Table 3. Evaluation of the mj gene In five cucumber backgrounds utilizing segregation ratios in the F,
and BC, to LJ 90430 generations*
Generation
No. observed
(R3)
No. expected
(RS)
Fitted ratio
(RS)
8:32
10-.30
8:32
13:27
8:32
10:30
10-30
10:30
1030
1030
1::)
1:3
1::)
1:3
lu )
053
0.00
0.53
1.20
0.53
47:153
50:150
1:.i
0.24
.48
28
.48
.65
11.-9
9:11
12:8
9:11
115
52:48
10:10
10:10
10:10
10:10
10:10
5050
1:
1:
1:
1:
1:
1:
0.20
0.20
0.80
0.20
0.20
0.16
.68
.68
.40
.68
.68
.71
F,
(Addis X LJ 90430)
(Sumter x LJ 90430)
(Gy 14 X LJ 90430)
(Polnsett 87 X U 90430)
(Gy 57u X U 90430)
Pooled
BC, to recessive parent
(Addis X LJ 90430)
(Sumter X LJ 90430)
(Gy 14 x U 90430)
(Polnsett 87 X LJ 90430)
(Gy 57u X LJ 90430)
Pooled
.48
1.00
Jewell DL, 1987. Agricultural statistics. Washington,
D.C.: VS. Government Printing Office; 157.
Netscher C and Slkora RA, 1990. Nematode parasites of
vegetables. In: Plant parasitic nematodes In subtropical
and tropical agriculture (Luc M, Slkora RA, and Bridge
J, eds). Walllngford, U.K.: CAB International; 237-283.
• Gall Index resistant ^35% of roots galled, susceptible >35% of roots galled.
tlble (129 expected of each; x2 = 0.04, P =
.86).
We propose that the single recessive
gene for resistance to M. javanica be designated mj. This is the first gene Identified
in cucumber that confers resistance to a
nematode (Pierce and Wehner 1990; Wehner 1993). The genotype for resistance in
LJ 90430 is therefore mj/mj. The simple nature of inheritance of resistance to M. javanica indicates that it could be incorporated easily into elite inbreds using backcross methods. Cucumber cultivars with
M. javanica resistance would benefit growers in the southeastern United States, as
well as in Texas, Arizona, and California,
because this nematode species is widely
distributed in those areas (Walters and
Barker 1994).
From the Department of Horticultural Science (Walters
and Wehner) and the Department of Plant Pathology
(Barker), North Carolina State University, Raleigh, NC
27695. The research reported In this article was funded
In part by the North Carolina Agricultural Research
Service and Pickle Packers International. The use of
trade names In this article does not Imply endorsement
by the NCARS of the products named, nor criticism of
similar ones not mentioned. This article was part of a
thesis submitted by the senior author In partial fulfillment of the requirements for a Ph.D. degree.
The Journal of Heredity 1997:88(1)
References
Amrltphale D, Dbdt S, and Singh B, 1993. Effect of acetone on the Induction and breakage of secondary dormancy In seeds of cucumber. J Exp Bot 44:1621-1626.
Barker KR, Townshend JL, Bird GW, Thomason LJ, and
Dlckson DW, 1986. Determining nematode population
responses to control agents. In: Methods for developing pesticides for control of plant pathogens (Hlckey
KD, ed). St. Paul, Minn.: APS Press; 283-287.
Byrd DW Jr, Ferris H, and Nusbaum a , 1972. A method
for estimating numbers of eggs of Meloidogyne spp. In
soil. J Nematol 4:266-269.
Hadisoeganda WW and Sasser JN, 1982. Resistance of
tomato, bean, southern pea, and garden pea cultivars
Table 4. Segregation of resistance to Meloidogyne javanica in F, families developed from selfpollination of resistant and susceptible F, plants from five crosses
Family
Addis X U 90430
Sumter X LJ 90430
Gy 14 X LJ 90430
Poliuett 87 x LJ 90430
Gy 57u x U 90430
Pooled
F,
parent
reaction
F, observed
R-
Sg*
S«
R'
S»
R
S
R
S
R
S
R
S
R
S
5
0
5
0
5
0
5
0
5
0
25
0
0
7
0
3
0
3
0
2
0
3
0
0
7
0
8
0
7
0
7
0
36
Fitted
3
0
14
(fcSgS)
AllR
0R:2Sg:l S
AUR
0R:2Sg:l S
All R
0 R:2 Sg:l S
AllR
0R:2Sg:l S
AllR
0R2Sg:lS
All R
0R:2Sg:l S
'All plants resistant.
•Some plants resistant, some susceptible.
'All plants susceptible.
' Fj families developed from resistant F, plants ( F ^ . Expect 5:0:0 (25:0:0 pooled).
• Fj families developed from susceptible F, plants ( F J . Expect 0:73 (033:17 pooled).
to root-knot nematodes based on host suitability. Plant
DIs 66:145-150.
Hartman KM and Sasser JN, 1985. Identification of Meloidogyne species on the basis of differential host test
and perineal-pattem morphology. In: Advanced treatise
on Meloidogyne, voL n. Methodology (Barker KR, Carter CC, and Sasser JN, eds). Raleigh, N.C.: North Carolina State University Graphics; 69-77.
X*
P
0.05
.84
0.05
—
0.80
—
.40
0.05
—
0.05
.84
—
M
0.64
.45
Pierce LK and Wehner TC, 1990. Review of genes and
linkage groups In cucumber. HortSclence 25:605-615.
St Amand PC and Wehner TC, 1991. Crop loss to 14
diseases In cucumber in North Carolina for 1983 to
1988. Cucurbit Genet Coop Rpt 14:15-17.
USDA (United States Department of Agriculture), 1993.
Agricultural statistics. Washington, D.C.: US. Government Printing Office.
Walters SA and Barker KR, 1994. Update on the distribution of five major Meloidogyne species In the United
States. Plant DIs 78:772-774.
Walters SA, Wehner TC, and Barker KR, 1993. Root-knot
nematode resistance In cucumber and homed cucumber. HortSclence 28:151-154.
Wehner TC, 1993. Gene list update for cucumber. Cucurbit Genet Coop Rpt 1652-97.
Weston LA, Geneve RL, and Staub JE, 1992. Seed dormancy in Cucumis satiuus var. hardwickii (Royle) Alef.
Scl Hort 50:35-46.
Whltaker TW and Davis GN, 1962. Cucurbits. London:
Leonard Hill.
Received July 21, 1995
Accepted May 24, 1996
Corresponding Editor James L Hamrick
Analysis of Flowering Time
in Ecotypes of Ambidopsis
thaliana
S. Sanda, M. John, and R.
Amasino
There exists variation in the timing of the
initiation of flowering among different ecotypes of Arabidopsis thaliana. We have
examined the basis of this variation between the early flowering Columbia (Col)
ecotype and the late flowering ecotypes
Coimbra (Co-4), Geneva (Ge-2), and Zurich (Zu-0). In crosses of Col to Co-4,
Ge-2, and Zu-0, the late flowering trait behaved as a single dominant gene: the F,
plants were late flowering and in segregating F2 populations a 3:1 ratio of late to
early flowering plants was observed. This
dominant gene resides in a region of chromosome 4 that contains a gene (FRIGIDA)
conferring late flowering in certain other
Arabidopsis ecotypes. AJIelism tests indicate that the same gene is responsible for
late flowering in Co-4, Ge-2, Zu-0, and the
Brief Communicalioru 6 9
San Feliu (Sf-2) ecotype. These and previous results indicate that FRIGIDA accounts for much of the later fbwering observed in various ecotypes of Arabidopsis.
(FRI) by Napp-Zinn (1985). Recent work
has shown that FRI resides in the same location on chromosome 4 as FLA and therefore these genes may be allelic (Clarke and
Dean 1994). The ecotypes Pitzal and Innsbruck also contain a dominant gene that
confers late flowering which is located in
this region (Burn et al. 1993). In this study
the late flowering behavior of three additional Arabidopsis ecotypes was analyzed.
The results demonstrate that late flowering was always associated with a locus in
the same region of chromosome 4. Allelism tests further indicate that in all of
these ecotypes late flowering is conferred
by the same gene.
The timing of the transition from vegetative to reproductive development is influenced by environmental cues in many
plant species. Two environmental variables that often influence flowering time
are photoperiod and temperature (VincePrue 1983). The ability to measure these
variables allows certain plant species to
initiate flowering at optimal times of the
growing period.
The flowering time of Arabidopsis thaliana is affected by both photoperiod and
temperature. The response to photoperiMaterials and Methods
od is quantitative; flowering Is promoted
by long days although most ecotypes of
Plant lines and Growth Conditions
Arabidopsis will eventually flower in non- Ecotypes Co-4, Ge-2, and Zu-0 were obinductive photoperiods (Napp-Zinn 1985). tained from the Arabidopsis Information
Flowering in Arabidopsis can also be pro- Service stock center (Frankfurt, Germamoted by prolonged exposure to low tem- ny). These lines originated from Coimbra,
peratures, a process known as vernaliza- Portugal (Co4); Geneva, Switzerland (Getion (Napp-Zinn 1985). Ecotypes of Arabi- 2); and Zurich, Switzerland (Zu-0). Seeds
dopsis commonly used for laboratory
were sown on 0.8% agar-solidified medium
studies, such as Columbia (Col) and
containing one-fourth of the recommendLandsberg erecta, flower rapidly under
ed level of minerals in Murashige-Skoog
long-day inductive photoperiods, and un- medium (Murashige and Skoog 1962). Afder these conditions vernalization has lit- ter imbibition for 24 h at room temperatle effect on flowering time (Koornneef et
ture, seeds were incubated at 4°C under 8
al. 1991; Lee and Amasino 1995). However,
h photoperiods of about 20 (i.M/m2/s of
if flowering Is delayed by induced muta- cool-white fluorescent light for 24 h to
tions in genes such as FCA or LUMINIDE- break dormancy or for longer periods for
PENDENS, vernalization can restore early vernalization studies, then cultured at
flowering (Koornneef et al. 1991; Lee et al.
25°C for an additional 5-7 days under
1993). Many ecotypes of Arabidopsis are
about 70 (j.M/m2/s fluorescent light prior
naturally late flowering under long days,
to transplanting. Seedlings were subseand flowering of these ecotypes is strongly
quently grown as previously described unpromoted by vernalization (Lee and Ama- der continuous light (Lee et al. 1993).
sino 1995; Napp-Zinn 1985).
The variation in flowering time among
DNA Analysis
late flowering Arabidopsis ecotypes has
DNA was extracted as described (Mibeen studied genetically by crossing late
chaels et al. 1994). The genotype at microflowering to early flowering ecotypes.
satellite loci was determined with primers
These studies generally show that late
from Research Genetics (Huntsville, Alaflowering is a dominant trait, but In cer- bama) as described by Bell and Ecker
tain crosses multiple genes can be respon- (1994) except that 26 thermal cycles were
sible for differences in flowering time (e.g.,
used to minimize secondary band formaHarer 1950; Karlovska 1974; Napp-Zinn
tion. RFLP analysis was performed as pre1985). The late flowering trait of the eco- viously described using "P-labeled rantypes San Fellu-2 (Sf-2) and Leiden be- dom-primed probes (Lee et al. 1993).
haves as a single dominant gene in crosses to the early flowering ecotype Col. This
gene was designated FLA and was located Results
near the end of the short arm of chromo- Three late flowering ecotypes of Arabidopsome 4 by restriction fragment length
sis were selected for genetic analysis of
polymorphism (RFLP) analysis (Lee et al.
flowering time: CoA, Ge-2, and Zu-0. All of
1993). A major determinant of late flower- these lines were relatively late flowering
ing in the ecotype Stockholm is a domi- as compared to the Col ecotype when
nant gene that was designated FRIGIDA grown in continuous light without vernal-
7 0 The Journal of Heredity 1997:88(1)
Table 1. Effect of vernalization on ro*ette leaf
number
Ecotype
C<v4
Ge-2
Zu-0
Col
Without
vernalization
With
vernalization
51.6 ±3.8
49.7 ± 5.3
84.1 ± 7.6
12.6 ± 1.1
125 ± 1.4
14.2 i 1.6
18.8 ± 2.1
11.6 ± 0.9
Plants were grown In continuous light except during
vernalization. For the vernalization treatment, imbibed
seeds were incubated at 4°C for 28 days In 8 h photoperiods. The values for each treatment represent an
average of 10 plants ± standard error.
ization (Table 1). However, vernalization
of imbibed seeds at 4°C for 4 weeks
caused much earlier flowering of the late
flowering ecotypes (Table 1). Vernalization had only a slight effect on the flowering time of Col.
To investigate the genetic basis for the
difference in flowering time, the flowering
behavior of crosses between the early
flowering ecotype Col and the late flowering ecotypes CoA, Ge-2, and Zu-0 was determined. All of the F, plants were as late
or later flowering than the late flowering
parental line (data not shown). In the F2
generation, resulting from self-pollination
of the F, plants, there were two distinct
classes of plants: early flowering plants
that had fewer than 18 leaves at the time
of flowering and late flowering plants that
had greater than 40 leaves (Figure 1). In
these F2 populations the ratio of late to
early flowering plants was 3:1; thus, the
late flowering trait segregated as a single
dominant gene (Table 2).
To determine whether the late flowering
locus in lines Co-4, Ge-2, and Zu-0 resides
in the same region of chromosome 4 that
had been previously shown to contain a
gene conferring late flowering in other ecotypes such as Sf-2 (Lee et al. 1993), the
genotypes of F2 plants in the populations
described in Table 2 were determined at
RFLP marker 6844 (Nam et al. 1989) and
microsatellite marker nga8 (Bell and Ecker
1994). In each segregating population, late
flowering was linked to these markers. Recombination percentages of 7.4% and
10.9% were observed between the late
flowering locus and 6844 and nga8, respectively. This places the late flowering locus
of these ecotypes in the same region as
previously described for FRI and FLA.
To determine the relationship among
the late flowering loci in Co4, Ge-2, Zu-0,
and Sf-2, a plant that was homozygous for
the late flowering locus from each of the
F2 populations described in Table 2 was
crossed to a line that contains the late
Figure 1. Representative late and early flowering plants from the F, populations of the late ecotypes crossed to Col. (A) Co-4 X Col late dowering F, plant, (B) Co-4 X Col
early flowering F, plant; (Q Ge-2 X Col late flowering F, plant; (D) Ge-2 X Col early flowering F, plant; (E) Zu-0 X Col late flowering F, plant; (F) Zu-0 X Col early flowering
F2 plant.
flowering locus from Sf-2 introgressed into
the Col genetic background [see Lee et al.
(1994) for a description of this line]. The
resulting F, plants were crossed to the early flowering Col ecotype. All of the progeny of these latter testcrosses were late
flowering (Table 3). The lack of any early
flowering recombinants in the testcrosses
indicates that late flowering is conferred
by the same gene in Sf-2, Co-4, Ge-2, and
Zu-0. It is possible that these genes are not
allelic, but the absence of recombinants
indicates that at a probability of 99% the
genes must be linked by fewer than 4 cm.
Discussion
The difference in flowering time between
the late flowering ecotypes Co4, Ge-2, and
Zu-0 and the early flowering ecotype Col
Table 2. Segregation data and cbl-sqnare
analyst! of late and early flowering In the F,
populations
Cross
Segregation
late*:early»
Co-1 X Col
Ge-2 x Col
Zu-0 X Col
143:42
117:39
103 30
0.521
0.000
0.424
• These plants formed greater than 40 rosette leaves.
•These plants formed fewer than 18 rosette leaves.
'X1 values for an expected ratio of 3 late:l early flowering plants.
results from a single dominant gene that
is present in the late flowering ecotypes
and that is allelic to a previously described locus conferring late flowering in
the Arabidopsis ecotype Sf-2 (Lee et al.
1993). Furthermore, late flowering in other
ecotypes (Stockholm, Pitzal, and Innsbruck) has been shown to be associated
with this region by RFLP analysis (Burn et
al. 1993; Clarke and Dean 1994), and allelism tests indicated that Zu-0 and the
Stockholm ecotype contain the same late
flowering locus (Napp-Zinn 1979). The inclusion of the Zu-0 ecotype in our experiments permits this work to be related to
previous studies. Thus, as previously discussed for the Sf-2 allele (Lee et al. 1994),
the late flowering genes from CoA, Ge-2
Table 3. Test of allellsm between the late
flowering loci In the Sf-2 ecotype and Co-4, Ge-2,
and Zu-0
Cross*
Late
flower- Early
Ing*
flowering
(Co-4 F, X Col FRI) F, X Col
(Ge-2 F, X Col FRI) F, X Col
(Zu-0) F, X Col FRI) F, X Col
285
298
234
0
0
0
• A late flowering F, plant from the crosses of Zu-0, Ge-2,
and Co-4 to Col was crossed to the Col strain containing FRI from Sf-2 [see Lee et al. (1994) for the derivation
of the Col line with FRLSI2] and the resulting F, was
crossed to Col.
• All plants flowered with greater than 30 leaves.
and Zu-0 will be referred to as alleles of
FRIG1DA (FRI).
/W-containing lines behave as winter
annuals which begin vegetative development in one growing season and then initiate flowering early in the subsequent
growing season (Laibach 1951; Napp-Zinn
1985; Ratcliffe 1961). Although recessive
mutations in genes such as FCA, FPA, FVE,
FY, and LD can result in a phenotype similar to that conferred by FRI (vernalizationreversible late flowering) (Martlnez-Zapater et al. 1994), mutations in these genes
have not been reported to occur in natural
populations. There are also examples of
other genes for which naturally occurring
alleles contribute to late flowering in certain crosses (e.g., Burn et al. 1993; Kowalski et al. 1994; Napp-Zinn 1979). However,
the delay of flowering due to these other
genes is not nearly as severe as that
caused by FRI. Recently it has been demonstrated that the Landsberg erecta allele
of FLC can suppress the late flowering effects of FRI (Koornneef et al. 1994; Lee et
al. 1994). FLC alleles that suppress FRI
have been found only In Landsberg and
the C24 strain of Arabidopsis (Sanda S, unpublished data); that is, FLC alleles that
permit the late flowering phenotype to be
expressed appear to be common. FRlXhus
appears to be a major determinant of flowering time variation and adaptation to spe-
Brief Communications 7 1
cific habitats in natural populations of Arabidopsis.
From the Department of Biochemistry, 420 Henry Midi,
University of Wisconsin, Madison Wl 53706-1569. This
work was supported by a grant from the U.S. Department of Agriculture to R.MA (95-37100-1614) and by
the College of Agricultural and life Sciences, University
of Wisconsin, Madison. We thank K. Omata, A. Masshardt and J. Pomerenlng for assistance with this work.
Address correspondence to R. Amaslno at the address
above.
The Journal of Heredity 1997*8(1)
References
Bell CJ and Ecker JR, 1994. Assignment of 30 mlcrosatelllte loci to the linkage map of Arabidopsis. Genomlcs
19:137-144.
Bum JE, Smyth DR, Peacock WJ, and Dennis ES, 1993.
Genes conferring late flowering In Arabidopsis thaliana
Genetlca 90:147-155.
Clarke JH and Dean C, 1994. Mapping FRI, a locus controlling flowering time and vernalization response In
Ambidopsis thaliana. Mol Gen Genet 242:81-89.
Harer L, 1950. Die Vererbung des Bluhalters fruher und
spater sommerelnjahriger Rassen von Arabidopsis thaliana (L) Heynh. Beltr Blol Pflanzen 28:1-35.
Karlovska V, 1974. Genotyplc control of the speed of
development In Arabidopsis thaliana (L.) Heynh. lines
obtained from natural populations Blol Plantarum 16:
107-117.
Koornneef M, Blankesti]n-de Vries H, Hanhart C, Soppe
W, and Peeters T, 1994. The phenotype of some late
flowering mutants Is enhanced by a locus on chromosome 5 that Is not effective In the Landsberg erecta
wild-type. Plant J 6:911-919.
Napp-ZInn K, 1979. On the genetlcal basis of vernalization requirement In Arabidopsis thaliana (L) Heynh.
In- La Physlologie de la Floraison (Champagnat P and
Jaques R, eds). Paris: Colloques Internatlonaux du Centre National de la Recherche Sdentlfique; 217-220.
Napp-ZInn K, 1985. Ambidopsis thaliana. In: CRC handbook of Dowering (Halevy AH, ed). Boca Raton, Florida:
CRC Press; 492-503.
Ratcllffe D, 1961. Adaptation to habitat in a group of
annual plants. J Ecol 49:187-203.
Vince-Pnie D, 1983. Photomorphogenesls and flowerIng. In: Encyclopedia of plant physiology, New Series:
Photomorphogenesls (Shropshire W and Mohr H, eds).
Berlin: Sprlnger-Verlag; 457^90.
Received August 7, 1995
Accepted March 11, 1996
Corresponding Editor Prem P. Jauhar
Molecular and Cytologlcal
Analysis of a Mariner
Transposon From
Hessian Fly
V. W. Russell and R. H. Shukle
Degenerate PCR primers for conserved
regions of the mariner transposase have
been shown to amplify DNA sequences
from the Hessian fly (Mayetiola destructor). Using one of these sequences as a
hybridization probe, a clone from an M.
destructor genomic library in phage lambKoomneef M, Hanhart CJ, and van der Veen JH, 1991.
A genetic and physiological analysis of late flowering
da was recovered and sequenced. A
mutants In Arabidopsis thaliana. Mol Gen Genet 229:57transposable element, Desmari, with per66.
fect inverted terminal repeats and an open
Kowalskl SP, Lan TH, Feldmann KA, and Paterson AH,
reading frame that encodes a mariner
1994. QTL mapping of naturally-occurring variation In
flowering time of Arabidopsis thaliana. Mol Gen Genet class transposase was found. When com245:548-555
pared to mariner sequences in the gene
Lalbach F, 1951. Uber sommer- und wlnterannuale Rasdatabase, the transposase proved to be
sen von Arabidopsis thaliana (L) Heynh. Eln Beltrag
similar to that of the active mariner Mos1
zur Atlologie der Blutenblldung. Beitr Blol Pflanzen 28:
73-210.
from the fruit fly (Drosophila mauritiana). In
situ hybridization of the transposon DNA
Lee I and Amaslno RM, 1995. Effect of vernalization,
photoperiod and light quality on the flowering phenosequence to salivary gland polytene chrotype of Arabidopsis plants containing the FRIGIDA gene.
mosomes revealed the general cytological
Plant Physlol 108.157-162.
locations of mariner elements. The distriLee I, Bleecker A, and Amaslno R, 1993. Analysis of naturally occurring late Dowering In Arabidopsis thaliana. bution of sequences with homology to the
Mol Gen Genet 237:171-176.
probe was predominantly, but not excluLee I, Michaels SD, Masshardt AS, and Amaslno RM, sively, in paracentromeric regions.
1994. The late flowering phenotype of FRIGIDA and LUMINIDEPENDENS Is suppressed in the Landsberg erecta
strain of Arabidopsis. Plant J 6:903-909.
Transposable elements are powerful reMartlnez-Zapater JM, Coupland G, Dean C, and Koornsearch tools for genetic manipulation and
neef M, 1994. The transition to flowering In Arabidopsis.
In: Arabidopsis (Meyerowltz E and SomerviUe CR, eds). investigation but such research has been
limited to only a few organisms. A search
Plalnvtew, New York: Cold Spring Harbor Laboratory
Press; 403-133.
for useful transposable elements has led
Michaels SD, John MJ, and Amaslno RM, 1994. Removal
to the discovery of numerous types of
of polysaccharides from plant DNA by ethanol precipthese elements across a wide phylogenic
itation. BloTechnlques 17:274-276.
range. One such element is mariner, a
Murashlge T and Skoog F, 1962. A revised medium for
short inverted repeat class, which transrapid growth and bioassay with tobacco tissue cultures. Physlol Plant 15:473-497.
poses via a DNA intermediate, and was
Nam H-G, Glraudat J, den Boer B, Moonan F, Loos WDB, first described in the fruit fly (Drosophila
Hauge BM, and Goodman HM, 1989. Restriction fragmauritiana) (Jacobson et al. 1986). The
ment length polymorphism linkage map of Arabidopsis
mariner element has since been detected
thaliana. Plant Cell 1:699-705.
7 2 The Journal of Heredity 1997.88(1)
in a wide variety of insects using PCR with
primers designed to conserved regions of
the transposase (Bigot et al. 1994; Robertson 1993; Robertson and MacLeod 1993).
Many of these mariner elements have been
shown to be defective, lacking a complete
transposase, and the role and specificity
of the Inverted terminal repeats is not
clear. To date only the mariner elements
from Drosophila have been shown to be
active, especially the Mosl mariner element from D. mauritiana, and have been
shown to be capable of transforming other
closely related Insects in the genus Drosophila (Maruyama et al. 1991). There is a
need to characterize other mariner elements in order to understand the potential of these elements for genetic studies.
We previously documented evidence of
mariner transposable elements from the
Hessian fly (Mayetiola destructor, family
Cecidomyiidae, order Diptera), a major
pest of wheat, using PCR to amplify fragments with degenerate primers designed
to conserved regions of the mariner transposase. One of these elements, Desmarl,
has now been sequenced and appears to
be in the subfamily of mariner elements
that was first characterized in D. mauritiana. This sequence, representing an intact
mariner element with perfect inverted repeats, and its Insertion site are presented
here. The distribution of this sequence in
the genome is described using in situ hybridization to salivary polytene chromosomes.
Materials and Methods
Experimental Insect
Hessian flies (white-eyed line; Shukle and
Stuart 1993) were reared on wheat cultivar
Blueboy (contains no known resistance
genes) in a growth chamber at 20°C with
a 16 h photoperiod (illumination 360 (xEin/
mVs).
Construction and Screening of Library
Hessian fly genomic DNA was prepared according to the method of Us et al. (1983).
A phage lambda library was constructed
from a partial Saul digest of Hessian fly
genomic DNA using the lambda BlueStar
Xhol half-site cloning system from Novagen (Madison, Wisconsin).
Desl, a 345 bp PCR product representing a conserved sequence of a mariner
transposase (Shukle and Russell 1995)
was labeled with [32P]dCTP (sp.act. 3,000
Ci/mmol; Amersham, Arlington Heights, Illinois) by ollgonucleotide random priming
reaction (Feinberg and Vogelstein 1983)