by means of the approach            Total Number of Chromosomes in BC1 Plants
method (Zemetra et al., 1998).
                                                                     The large number of univalents in the wheat jointed
                                                                   goatgrass hybrids because of having single copies of the
                Chromosome Preparation                             A, B, and C genomes resulted in great variation in the
   Ten BC1 seeds of each parental type were sampled ran-           total number of chromosomes in BC1 plants. Although
domly. BC1 seeds were germinated on water saturated filter         the hybrid plant always had 35 chromosomes, its ga-
paper in petri plates. When the primary roots reached 1 to         metes could have a very wide range of chromosomes
1.5 cm long, the root tips were collected and immediately          because of the irregular segregation of univalents. The
pretreated in 1 C water for 24 h. Root tips were then fixed
in Farmer’s solution (95%, v/v, ethanol–glacial acetic acid,       chromosome number of BC1 plants whose paternal par-
3:1) for 2 d before use (Tsuchiya, 1971). After root tip collec-   ent was jointed goatgrass varied from 34 to 49, while
tion, the seedlings were transplanted to peat pellets and grown    that of BC1 plants whose paternal parent was wheat
in the greenhouse. Secondary roots were also collected from        varied from 40 to 56 (Table 1). Since the chromosome

Table 1. The total number of chromosomes and the number of C-genome chromosomes in wheat        jointed goatgrass BC1 plants where
  the parental parent was either wheat or jointed goatgrass.
                     Backcross to wheat                                               Backcross to jointed goatgrass
                    Chromosome                  Number of                                 Chromosome                      Number of
Plant no.              count                 C-genome chrom.          Plant no.              count                     C-genome chrom.
BC1-915                  40                         5                  BC1-784                 37                            12
BC1-916                  40                         4                  BC1-786                 35                            10
BC1-917                  46                         6                  BC1-855                 36                            12
BC1-918                  42                         7                  BC1-857                 45                            13
BC1-919                  49                         6                  BC1-859                 49                            14
BC1-930                  42                         5                  BC1-920                 34                             9
BC1-931                  54                         6                  BC1-921                 42                            14
BC1-932                  42                         6                  BC1-922                 37                            13
BC1-933                  48                         7                  BC1-923                 44                            13
BC1-934                  56                         7                  BC1-924                 43                            12

number of the two types of BC1 plants overlapped, the                  somes, which may have resulted from chromosome
paternity of field derived BC1 plants could not be deter-              restitution. In the tribe Triticeae, meiotic restitution has
mined simply by counting the total number of chromo-                   long been reported in some intergeneric hybrids, includ-
somes. Some BC1 plants had a high number of chromo-                    ing Triticum dicoccoides (Koern. ex Asch. & Graebner)

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.
942                                        CROP SCIENCE, VOL. 42, MAY–JUNE 2002

Aarons.      Ae. squarrosa L. (Kihara and Lilienfeld,          nal parent was wheat, the number of C genome chromo-
1949); T. crassum Boiss       T. turgidum L. (Wagenaar,        somes ranged from four to seven. Although the chance
1968a,b); T. aestivum Hordeum vulgare L. (Islam and            of producing a gamete with seven C genome chromo-
Shepherd, 1980); and T. turgidum Ae. squarrosa (Xu             somes is as low (0.78%), theoretically, as the chance of
and Joppa, 1995). In intergeneric hybrids with unpaired        producing a gamete without any C genome chromo-
chromosomes or low meiotic pairing, the dyad may con-          somes in hybrids, three of the wheat backcrosses were
tain a complete set of chromosomes from the parental           found to have seven C chromosomes Since the average
species and produce 2n gametes (Xu and Joppa, 1995).           number of C chromosomes transferred by the hybrid
The BC1 plant, BC1-934, had 56 chromosomes (Ta-                gamete in the wheat backcrosses was 5.9 and 5.2 in the
ble 1, Fig. 1C). Since its paternal parent (wheat) pro-        jointed goatgrass backcrosses, it is reasonable to assume
vides 21 chromosomes, its maternal parent (hybrid)             that a BC1 plant with seven C genome chromosomes
must have provided the remaining 35 chromosomes.               originated from a backcross to wheat. Therefore, the
That is to say, the hybrid produced a 2n female gamete         number of C genome chromosomes appears to provide
that had as many chromosomes as its somatic (2n) cells         a reliable criterion to differentiate the paternity of BC1
because of meiotic chromosome restitution. The BC1             plants and could be used on field-derived BC1 plants
plant, BC1-934, had all 7C genome chromosomes from             to estimate better the potential for gene flow between
the hybrid (Fig. 1C), further indicating chromosome res-       herbicide resistant wheat and jointed goatgrass.
      The Number of C Genome Chromosomes                          The Ae. markgrafii seeds were kindly provided by Dr. Har-
                  in BC1 Plants                                old Bockelman, USDA-ARS, Aberdeen, ID 83210, USA. This
                                                               research was supported in part by grant from the USDA-
   Wheat jointed goatgrass BC1 plants could have the           CSREES National Jointed Goatgrass Initiative Program No.
chromosomes from four genomes (A, B, C and D).                 97-34327-3965, and USDA-NRICGP No. 98-35315-6774. Con-
Upon visualization, the A-, B-, and D-genome chromo-           tribution No. 00728 from the Idaho Agricultural Experiment
somes blocked by wheat DNA were red, while the C-              Station, University of Idaho, Moscow, ID.
genome chromosomes probed by biotin-labeled Ae.
markgrafii DNA were yellow-green (Fig. 1). The num-                                    REFERENCES
ber of C-genome chromosomes of the BC1 plants is               Allan, R.E., C.J. Peterson, Jr., G.L. Rubenthaler, R.F. Line, and
shown in Table 1. If the paternal parent was wheat, the           D.E. Roberts. 1989. Registration of ‘Madsen’ wheat. Crop Sci. 29:
number of C-genome chromosomes ranged from four                   1575–1576.
to seven. If the paternal parent was jointed goatgrass,        Anamthawat-Jonsson, K., T. Schwarzacher, A.R. Leitch, M.D. Ben-
                                                                  nett, and J.S. Heslop-Harison. 1990. Discrimination between
the number of C-genome chromosomes ranged from                    closely related Triticeae species using genomic DNA as a probe.
nine to fourteen (Table 1, Fig. 1). The lack of an overlap        Theor. Appl. Genet. 79:721–728.
in the number of C chromosomes between the two pa-             Cai, X., S.S. Jones, and T.D. Murray. 1998. Molecular cytogenetic
ternal parent types demonstrates that the paternity of            characterization of Thinopyrum and wheat-Thinopyrum translo-
BC1 plants could be determined from the number of C-              cated chromosomes in a wheat-Thinopyrum amphiploid. Chromo-
                                                                  some Res. 6:183–189.
genome chromosomes.                                            Chen, Q., R.L. Conner, and A. Laroche. 1995. Identification of the
   In theory, if a BC1 plant has seven C genome chromo-           parental chromosomes of the wheat-alien amphiploid Agrotana by
somes, then its paternal parent could be either wheat             genomic in situ hybridization. Genome 38:1163–1169.
or jointed goatgrass, causing ambiguity in paternity. In       Craighead, L., D. Paetkau, and H.V. Reynolds. 1995. Microsatellite
                                                                  analysis of paternity and reproduction in Arctic grizzly bears. J.
fact, for all the 20 BC1 plants observed, the number of           Hered. 86:255–261.
C genome chromosomes did not overlap for the two               Dewey, S. 1996. Jointed goatgrass—an overview of the problem. Pages
types of BC1 plants. For the 10 BC1 plants whose pater-           1–2 in proceedings of the Pacific Northwest Jointed Goatgrass
nal parent was jointed goatgrass, the minimum number              Conf., Pocatello, ID. Lincoln, NE: Univ. of Nebraska.
                                                               Donald, W.W., and A.G. Ogg, Jr. 1991. Biology and control of jointed
of C-genome chromosomes was nine, with 80% (8 of                  goatgrass (Aegilops cylindrica ), a review. Weed Technol. 53:3–17.
10) having more than 11 C genome chromosomes (Ta-              Dow, B.D., and M.V. Ashley. 1998. High levels of gene flow in bur
ble 1). Only when the female gamete from the hybrid               oak revealed by paternity analysis using microsatellites. J. Hered.
does not have any C genome chromosomes would the                  89:62–70.
number of C-genome chromosomes in a BC1 produced               Islam, A.K.M.R., and K.W. Shepherd. 1980. Meiotic restitution in
                                                                  wheat-barley hybrids. Chromosoma. 79:363–372.
with jointed goatgrass as the paternal parent be seven.        Johnston, C.O., and J.H. Parker. 1929. Aegilops cylindrica Host. A
Theoretically, the chance for a hybrid to produce a fe-           wheat-field weed in Kansas. Trans. Kans. Acad. Sci. 32:80–84.
male gamete without any C genome chromosomes is                Kihara, H., and F. Lilienfeld. 1949. A new synthesized 6x-wheat.
very low, being (1/2)7 0.78%. The presence of at least            Hereditas, (Suppl.): 307–319.
                                                               Kimber, G., and E.R. Sears. 1987. Evolution in the genus Triticum
2 C genome chromosomes in either the wheat or jointed             and the origin of cultivated wheat. p. 154–164. In E.G. Heyne
goatgrass backcrosses (Table 1) may indicate that the             (ed.) Wheat and wheat improvement. Second edition. ASA, Madi-
presence of at least some of the C genome chromosomes             son, WI.
are required to produce a viable female gamete in the          Linc, G., B.R. Friebe, R.G. Kynast, M. Molnar-Lang, B. Koszegi, J.
                                                                  Sutka, and B.S. Gill. 1999. Molecular cytogenetic analysis of Aegi-
hybrid. Further work involving additional backcrosses             lops cylindrica Host. Genome 42:497–503.
and C chromosome specific markers would be necessary           Mayfield, L. 1927. Goatgrass—a weed pest of central Kansas wheat
to confirm this observation. For BC1 plants whose pater-          fields. Kans. Agric. Student 7:40–41.

Miller, S.D. 1995. An integrated approach to jointed goatgrass control.     special reference to the modified Rattenbury’s method of making
   p. 27–29. In Proc. of the Central Great Plains Jointed Goatgrass         a preparation permanent. Barley Genet. Newsl. 1:71–72.
   Conf., Ogallala, NE, Colby, KS, and Sterling, CO. Univ. of Ne-         Wagenaar, E.B. 1968a. Meiotic restitution and the origin of polyploid.
   braska, Lincoln, NE; Kansas State Univ., Manhattan, KS; Colorado         I. Influence of genotype on polyploid seed-set in a Triticum
   State Univ., Fort Collins, CO; and USDA-Cooperative State Re-            crassum      T. turgidum hybrid. Can. J. Genet. Cytol. 10:836–843.
   search Service.                                                        Wagenaar, E.B. 1968b. Meiotic restitution and the origin of polyploid.
Mukai, Y., Y. Nakahara, and M. Yamamoto. 1993. Simultaneous                 II. Prolonged duration of metaphase I as causal factor of restitution
   discrimination of the three genomes in hexaploid wheat by multi-         induction. Can. J. Genet. Cytol. 10:844–852.
   color fluorescence in situ hybridization using total genomic and       Wang, Z., A. Hang, J. Hansen, C. Burton, C.A. Mallory-Smith, and
   highly repeated DNA probes. Genome 36:489–494.                           R.S. Zemetra. 2000. Visualization of A- and B-genome chromo-
Priadcencu, A.L., C. Miclea, and L. Moisescu. 1967. The local form          somes in wheat (Triticum aestivum L.) jointed goatgrass (Aegi-
   of the species of Aegilops cylindrica Host. and its genetic impor-       lops cylindrica Host) backcross progenies. Genome 43:1038–1044.
   tance. Rev. Roum. Biol-Botan. 6:421–425.                               Wang, Z., R.S. Zemetra, J. Hansen, and C. Mallory-Smith. 2001. The
Riede, C.R., and Anderson, J.A. 1996. Linkage of RFLP markers to            fertility of wheat       jointed goatgrass hybrid and its backcross
   an aluminum tolerance gene in wheat. Crop Sci. 36:905–909.               progenies. Weed Sci. 49:340–345.
Snyder, J., C. Mallory-Smith, S. Balter, J. Hansen, and R.S. Zemetra.     Xu, S.J., and L.R. Joppa. 1995. Mechanism and inheritance of first
   2000. Seed production on Triticum aestivum by Aegilops cylindrica        division restitution in hybrids of wheat, rye, and Aegilops squarrosa.
   hybrids in the field. Weed Sci. 48:588–593.                              Genome 38:607–615.
Tegelstrom, H., and M. Hoggren. 1994. Paternity determination in          Zemetra, R.S., J. Hansen, and C.A. Mallory-Smith. 1998. Potential
   the adder (Vipera berus ) -DNA fingerprinting or random amplified        for gene transfer between wheat (Triticum aestivum ) and jointed
   polymorphic DNA. Biochem. Genet. 32:249–256.                             goatgrass (Aegilops cylindrica ). Weed Sci. 46:313–317.
Tsuchiya, T. 1971. An improved aceto-carmine squash method, with

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