Chromosomal imbalances in Douglas-fir (Pseudotsuga menziesii)

GMALD E. REHFELDT, STEPHEN P. WLLLS, A N D JOHN Y. WOO 1rlter.r~lorrrlttrirl Fowsr rrrlrl Rtrrlg~ E.rpr-,r.r-irnc.rlr Srrrriorl. Ogtlerl. UT, U.S.A. 84401

Received November 26. I982

REHFELDT. G . E. . S. P. WELLS. and J . Y . Woo. 1983. Chromosomal imbalances in Douglas-fir (P.serrdnrsugrr men:ie.sii). Can. J . Genet. Cytol. 25: 11.3- 116.

Karyotypeg ot tour dwarf Dougalg-t~r tree4 ot glm~lar abnormal morphology wcre compared with the karyotype of 41x trees ot typ~cal phenotype All morphological variant4 were heteroplo~d None exhlb~ted the game ~rregular~t~eg, but all anomal~es ~nvolved one of the f ~ v e long metacentrlc chromogomeg

REHFELDT. G . E . , S. P. WELLS et J . Y . Woo. 1983. Chromosomal imbalanccs in Douglas-fir (P.serrrlor.sir,qtr nz~rlziesii). Can. J . Genet. Cytol. 25: 1 1 3- 1 16.

On a compark les caryotypes de quatre sapins de Douglas nains prkscntant unc morphologie anormale semblable avec le caryotype de six arbrcs dc phenotype typique. Tous les variants morphologiques btaient heteroplo'ides. Aucun ne montrait Ics mtmcs irrkgularit6s mais toutcs Ics anomalies impliquaient un des cinq longs chrornosomcs mktacentriqucs.

[Traduit par Ic journal]

Morphological variants in Douglas-fir. Psc~~rtiots~rgtr rncvlzic..sii. have been described cytologically in trees from British Columbia (Owens 1967) and Oregon (Ching and Doerksen 197 1 ) . Although differing in several characteristics, the variants generally have ( i ) short internodes and many branches: ( i i ) short. thick. and tleshy leaves; and ( i i i ) co~npact leaves that are evenly distributed around the stem. Consequently. these variants. in coniparison to trees of typical pheno- type. are decided dwarfs and have leaves that do not appear to be in two ranks along the branch (Fig. I ) .

Ching and Doerksen ( 197 1 ) also described a chimera which exhibited a branch of the typical phenotype on an otherwise variant seedling (Fig. 2 ) .

The chromosonial complement of variant seedlings deviates from the typical. The karyotype (2n = 26) of both the coastal variety ( P . rn. var. rnc~rlzie.sii) and the Rocky Mountain variety ( P , rn. var. gicurc.(r) includes five metacentric. six submetacentric. and two telo- centric chromosome pairs (Barner and Christiansen 1962; Thomas and Ching 1968: Livingston 197 1 : de Vescovi and Sziklai 1975 ). Owens ( 1967) described a cell of a variant seedling as lacking one submetacentric homologue, but containing two metacentric chromo- somes, one shorter and one longer .than those of the typical karyotype. Owens also reported chromosomal abnormalities in four additional variants, but no vari- ants exhibited the same abnormalities. The anomalous cell described by Ching and Doerksen ( 197 I ) contained a short metacentric chromosome in addition to the typ- ical complement. In the present note. the chromosomal complement of four variant trees is compared with that of six trees phenotypically representative of the Rocky Mountain variety of northern Idaho.

Meristematically active root tips or, for tree 5 (Table I ) , buds were collected from 3- to 8-year-old

trees growing in pots in a shadehouse at Moscow, Idaho. Meristems were immersed in 0.3% colchicine for 2 h. rinsed in distilled water for I0 niin. hydrolyzed in 1 N HCI at 55°C for about 30 min. rinsed in distilled water for several hours. squashed with I drop of aceto- orcein stain. and mounted. Suitable cells were photo- graphed at 900X magnification. and the chroniosomal coniplenient was studied froni enlarged photographic prints (20 x 25 cni).

The typical karyotype was developed froni photo- graphs of 37 cells representing six trees of typical phe- notype. Because of a variety of chromosome mor- phologies (Fig. 5 ). individual chromosomes could be identified with confidence. For only the largest of the submetacentric chromosomes was identification ambig- uous. Karyotypes of the typical phenotype were ex- pressed as the ratio of the length of a particular chromo- some to the length of the longest chromosome in each cell. In addition, the ratio of the length of the short arm to that of the long arm was determined for each chromo- somal pair. For none of the 47 cells examined in detail. and for none of the innumerable cells observed micro- scopically, could chromosomal abnormalities be veri- fied in phenotypically normal trees.

The six trees of typical phenotype provided the basis for karyotypic analyses of variant trees. From the ratio of either chromosome or arm lengths within the typical karyotype, missing homologues or additional chromo- somes not represented in the typical karyotype could be identified in variant trees. Chromosomal complements of as many as eight cells are summarized in Table 1 for each variant. Chromosomal nomenclature follows Barner and Christiansen ( 1962) who sequentially num- bered the chromosome pairs of Douglas-fir from the longest to the shortest.

Karyotypes of variant trees were highly variable

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a single branch has reverted to the typical phenotype. FIG. 3. Chromosomal complement of cell 1 , tree 2 (Table 1 ) containing all chromosomes of the typical karyotype plus a dicentric chromosome. FIG. 4. Chromosomal complement of cell 4, tree 1 , which lacks a homologue for chromosomes I and V but contains three atypical chromosomes or fragments.

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KEHFELDT ET AL

TABLE I . Chromosomal composition of cells from variant seedlings

No. of No. of Homologues atypical chromosomes

Tree Cell chromosomes missing or fragments

v v

None 1. v v

None None

1

None Both of I Both of 1

both of 1V and V 1V. both of V

None Both of I

I

Both of I 1

None I I

Both of 1 None

1 v

among trees and among cells within a tree (Table 1 : Figs. 3 to 5 ) . Consequently. all variants were hetero- ploid. Some cells exhibited the typical karyotype. Most. however, lacked one to several of the typical chromosomes while exhibiting various chromosomal abnormalities. All of the irregularities involved one or more of the five relatively long nietacentric chromo- somes. particularly 1 and V . Since the irregular cell described by Owens (1967) lacked a complete homo- logue of a submetacentric chronlosome. it seems that irregularities may involve any chromosome but most frequently involve the long chromosomes. In some cells, missing homologues seemed to be represented exactly by the extra chromosomal segments. In most cases, however, a chromosomal surplus seemed evi- dent. No cells exhibited a marked deficiency in total chromosome volume.

One of the irregular cells (cell 1 , tree 2. Figs. 3 and 5 ) contained a dicentric chromosome. Even though sec- ondary constrictions are common in the long chromo- somes of Douglas-fir, the long chromosome in Fig. 3 is recognizable as a dicentric by its size and morphology. For this chromosome (Fig. 3) to be exhibiting second- ary constrictions means that all other chromosomes within the cell would be 5- 10% shorter than expected

(Fig. 5 ) . Moreover, secondary constrictions. apparent in Figs. 3 and 4, are readily distinguished.

There is little doubt that the variant phenotype is associated with heteroploid chromosonlal imbalances that recur naturally. Heteroploidy likely arises from breakage- fusion- bridge cycles that invclve intra- and inter-chromosomal translocations either during meiosis or during mitosis in early embryogenesis. This conclu- sion is supported by the dicentric chromosome in Figs. 3 and 5; by Livingston's (1971) observation of chromosomal bridges during meiosis in Douglas-fir; and by the occurrence of imbalances in both the shoot and root. In addition. natural recurrence of variant seed- lings was estimated by Owens ( 1967) as 1 in 5000 at a nursery in British Columbia. In fact, horticulturists rec- ognize 14 varieties of dwarf Douglas-fir (Welch 1979); most varieties exhibit phenotypes similar to those of the variants that have been described cytologically.

If all variants are recurring heteroploids, all may have inherited a predisposition to chromosome break- age. German (1972) reviewed several cases of hetero- ploid plants and animals in which Mendelian factors exert an effect on chromosomal phenotype. Con- sequently, it is tempting to attribute heteroploidy in Douglas-fir similarly to McClintock's (1956) "dis-

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116 C A N J GENET CYTOL V O L 25. 19x3

FIG. 5. ldiograms of cells illustrating typical and aberrant karyotypes. The typical idiogram is the mean of six trees. Chromosome lengths arc relative to either the observed or expected length of chromosome I.

sociator" and "activator" genes of maize: "activator" controls the frequency at which "dissociator" causes the chromosomal breakage prerequisite to translocation. Regardless, further speculation on the origin and extent of chromosomal abnormalities in variant Douglas-fir seedlings must be delayed until cytological studies of either meiosis or sister chromatid exchanges are possible.

Acknowledgments We thank Dr. G. H. Thorgaard for stimulating cyto-

genetic interpretations. Drs. D. T. Lester, R. J . Naskali, and E. D. McArthur also provided thoughtful

CHIN(;, K . K . . and A. DOERKSEN. 197 1 . A natural chimera of Douglas-fir. Silvae Genet. 20: 209-210.

DE VESCOVI, M. A. . and 0 . SZIKLAI. 1975. Comparative karyotype analysis of Douglas-fir. Silvae Genet. 24: 68-73.

GERMAN, J . 1972. Genes that increase chromosomal in- stability and predispose to cancer. Prog. Med. Genet. 8: 61-101.

LIVINGSTON, G. D. 197 1 . The morphology and behavior of meiotic chromosomes of Douglas fir. Silvae Genet. 20: 75-82.

MCCLINTOCK. B. 1956. Controlling elements and the gene. Cold Spring Harbor Symp. Quant. Biol. 21: 197 -2 16.

OWENS, V. N . 1967. Chromosome abberations in Douglas- fir. Can. J . Bot. 45: 1910-1913.

criticism. THOMAS. G. , and K . K. CHING. 1968. A comparative karyo- type analysis of Pseudotsugc~ menziesii (Mirb.) Franco and

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