Phenotypic Instability and Rapid Gene Silencing in Newly Formed … · 1997); novel homeotic phenotypes in Digitalis (Schwanitz, 1957), Gilia (Grant, 1956), and cotton (Meyer, 1970);

The Plant Cell, Vol. 12, 1551–1567, September 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

Phenotypic Instability and Rapid Gene Silencing in Newly Formed Arabidopsis Allotetraploids

Luca Comai,

a,2

Anand P. Tyagi,

b,1

Ken Winter,

a

Rachel Holmes-Davis,

a

Steven H. Reynolds,

a

Yvonne Stevens,

a

and Breck Byers

b

a

Department of Botany, Box 355325, University of Washington, Seattle, Washington 98195

b

Department of Genetics, Box 357360, University of Washington, Seattle, Washington 98195

Allopolyploid hybridization serves as a major pathway for plant evolution, but in its early stages it is associated withphenotypic and genomic instabilities that are poorly understood. We have investigated allopolyploidization between

Ar-

abidopsis thaliana

(2

n

5

2

x

5

10;

n

, gametic chromosome number;

x

, haploid chromosome number) and

Cardaminop-sis arenosa

(2

n

5

4

x

5

32). The variable phenotype of the allotetraploids could not be explained by cytologicalabnormalities. However, we found suppression of 20 of the 700 genes examined by amplified fragment length polymor-phism of cDNA. Independent reverse transcription–polymerase chain reaction analyses of 10 of these 20 genes con-firmed silencing in three of them, suggesting that

z

0.4% of the genes in the allotetraploids are silenced. These threesilenced genes were characterized. One, called K7, is repeated and similar to transposons. Another is

RAP2.1

, a mem-ber of the large

APETALA2 (AP2)

gene family, and has a repeated element upstream of its 5

9

end. The last, L6, is an un-known gene close to

ALCOHOL DEHYDROGENASE

on chromosome 1. CNG DNA methylation of K7 was less in theallotetraploids than in the parents, and the element varied in copy number. That K7 could be reactivated suggests epi-genetic regulation. L6 was methylated in the

C. arenosa

genome. The present evidence that gene silencing accompa-nies allopolyploidization opens new avenues to this area of research.

INTRODUCTION

Allopolyploids are hybrids whose genomes contain a com-plete diploid set of chromosomes from each parental spe-cies. Both parental genomes are maintained with littlechanges through successive generations by limiting meioticpairing to homologous chromosomes (i.e., from the samegenome, in contrast to homeologous, from different ge-nomes). Although many wild and cultivated allopolyploidsthat originated in prehistoric times are fertile, well adapted,and genetically stable, allopolyploids of more recent origincommonly display genomic and phenotypic instability (Popeand Love, 1952; Allard, 1960; Gupta and Reddy, 1991). Ge-nomic instability has been shown to involve changes inchromosome structure, including the appearance (Burnsand Gerstel, 1967) or disappearance (May and Apples,1980) of heterochromatic blocks, the loss of nucleolar orga-nizing regions (Vaughan et al., 1993), rearrangements of re-peated DNA (Kamm et al., 1995; Zhao et al., 1998), andfrequent changes in restriction fragment length polymor-phism patterns (Song et al., 1995). Phenotypic variability ofsynthetic allotetraploids has been shown to involve numer-

ous abnormalities, including sterility (Leitch and Bennett,1997); novel homeotic phenotypes in Digitalis (Schwanitz,1957), Gilia (Grant, 1956), and cotton (Meyer, 1970); flowervariegation in Nicotiana (Gerstel and Burns, 1966); and theglobal dominance of one parental phenotype (Heslop-Harrison,1990).

The causes of these extreme phenotypes are largely un-known. McClintock (1984) invoked the concept of genomicshock, which she defined as a preprogrammed response toan unusual challenge that led to extensive restructuring ofthe genome. A possible contributor to the “unusual chal-lenge” is epigenetic gene silencing, which is triggered byhomologous gene–gene interactions (Meyer and Saedler,1996; Matzke and Matzke, 1998). The sudden union of re-dundant and diverged homeologous sets of genes in allo-polyploids could trigger widespread gene silencing (Leitch andBennett, 1997; Henikoff and Comai, 1998b; Rieseberg andNoyes, 1998) with accompanying changes in chromatinstructure and DNA methylation (Henikoff and Matzke, 1997).Whatever its cause, the greater instability of syntheticallopolyploids than of established allopolyploids suggeststhat the latter have evolved mechanisms to gain fertilityand stabilize their phenotypes and genomes (Sears, 1976;Eckenwalder and Brown, 1986; Gupta and Reddy, 1991).The importance of allopolyploid hybridization to basic andapplied botany makes this area worthy of further investigation.

1

Current address: Department of Biology, University of the SouthPacific, P.O. Box 1168, Suva, Fiji.

2

To whom correspondence should be addressed. E-mail [email protected]; fax 206-685-1728.

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ic.oup.com/plcell/article/12/9/1551/6009349 by guest on 03 August 2021

1552 The Plant Cell

The model plant

Arabidopsis thaliana

should prove valuablefor a detailed analysis of allopolyploidy. This species is thematernal parent of the allotetraploid

A. suecica

, a selfing spe-cies native to northern Europe. The parental origin of the

A.suecica

chromosomes was demonstrated by DNA sequenceanalyses (Price et al., 1994; Kamm et al., 1995; O’Kane et al.,1996) and by in situ hybridization to the repeat sequences ofeither

A. thaliana

or

Cardaminopsis arenosa,

which mark 10and 13 centromeres, respectively (Kamm et al., 1995). The 26chromosomes of

A. suecica

(2

n

5

26, set A

9

A

9

C

9

C

9

;

n

, ga-metic chromosome number;

x

, haploid chromosome number)make up the two sets depicted in Figure 1A: 10 from the dip-loid selfing species

A. thaliana

(2

n

5

2

x

5

10, set AA) and 16from the tetraploid outcrossing species

C. arenosa

(2

n

5

4

x

5

32, set CCCC). Although very closely related in some as-pects, these two taxa nonetheless exhibit 5 to 8% divergenceof nucleotide sequence in protein-coding genes (Hanfstingl etal., 1994; Henikoff and Comai, 1998a) and 30 to 40% diver-gence in the 180-bp centromeric repeats (Martinez-Zapater etal., 1986; Vongs et al., 1993; Round et al., 1997).

The availability of allopolyploids of Arabidopsis could helpus address genetic and evolutionary aspects of allopoly-ploidy. However, the utility of

A. suecica

for this purpose islimited by the probable genotypic divergence of any con-temporary stocks from plants that were parental to the al-lopolyploidization. Nonetheless, synthetic allotetraploidsgenerated from our current stocks should approximate theancestral hybridization and permit precise comparison ofparental and allotetraploid phenotypes as well as provide aperspective on what changes may occur early in allotetra-ploid evolution.

Our characterization of synthetic allotetraploids between

A. thaliana

and

C. arenosa

indicates that they are indeedphenotypically unstable and less fit than the parents andthat this instability cannot be explained by the meiotic be-havior of the allotetraploids. Interestingly, analysis of the ex-

pression of random genes demonstrated gene silencing inthe allotetraploids. The characterization of these silencedgenes revealed a role for epigenetic regulation and repeatedsequences in silencing.

RESULTS

Hybridization and Seed Development

To reconstruct an Arabidopsis allotetraploid similar to

A.suecica

, we performed interspecific crosses in both direc-tions between diploid or tetraploid

A. thaliana

Landsberg

erecta

(L

er

)

and

C. arenosa

Care-1 (all

C. arenosa

isolatesare tetraploid). The schematic karyotypes are represented inFigure 1. On cross-pollination of

C. arenosa

, most siliquesfailed to develop. The seeds of cross-pollinated diploid

A.thaliana

plants enlarged at the normal rate for 10 days, butthe embryos within these seeds became arrested at theglobular stage; subsequently, the seeds turned brown andcollapsed (Table 1). However, when we cross-pollinated atetraploid

A. thaliana

, the seeds contained embryos at vari-ous more advanced stages of development, and althoughthe majority of the seeds turned brown and collapsed, asmall fraction matured. The development of the embryos inthese crosses was scored at silique maturity and assignedthe following stages: 70% globular, 7% heart to torpedo,6% walking stick, 10% mature (including normal and mal-formed), and 7% viviparous. Approximately half of the ma-tured seeds produced hybrid plants. The other half of thematured seeds, as well as a few rare seeds obtained bycross-pollination of

C. arenosa

, produced plants that resem-bled the maternal parent and lacked the paternal genome,as determined by random amplified polymorphic DNA(RAPD) analysis (data not shown). Clearly, hybridization be-tween these species is severely challenged but can succeedwhen parental genomic ratios are balanced.

F

1

Phenotype

A. thaliana

and

C. arenosa

differ in overall size (small versuslarge), leaf shape (smooth versus serrated), mating habit(selfing versus outcrossing), and flower size, shape, andpetal color. Various phenotypic features of

A. thaliana

arecompatible with a selfing habit, whereas the outcrossinghabit of

C. arenosa

depends on both morphological featuresand self-incompatibility. The rosette and cauline leaves ofthe hybrid plants resembled those of

C. arenosa

and

A.suecica

in serration and size (Table 2). Although several ofthe hybrids died for unknown reasons before flowering, fourhybrids (of

z

300 original interspecific zygotes) flowered andset seed, establishing hybrid lines. Replicated crosses havedemonstrated the reproducibility of the above method forgenerating allotetraploid lines (data not shown).

Figure 1. Genomic Structure of Parents and the Outcome ofCrosses.

Shown are schematic karyotypes in which each dot represents achromosome. A. t, A. thaliana; C. a., C. arenosa.

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Gene Silencing in Allopolyploids 1553

Figure 2 illustrates the hybrid phenotype: three of the fourhybrids (605A, 605B, and 49-2B) had flowers that were in-termediate in size and color to those of the parents (Figure2H). In addition, the flowers were scented, had well-devel-oped nectaries, and were slightly zygomorphic. Althoughpollen production was poor, manual pollination resulted instrong carpel elongation. Microscopic examination of theovules within these carpels revealed developing embryosand endosperm, indicating self-compatibility and good fer-tilization. Nevertheless, many embryos then arrested at vari-ous developmental stages, and only

z

20% of the seedswere viable (Figure 2D). In most cases, as shown in Table 3,cross-pollination with pollen from

A. thaliana

(either diploidor tetraploid),

C. arenosa,

or

A. suecica

led to better seed

set than did selfing. The hybrids resembled

C. arenosa

and

A. suecica

in their perennial growth habit. Whereas

A.thaliana

undergoes senescence and dies after a majority ofsiliques have matured,

C. arenosa

,

A. suecica,

and thesethree hybrids displayed abundant new basal growth andproduced many flowers.

The fourth hybrid, 49-2A, behaved differently. Although itformed a rosette resembling those of the other hybrids, itgrew more slowly and flowered later. Its inflorescence re-sembled that of

A. thaliana

, the cauline leaves were smooth-margined instead of serrated, and the flowers were smallerand had shorter petals. Although these flowers were effi-cient at self-pollination, the seeds produced were mostlyshrunken and inviable. Molecular marker analysis, in addi-tion to the progeny phenotype and karyotype (see below),confirmed the hybrid nature of this plant, which died afterforming

,

30 flowers.

Molecular Markers

We confirmed the genomic constitution of the hybrids, theirparents, and

A. suecica

Sue-2 by DNA fingerprinting withRAPD (Williams et al., 1990) and microsatellite (Bell andEcker, 1994) markers (http://faculty.washington.edu/comai/molmarks.htm). These assays suggested a considerableheterozygosity in the

C. arenosa

genome. For example,analysis of the inheritance of the chromosome 1 microsatel-lite

nga111

in the F

2

plants derived from the dimorphic F

1

Table 1.

Production of Viable Hybrid Seed by Different Crosses

Cross

Mother x

a

Father x Hybrid Seed % Stage of Failure

b

A. t.

c

2 C. a.

d

4 0 GlobularC. a. 4 A. t. 2 0 Pre-globularA. t. 4 C. a. 4 5 Various (see Results)C. a. 4 A. t. 4 0 Pre-globular

a

Monoploid (haploid) number.

b

Stage of embryo in failed seed.

c

A. t.,

A. thaliana

.

d

C. a.,

C. arenosa

.

Table 2.

Characteristics of Parents and F

1

Hybrids

Line or Plant

Character

A. thaliana

LC612 Care-1 Sue-2 49-2A 49-2B 605A 605B

n

(no. of gamete chromosomes) 5 16 13 13

a

13 13 13Leaf length (cm) 3.8

6

0.3 9.7

6

0.8 8.8

6

1 ND

b

8.8

6

0.6 11

6

0.8 9.5

6

1.4Leaf serration

c

2 11 11 1

to

2

d

11 11 1

Weeks to bolting 3 5 6 20 5 6 5Secondary rosettes

e

No Yes Yes No Yes Yes YesPetal length (mm) 3.7

6

0.04 7.1

6

0.04 6 6 0.05 3 6 0.03 5.2 6 0.04 5.8 6 0.05 5.2 6 0.04Corolla diameter (mm) 3.3 6 0.03 9.7 6 0.07 6.1 6 0.07 ND 6.2 6 0.03 7.2 6 0.03 6 6 0.03Anther orientationf In Out In In Out Out OutSelf-compatibility Yes No Yes Yes Yes Yes YesPolleng ND/ND/92 100/94/98 86/88/81 ND 29/17/13 76/24/6 NDSeed set Excellent Very good Excellent Poor Poor Poor Poor

a Chromosomes counts were performed in F2 progeny.b ND, not determined.c (2), none; (1), moderate; (11), strong.d Due to “reversion” to an A. thaliana–like phenotype in the inflorescence (see text).e Rosettes formed in cauline leaf axils.f Anthers dehisced toward (in) or away from (out) stigma.g Percentage of viable pollen at three stages (Regan and Moffatt, 1990): after meiosis (stage 10), during maturation (stage 11), and at dehiscence(stage 13).

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1554 The Plant Cell

Figure 2. Morphology and Phenotype of Synthetic Hybrids.

(A) A. thaliana LC612 (left) and C. arenosa Care-1 (right).(B) F2 allotetraploids.(C) to (F) Seed from A. thaliana LC612, an F1 allotetraploid, C. arenosa Care-1, and Arabidopsis suecica Sue-2, respectively. Embryo mortalitywas predominant in the synthetic hybrid, as shown by the shrunken seeds.(G) Rosette leaves of A. thaliana (far left), C. arenosa Care-1 (far right), and F2 allotetraploids (center). The latter display the range of variation.(H) to (N) Flowers of A. thaliana LC612 (H), an F1 allotetraploid (L), C. arenosa Care-1 (K), A. suecica Sue-2 (J), and three F2 allotetraploids: (I),(M), and (N). Many traits, including rosette leaf and flower morphology, were highly variable in the F2 individuals of the synthetic hybrids.The pot size in (A) and (B) is 10 cm. The flower in (H) is 4 mm in diameter, and all flowers are to the same scale.

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hybrid 49-2B and the trimorphic hybrid 605B showed that allF2’s had the A. thaliana allele (162 bp) but differed in the alle-les inherited from C. arenosa. The 49-2B progeny had a sin-gle 116-bp form, whereas the 605B progeny showedsegregation of the 116 and 132 forms (data not shown here,but see http://faculty.washington.edu/comai/nga111.htm).Of 44 individuals scored, 21 were homozygous for the 116allele, four were homozygous for the 132 allele, and 19 wereheterozygous. Using the x2 test, the probability of the null hy-pothesis for a 1:2:1 ratio is P 5 0.0009; therefore, the resultsfor F2 scoring indicate strong segregation distortion. Segre-gation distortion in hybrids has been attributed to the inter-action of incompatible genic systems such as those resultingin gametocidal action (Endo, 1990).

Cytology

We examined the karyotype of pollen mother cells from hy-brid and parental plants to establish their expected chromo-some number and to assess whether they displayed normalmeiosis. Figure 3 provides examples of these analyses forhybrid 605B, and Figure 4 provides examples of parentalkaryotypes and other hybrid karyotypes. We anticipated thatthe hybrids would have 26 chromosomes—the 10 chromo-somes from A. thaliana and 16 from C. arenosa. Indeed, weobserved this number in all three of the F1 hybrids that wereavailable for analysis, as well as in the F2’s of hybrid 49-2A,whose F1 parent had died prematurely. Thirteen bivalentswere visible at metaphase I (e.g., Figure 3A), and 13 chro-mosomes (chromatid pairs) could be seen in the metaphaseII plates (Figures 3F and 3G). Thus, the allotetraploids hadthe expected chromosome numbers.

Homeologous pairing and recombination in the hybridsbetween A. thaliana and C. arenosa might have been ex-pected to result in the formation of multivalents and uni-valents during meiosis I and, if chromosomal inversionsdifferentiated the A. thaliana and C. arenosa genomes, in theformation of anaphase bridges. Indeed, some of the pollenmother cells displayed meiotic abnormalities such as lag-gard chromosomes and chromosomal bridges. Although thesmall chromosome size made it difficult to count pairedchromosomes in many cells, all genotypes (except diploidA. thaliana; Figures 4A to 4D) clearly formed some multi-valents, which were evident as figures of greater complexity(Figures 3B, 3C, 4G, and 4I) than were the normal rod andring bivalents (Figures 3A, 4A to 4C, and 4J). In addition, theabnormal segregations seen as bridges and lagging chro-mosomes were observed in anaphase cells of both C.arenosa (Figure 4L) and A. suecica (Figures 4N and 4O), aswell as in those of the synthetic hybrids. The incidence ofmeiotic abnormalities per observed meiosis was 5 to 15%for the synthetic hybrids, 10% for one C. arenosa plant and0% for a second one, and 2 to 25% for A. suecica (seeMethods for details). In conclusion, meiotic abnormalitieswere evident in the hybrid but were not demonstrably more

extreme than those seen in the C. arenosa parent or in thenatural allotetraploid.

Phenotypic Variation and Instability among Allotetraploid F2 Progeny

The allotetraploid F2’s displayed considerable variation inmorphology, flowering time, and fertility—in contrast to theuniform phenotypes of Sue-1, Sue-2, and several A. thalianaecotypes grown in the same environment. Leaf and flowerphenotypes are illustrated in Figure 2, and the variation inseveral characters is indicated in Table 4. These striking pat-terns of variation could be caused by segregation of alleleswithin the C. arenosa genome, which is highly heterozygous.

In addition to phenotypic variation, phenotypic instabilitywas observed in several instances: First, the phenotype ofall 12 of the 49-2A F2 plants grown was distinctly allotetra-ploid (having large pink flowers and serrated leaves), eventhough the plants were derived from seeds produced byflowers similar to those of A. thaliana. In the F3 and F4 gener-ations, the allotetraploid phenotype was inherited exclu-sively. The phenotype of the F1 and its progeny cannot bereadily explained by Mendelian genetics. Second, reversionto an A. thaliana–like phenotype was again observed in theF2 of 605B, as exemplified in Figure 5. The switch to the Ar-abidopsis parental phenotype affected the first few flowersof basal lateral inflorescences. The subsequent flowers pro-duced by this individual reverted progressively to the allotet-raploid phenotype (Figures 5A to 5D). Eight of 13 F2

individuals in this family displayed dimorphic flowers: normalallotetraploid flowers and flowers with shorter petals. Third,variegated epidermal characters were observed, such as thestem anthocyanin shown in Figure 5E. Fourth, in .50% ofthe F2’s, the first one to three flower pedicels were aberrant;some formed a bifurcation (Figures 5F and 5G), whereasothers were sharply basipetal rather than orthogonal liketheir parents (Figures 5I and 5J). We hypothesize that thesestructures resulted from partial arrest of the shoot apicalmeristem, followed by switching the main growth axis to alateral shoot and converting the original shoot apical mer-istem to a lateral floral shoot.

Table 3. Embryonic Lethality in Two Hybrid F2’s and Crosses

Maternal Parent

Cross 605B 49-2B

Selfed 80a 803 C. arenosa 30 313 A. thaliana tetraploid 37 13 A. thaliana diploid 99 373 A. suecica 22 35

a % lethality.

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1556 The Plant Cell

Figure 3. Karyotypic Analyses of Meiosis in a Synthetic Hybrid.

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Gene Silencing and Reactivation in the Allotetraploids

In the absence of any evidence that the allotetraploids wereunusually unstable in their karyotypes, we sought an expla-nation for their phenotypic instability in gene silencing orectopic gene expression. To examine this possibility, wecompared gene expression in the allotetraploids with thatin the parents. Because changes in ploidy have been asso-ciated with changes in gene expression (Guo et al., 1996;Mittelsten Scheid et al., 1996), we undertook these compar-isons in lines of the same ploidy, the parents being autotet-raploid and the progeny allotetraploid. Using these strains,we first screened for differences in gene expression bymeans of amplified fragment length polymorphism (AFLP)analysis (Bachem et al., 1996), a polymerase chain reaction(PCR)–based method that displays random restriction en-zyme fragments of cDNA (heretofore referred as “cDNAs”)on denaturing polyacrylamide gels. Analysis of z700 cDNAsderived from leaf or flower mRNA (see Methods) revealedfrequent differences between F2 allotetraploids and theparents, as shown in Figure 6. Specific cDNAs varied in in-tensity between genotypes and, interestingly, were undetec-table from some genotypes (Figure 6A). In several cases,cDNAs that could be detected in the parents were absent incertain allotetraploids. On the other hand, in two cases, cDNAswere present only in the allotetraploids. Because of their rel-ative abundance, we focused on those cDNAs that were ab-sent in the allotetraploids. We identified nine ArabidopsiscDNAs that were absent in all tested allotetraploids andeight that were absent in a subset of the allotetraploids.Similarly, eight Cardaminopsis cDNAs were absent in all testedallotetraploids, whereas z10% of Cardaminopsis cDNAswere absent in some but not all of the allotetraploids. Be-cause heterozygosities are common within the C. arenosagenome because of its outcrossing habit (see “MolecularMarkers”), absence from the allotetraploids of cDNAs spe-cific to C. arenosa might reflect the fact that a putativelypolymorphic allele was not inherited. Therefore, we chose ascandidates for silenced alleles only those eight cDNAs thatwere absent in all tested allotetraploids.

To verify the reliability of the AFLP-cDNA analysis and es-tablish whether a silenced gene was present, we clonedDNAs from 10 differential gel bands, sequenced them, anddesigned primers (18 to 22 nucleotides long) specific foreach gene. Using these primers to conduct reverse tran-scription (RT)–PCR, we tested the expression of each candi-

date gene in new RNA preparations, both from theallotetraploids that were used in the original AFLP-cDNAanalysis (Figure 6C, individuals 1 and 12) and from the 10 F2

siblings of each allotetraploid, and from selected F3 progeny(Figures 6B and 6C). We confirmed that three of the candi-date genes, designated K7, K9, and L6, were silenced; atthe same time, we confirmed the presence of the relevantgenes by PCR of genomic DNA. Expression of gene K7 wasdetected in C. arenosa but was absent in the two allotetra-ploids (Figure 6). It was also absent in 10 of 10 siblings of in-dividual 12 and in eight of 10 siblings of individual 1. Itreappeared in one of eight F3 progeny. This positive F3 indi-vidual was produced by individual 3, a nonexpressor (Fig-ures 6B and 6C), thus indicating reactivation of this element.

We also confirmed the silencing of two other genesamong those that had provisionally been identified by AFLP-cDNA analysis as candidates for silencing. Both genes weresilenced in a single allotetraploid individual but remained ac-tive in its siblings (Figure 6A; data not shown). Several othercloned cDNAs that seemed likely to represent silencedgenes on the basis of the AFLP-cDNA analysis were shownby RT-PCR to be positive for expression. The differential be-havior of these cDNAs during the AFLP-cDNA analysiscould have had any of several causes. For example, thesecDNAs could be subject to undetermined PCR artifacts, or ifthese cDNAs were derived from C. arenosa, they could rep-resent alleles that were polymorphic in one of the tworestriction sites defining each AFLP-cDNA product. Alterna-tively, they could represent minor background cDNAs thatwere coelectrophoresed and coeluted from the gel bandwith a cDNA that showed differential activity. Finally, theycould represent genes that were only partially silenced butretained sufficient activity to score as positive under ourRT-PCR conditions. Although seven of these isolates failedto pass the RT-PCR test for silencing, three of the original10 cDNAs were confirmed to represent bona fide silencedgenes in the allotetraploids. Given that these three wereidentified from z700 that were sampled, we estimate that atleast z0.4% of the genes may be silenced in allotetraploids.

Characterization of the Silenced Loci

The cloned differential AFLP-cDNAs were derived fromgenes of either C. arenosa (K7 and L6) or A. thaliana (K9).The Arabidopsis genome should contain the K7 and L6

Figure 3. (continued).

Meiotic figures in pollen mother cells of the F1 hybrid 605B. Those in (A), (E) right, (F), (G), and (H) left are apparently normal; those in (B) to (D),(E) left, (H) right, and (I) are abnormal. Thirteen chromosomes are visible (some overlapping) within the metaphase II plates ([F] to [H]). A three-pointed multivalent chromosomal association is visible in the center of the cell in (B). The meiotic phase is indicated in the upper right corner ofeach figure (A, anaphase; M, metaphase; I and II, meiotic division numbers; MI/AI, transition between phases). The arrows indicate laggards, andthe arrowhead indicates a chromosomal bridge. The bar in (A) 5 5 mm for (A) to (I).

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1558 The Plant Cell

homeologous genes (expected to have 92 to 95% identity)and the K9 gene. We investigated the nature of the loci en-coding these transcripts by a combined approach that in-cluded similarity searches of DNA databases, cloning of theDNA flanking the AFLP-cDNAs, and gel blot analysis. Ourresults are summarized in Figure 7.

We searched the database of sequenced A. thaliana DNAby BLASTN (Altschul et al., 1990) analysis with the K7 se-quence but detected no match of the expected homeology,indicating that this DNA element is absent from the se-quenced genome database. We found a match of low butsignificant similarity between K7 and the long terminal re-peat (LTR) of a copia-like retrotransposon found in bacterialartificial chromosome F28H18 (nucleotides 49,801 to 56,100,E 5 4.5E24), which we named Brewmeister1. Brewmeister1is probably inactive, having several lesions in its coding re-gion. K7 was even more similar to “solo” copies of this LTR(found at loci F14P22 and T3E15) that occur within genesputatively encoding products related to those of DNA trans-posons. Thus, K7 could be expressed from a gene related toeither a retroelement or a DNA transposon. To distinguishbetween these and other possibilities, we isolated DNAflanking the K7 sequence from the genomes of A. thalianaand C. arenosa by inverse PCR (IPCR) (Ochman et al.,1988). Regardless of whether the template genomic DNAwas undigested (and unligated) or had been digested withany of several restriction enzymes before ligation, the IPCRproducts were of similar size (2.2 kb), suggesting that circu-larization of a restriction fragment carrying K7 was not nec-essary for successful PCR with diverging IPCR primers. Weassumed, therefore, that tandem repeats containing K7were present in both parental genomes. The K7-flanking se-quences isolated from both species were similar and de-fined several repetitive DNA elements. Their ensemble isdefined here as the K7 repeat. To determine whether the K7repeat is a gene, we subjected its sequence to BLASTX andgene prediction analysis (see Methods). The results indicatethat a gene or pseudogene (Figure 7, G7) spans the originalK7 AFLP-cDNA. This putative gene encodes a protein re-lated to the transposon En/Spm hypothetical protein 1(pirS29329; E 5 0.083). Similar proteins are encoded by theF14P22 and T3E15 loci. Additional repetitive elements arefound 59 and 39 of the putative gene (Figure 7, U7, D7, andGnomo) but do not occur in loci F14P22 and T3E15. In-stead, they are found separately and individually in the A.thaliana genome (e.g., in the A. thaliana genome, U7

Figure 4. Karyotypic Analyses of Meiosis.

Apparently normal ([A] to [D], [F], [H], [J], [K], and [M]) and abnor-mal ([E], [G], [I], [L], and [N] to [Q]) meiotic figures of pollen mothercells from diploid A. thaliana ([A] to [D]) and C. arenosa ([F], [G], [K]

to [M]), from tetraploid A. thaliana ([H] and [I]), from F1 hybrids 49-2B([E], [P], and [Q]) and 605A (J), and from A. suecica ([N] and [O]).The meiotic phase is indicated in the upper right corner of each figure(A, anaphase; M, metaphase; T, telophase; I and II, meiotic divisionnumbers. The arrows indicate laggards or a micronucleus formedaround laggards (E), and the arrowheads indicate chromosomalbridges. The bars in (A) to (Q) 5 5 mm.

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matched LERJF13TF; Gnomo matched 77 elements), andthe element called Gnomo is found in the genomes of othertaxa such as mulberry and Brassica juncea.

Figure 8 shows gel blot analysis of genomic DNA probedwith the K7 cDNA (Figure 8A) or with the K7 repeat (Figures8C and 8D; see legend). Sequences showing high similarityto the K7 repeat are present in two to five copies in the A.thaliana genome but in higher copy numbers (20 to 50) in theC. arenosa genome. This indicates that many copies havebeen gained by C. arenosa or eliminated by A. thaliana,given that these two species diverged from a common an-cestor. Surprisingly, K7 elements are present in variablecopy numbers in the allotetraploids. For example, two F3 al-lotetraploids displayed a higher copy number than the C.arenosa parent (Figures 8A and 8B). Most copies were re-fractory to HpaII digestion but were partially digested byMspI, indicating heavy CG methylation and partial CNG me-thylation. In the allotetraploids, the K7-related elementswere more susceptible to MspI digestion than were those inC. arenosa, implying less extensive CNG methylation of theK7 elements. Taken together, these data indicate that theK7 gene is related to transposable elements and that it maybe heterochromatic in both parental genomes.

K9, one of the other two AFLP-cDNAs, is transcribed fromthe A. thaliana genome and matches a partial cDNA encod-ing the RAP2.1 protein of the APETALA2 (AP2) family(Okamuro et al., 1997). Approximately 10 genes in the Arabi-dopsis DNA database shared 50 to 75% identity with someportion of the RAP2.1 cDNA. The genomic regions flankingRAP2.1, which we isolated by IPCR as a 5-kb fragment,were not represented in the database. An ATG codon ispresent a few codons upstream of the available cDNA entry.Further upstream and downstream of the RAP2.1 cDNAsequence are regions that were assigned low coding proba-bility by the gene prediction program NetPlantGene2(Hebsgaard et al., 1996). Approximately 0.7 kb upstream ofthe putative RAP2.1 start codon, an open reading frame di-verges from RAP2.1 and may represent a different gene,here called G9. G9 is similar to z100 A. thaliana sequences

(65 to 90% identity) and is therefore repeated (some G9 re-peats have 8-bp inverted repeats, suggesting that they aremobile elements; B. Belknap, personal communication). Todetermine whether mutations in the promoter region couldhave caused the silencing, we sequenced 800 bp of theRAP2.1 59 region, which presumably includes the promoter,from both the parent A. thaliana and the silenced allotetrap-loid and found no changes. Hybridization of the sequencesof RAP2.1 isolated by IPCR to A. thaliana DNA confirmedthe existence of closely related repeated sequences in theA. thaliana genome (Figure 8G) that were at least partiallymethylated at CG sites (data not shown). The K9 flanking re-gions did not hybridize to the C. arenosa DNA (Figure 8G),which suggests that the regions were absent or that consid-erable divergence had occurred.

The last AFLP-cDNA, L6, is from C. arenosa and displayshigh similarity to a sequenced region of the A. thaliana ge-nome adjacent to ALCOHOL DEHYDROGENASE 1 on chro-mosome 1. This relationship was confirmed by analysis ofthe flanking DNA isolated by IPCR from both A. thaliana andC. arenosa, which verified that L6 is the transcription prod-uct of the C. arenosa homeolog. A small gene, F22K20.6, ispredicted around nucleotide 39,000. Both F22K20.6 and L6are moderately well represented in the database of expressedsequence tags and could be part of the same transcriptionalunit. The L6/F22K20.6 region has limited similarity to threegenomic DNAs in the Arabidopsis database. When severalrandomly chosen A. thaliana genes were tested by BLASTNanalysis, they showed database matches of comparablesimilarity and frequency (L. Comai, unpublished observa-tions). Therefore, unlike K7 and K9, the L6 locus does not showan unusual association with repeated DNA. Gel blot analysisof genomic DNAs probed with L6 revealed an unexpectedHindIII digestion pattern (Figure 8F): a 0.9-kb digestion prod-uct of A. thaliana DNA was absent in the allotetraploid and inC. arenosa DNA lanes, which instead showed an additional2-kb band. Perhaps asymmetric methylation of the HindIIIsite (Nelson et al., 1993) might have prevented completedigestion of the allotetraploid and of the C. arenosa DNA.

Table 4. Variation in the Hybrid F2’s

Character Description

Leaves From smooth-margined (only seen in the 605B F2) to different degrees of serration. Varying width, length, curvature, midribwidth, epidermal hair density, and anthocyanin pigmentation.

Lethality 5 to 10% of the F2’s died at the rosette stage.Flowering Varying from very early (6 weeks) to very late (several months).Height The F2’s of 605B were shorter than the F2’s of 49-2B and 605A.Apical dominance The apical dominance phenotype of the main inflorescence on the rosette coflorescences varied from strong to none.Pollen production Moderate to none.Flowers Flower types varied greatly. Most distinctive were differences in size (ranging among the parental sizes), petal color (white

to darker pink than in C. arenosa), width, and position (radial to zygomorphic). Stamens and pistils varied in length, withcertain individuals exhibiting stamens longer than the pistil or vice versa.

Aberrations Approximately 5% of the F2 plants exhibited altered epidermal cell patterns, fasciation, or homeotic conversions.Variegation Approximately 5% of the F2 plants exhibited leaf or stem variegation for either photosynthetic or anthocyanin pigments.

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Methylation of the C. arenosa homeolog was detected in theDNA of at least one tested allotetraploid and in one C.arenosa individual.

DISCUSSION

The causes of the phenotypic and genomic instabilities oc-curring in synthetic allopolyploids have long been mysteri-ous. In this article, we show that gene silencing can takeplace in the F2 generation after allopolyploid hybridization ofA. thaliana and C. arenosa, affecting both putative euchro-matic genes and a repeated gene related to transposons.This discovery was made possible by the analysis of syn-thetic allotetraploids, which were generated through sexualcrosses. Consistent with the instability of gene expression,these allotetraploids displayed phenotypic instability, al-though their rate of meiotic dysfunctions did not vary appre-ciably from the C. arenosa parental rate.

Synthesis and Characteristics of theAllotetraploid Hybrids

Synthesis of these allotetraploid hybrids was simple and re-producible, despite several barriers to hybridization. On av-erage, pollination of just two to three flowers (z75 zygotes)of tetraploid A. thaliana with C. arenosa (a natural tetraploid)was sufficient to produce each viable allotetraploid. Thesefindings are relevant to the manner of hybridization that gen-erated A. suecica, which has been the subject of debate(Redei, 1974). Our data suggest that this natural allotetra-ploid originated from pollination of a diploid egg of A. thalianaby C. arenosa pollen. Diploid A. thaliana fails to hybridizesuccessfully with C. arenosa, because the triploid hybridembryos (ACC genomes) arising from this cross arrest at theglobular stage, even though triploid A. thaliana embryos(AAA genomes) develop relatively normally (Scott et al.,1998). Consistent with the A. thaliana–like cytoplasm of A.suecica (Price et al., 1994), hybridization between A. thalianaand C. arenosa was successful only if A. thaliana was the fe-male parent. Failure of the reciprocal cross suggests thatcytoplasmic–nuclear incompatibilities or parent-specific DNAimprinting (Scott et al., 1998) may affect the outcome.

Barriers to hybridization between these species were evi-dent not only from the poor seed set and inviability but alsofrom the phenotypic variability and instability of those hy-brids that germinated. Although our cytological analysis ofFigure 5. Abnormal Phenotypes in the Hybrid F2.

(A) to (D) Switch in flower morphology. The main inflorescence pro-duced typical hybrid flowers (A), but flowers on basal coflores-cences varied from a small-petaled form (C) to a large-petaled one(B). All flowers are shown at anthesis. The position of the differentflower types on the plant body is illustrated in (D).(D) and (E) Variegation. Anthocyanin-free regions were present alongan inflorescence stem (in the position indicated by the rectangle).

(F) to (J) Irregular transition. Irregular transition between vegetativeand floral stages resulted in bifurcation of the growth axis. The nor-mal A. thaliana phenotype is shown in (H). A related alteration led toformation of basipetal flower pedicels ([I] and [J]).

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meiosis revealed some of the karyotypic abnormalities thatare expected to cause inviability and instability (such asanaphase bridges and laggard chromosomes), similar de-fects were also seen at comparable frequencies in the C.arenosa parent as well as in the natural allotetraploid A.suecica. Furthermore, we found no evidence for any strik-ing chromosomal instability that might explain stochasticchanges in phenotype. More compellingly, the observedphenotypic instability often involved reversion between twostates in a manner that would be inconsistent with any ir-reversible changes of chromosomal complement. Moreprobably, the phenotypic instabilities seen in these allotetra-

ploids result from regulatory dysfunctions such as epige-netic gene silencing (Meyer and Saedler, 1996; Matzke andMatzke, 1998).

Gene Silencing

A well-established case of gene silencing in interspecific hy-brids—nucleolar dominance—is triggered in some mannerby interactions between homeologous sets of ribosomalgenes (Navashin, 1934; Durica and Krider, 1977). Our paral-lel investigation of nucleolar dominance in the hybrids used

Figure 6. Analysis of Silenced Genes.

Comparison of gene expression in the parents, tetraploid A. thaliana (At) and tetraploid C. arenosa (Ca), and in synthetic F2 and F3 allotetraploids(1 to 24).(A) Portions of an AFLP-cDNA gel displaying random leaf RT-PCR products from parental and filial genotypes (1 and 12) and an artificial recon-struction of the expected allotetraploid expression pattern (AC) made by mixing the cDNAs. The product corresponding to product K7 is indi-cated in the top section. Examples of AFLP-cDNA patterns are shown in the sections below.(B) RT-PCR analysis of gene expression performed with mRNA preparations from allotetraploids and parents. Amplification of K9, K7, and L6mRNAs is compared with that of the control ROC1 (CYC, cyclophilin) mRNA (Lippuner et al., 1994) or that of the control actin ACT2 mRNA. Theunmarked lanes contain molecular size standards (25-bp ladder; the strongest band is a 125-bp or 100-bp ladder). Lanes labeled DNA displaycontrol amplification products from genomic DNAs. Allotetraploids 2 to 11 and 13 to 22 were positive for the K7 gene (data not shown). Bl, “notemplate” control; P, K7 PCR product from a plasmid clone. The low CYC signal in the DNA control lane marked Ca (second gel from the top) isprobably attributable to a low input of DNA, which would affect the repetitive K7 signal to a lesser degree.(C) Genealogical tree showing three generations of the tested hybrids. Individuals (rounded squares) are numbered corresponding to the gellanes in (A) and (B). The RT-PCR results for unnumbered individuals without the (?) mark are not shown.

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in the present study revealed differences in rRNA gene ex-pression among the F1 hybrids, thus showing variation be-tween codominance and dominance of the C. arenosa rRNAgenes (Chen et al., 1998). This instability was resolved in theF2 plants, which consistently showed silencing of the A.thaliana genes. This dominance relationship could be re-versed, however, by changing the parental genomic ratiofrom 1:1 (AACC) to 3:1 (AAAC). In this article, we extend theanalysis of phenotypic instability in these allotetraploids toestablish whether the genes that are transcribed by RNApolymerase II to generate mRNAs are also subject to silencing.

The present comparison of AFLP-cDNA products in theparents and the allotetraploids suggested the occurrence ofgene silencing but also, though less frequently, gene activa-tion in the allotetraploids. Sequencing candidate cDNAs fol-lowed by RT-PCR provided definitive evidence for genesilencing in three instances. The silenced K7 gene (the K7repeat) is related to transposable elements; it appears toconsist of a solo LTR embedded inside a putative transpo-

son gene. In addition, the K7 locus is methylated, and itscopy number differs between the parental genomes, beinglow in A. thaliana and higher in C. arenosa. Its chromosomallocation in the A. thaliana genome is unknown. A veryclosely related element (T3E15) resides in a region of chro-mosome 4 that has been cytologically identified as paracen-tromeric heterochromatin and is densely populated byAthila, MuDR-like sequences, and retroelements (Fransz etal., 2000). Although silenced in most allotetraploids tested,the K7 gene was active in a few of them. One of these activeindividuals was the progeny of an F2 allotetraploid silencedfor K7. In this lineage, therefore, the K7 gene had undergonereactivation, indicating that its initial silencing must not havebeen caused by a permanent genetic lesion. The two othersilenced loci have no obvious association with transposons,although the K9 locus contains repeated DNA. K9, identifiedas RAP2.1, is a member of a large gene family that encodesproteins related to AP2; its chromosomal location is un-known. L6 is located at 114 centimorgans (from the top) onchromosome 1 close to ALCOHOL DEHYDROGENASE, in aregion presumed to be euchromatic, although its homeologin C. arenosa, unlike that in A. thaliana, was methylated atHpaII sites. L6 may be part of gene F22K20.6, which is an-notated as encoding a protein of unknown function.

These results reveal the occurrence of rapid silencing ofpolymerase II–transcribed genes in interspecific hybridiza-tion. Such silencing has important implications for pheno-types of hybrid organisms. The conjunction of two divergedgenomes into a hybrid individual may reveal incompatibili-ties between biochemical, regulatory, or developmentalpathways that are controlled by genes segregating in theusual Mendelian manner (Dobzhansky, 1937). If this hybrid-ization is also accompanied by gene silencing, then therange of phenotypes exhibited by the hybrid could vary evenmore widely. On one hand, homology-dependent silencingmay completely prevent the expression of an important ge-netic function that otherwise would have been conferred byhomeologous genes from both parental genomes; in thatcase, this silencing would strengthen the barrier to hybridiza-tion. Or gene silencing might improve the fitness of the hy-brid individual by preventing the simultaneous expression ofhomeologous functions for which coexpression is incompati-ble with normal development. Given the instability of silenc-ing interactions, this latter advantageous result might benefitthe hybrid only transiently and have to be replaced in subse-quent generations by stable genetic changes conferred bymutation or gene conversion. Thus, gene silencing might ei-ther impede hybridization or provide a plasticity of gene ex-pression that could foster the vitality of allotetraploids.

Gene silencing appears to have an especially crucial rela-tionship, both as a cause and an effect, to the genomic in-stability that often accompanies allopolyploid hybridization(Song et al., 1995; Feldman et al., 1997; Liu et al., 1998). In-stability of this sort might derive from the breakdown of ge-nomic surveillance systems, for example, if the genessilenced encode functions in DNA repair or in chromatin

Figure 7. Structure of the Silenced Gene Loci.

The schematic drawing represents the structure of the three si-lenced loci, showing the elements identified by BLAST analysis, al-gorithm prediction (see Methods), or transcription analysis. Gnomo,G7, K9, and G9 are highly to moderately repetitive elements. Theputative repeated unit of K7 is indicated. The GenBank accessionnumbers are given in the right lower box. chr., chromosome; cM,centimorgan; Seq., sequence.

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structure. In marsupial hybrids, for example, chromosomeremodeling resulting from runaway transposition of retroele-ments is associated with hypomethylation of the hybrid ge-nome (O’Neill et al., 1998). Similarly, reactivation of dormanttransposons may be responsible for the frequent changes inrepeated DNA elements that are often observed in allopoly-ploid genomes (Burns and Gerstel, 1967; Zhao et al., 1998).

Although gene silencing in the Arabidopsis allotetraploidswe examined affected euchromatic genes, such as K9 andL6 may be, it was more frequently directed against the K7gene, a repeated element related to transposons, the copynumber of which changed in some of the allotetraploid ge-nomes. Silencing of the K7 gene may be related to a de-fense response against transposons (Comai, 2000). The

Figure 8. Genomic DNA Gel Blot Analysis of DNA Methylation: Parental and Allotetraploid DNAs Probed with the Silenced Genes.

(A) K7 hybridization to HpaII (H)-resistant DNA revealed heavy cytosine DNA methylation at CG sites. Partial resistance of hybridizing DNA toMspI (M) revealed reduced but still substantial methylation of CNG sites. These sites of the C. arenosa K7 sequences were less methylated in thehybrids. The copy number of K7 varied in different hybrids: the F2 hybrids of the 605B family displayed strong hybridization even though com-paratively less DNA was loaded, as shown in (B). The probe used was a 200-bp PCR product corresponding to the K7 AFLP-cDNA amplifiedfrom the genomic DNA of C. arenosa. This probe distinguished the C. arenosa K7 locus from the A. thaliana homeolog.(B) Gel blotted in (A).(C) The A. thaliana K7 homeolog hybridized to HpaII-resistant DNA. The probe used was the K7 IPCR product (2.2 kb) isolated from A. thaliana.(D) K7 hybridized to HindIII-digested DNA. The probe used was the K7 IPCR product (2.3 kb) isolated from C. arenosa.(E) and (F) L6 hybridized to DNA digested with HpaII (E) and HindIII (F); the DNA used was prepared from different individuals. Asterisks showpartial HpaII digestion products protected by methylation. The probe was the L6 1-kb IPCR product obtained from A. thaliana DNA.(G) K9 hybridized to EcoRI-digested DNA. The lack of hybrididization to the C. arenosa DNA is explained by the use of a probe (K9/RAP2.1IPCR; see Methods) representing the flanking sequences and covering only a few codons of the RAP2.1 transcribed region.Gel lanes were loaded with equal amounts of DNA. Therefore, the lanes with hybrid DNA contain approximately half the amount of each parental DNA.In (C) to (G), the size in kilobases of molecular size standards is shown at right of the gel. At, A. thaliana; Ca, C. arenosa; Hy, hybrid or allotetraploid.

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hypothesis that K7-related elements become active in al-lopolyploidization, thereby triggering a genomic defense re-sponse, could be explored in greater detail by analyzing theexpression, copy number, and chromosomal distribution ofthese elements in F1 and successive allotetraploid genera-tions. On the other hand, silencing of K9 (RAP2.1) and L6(perhaps F22K20.6) may well depend on paramutagenic in-teractions between homeologous genes. For example, giventhe methylated state of L6 in C. arenosa, this locus is possi-bly paramutable and perhaps paramutagenic.

In conclusion, the discovery of rapid gene silencing insynthetic allotetraploids provides a new avenue of investiga-tion into the molecular events that shape the outcome of al-lopolyploidy. Uncovering the causes of silencing will furtherour understanding of how related genomes that have di-verged since different species arose from a common ances-tor then interact when reunited. This task will be facilitatedby the powerful tools available in Arabidopsis for geneticand genomic analyses.

METHODS

Plant Lines, Growth, and Hybridization

Arabidopsis thaliana diploid ecotypes Nossen (No-0) and Landsbergerecta (Ler) were used (2n 5 2x 5 10; n, gametic chromosome; x,haploid chromosome). A tetraploid of Ler (LC612; 2n 5 4x 5 20) wasidentified among plants obtained by regeneration of roots (Valvekenset al., 1988) and designated 612. Cardaminopsis arenosa Care-1(WU9509; 2n 5 4x 5 32; also known as A. arenosa) and A. suecicaSue-2 (WU9510; 2n 5 26) were obtained from Craig Pikaard (Wash-ington University, St. Louis, MO). A. suecica Sue-1 (2n 5 26) was ob-tained from Fumiaki Katagiri (MIT, Cambridge, MA) (Hanfstingl et al.,1994). Plants were grown in a growth room at 22 6 38C and in 16 hrof daily artificial daylight provided with fluorescent bulbs (modelTL80; Philips, Sunnyvale, CA). For hybridization, the three largestclosed buds were emasculated by removing the immature antherswith the help of microdissecting forceps and a jeweler’s magnifyingglass. The stigma of emasculated buds was immediately fertilizedwith pollen from mature anthers taken from freshly opened flowers.Crosses were reciprocal and were repeated at least 10 times. The C.arenosa 3 A. thaliana (4x) cross was repeated 30 times. Pollinationswere usually performed around noon.

Embryo Development

To monitor the fate of each cross, we followed seed development byscoring specific stereotypical, well-characterized events (Bowman,1994). At several different times after fertilization, immature siliqueswere dissected, and the developing seeds were placed in a drop ofwater on a slide. An average of 150 seeds of each available genotypewas examined microscopically by either dissecting them individuallywith a teasing needle or by expressing their content with a cover slip.The measurements performed during development were used to es-timate the embryonic fate in mature siliques because collapsed and

dried seeds in mature siliques could no longer be dissected. Inviable(lethal) embryos (brown, red, collapsed, or very small seeds) werescored by dissecting 10 green siliques approaching maturity.

DNA Analysis

DNA was prepared using the LPG prep. The extraction buffer con-sisted of 0.1 M Tris, pH 8.0, 50 mM EDTA, 0.5 M NaCl, and 0.7% (w/v)SDS; to this, proteinase K was added to 50 mg/mL (final concentra-tion) just before extraction. Fifty to 100 mg of tissue was ground for10 sec in a plastic 1.5-mL tube with a disposable plastic pestle (Kon-tes, Vineland, NJ), 150 mL of extraction buffer was added, and thesample was ground for another 20 sec. Finally, 700 mL of extractionbuffer was added, and if tissue fragments were still visible, the sam-ple was further dispersed with the pestle. The lysate was incubatedat 558C for 1 to 5 hr, mixed with 520 mL of a saturated NaCl solution,and centrifuged at 12,000g for 20 min. The nucleic acids in the su-pernatant were precipitated by adding 1.7 mL of 85% isopropanoland collected by centrifuging at 10,000g for 10 min. The pellet waswashed with 70% ethanol and resuspended in 200 mL of 10 mM Tris,pH 8, containing 1 mM EDTA (TE buffer). The RNA was precipitatedby adding 133 mL of 5 M LiCl, incubating at 48C for 5 hr, and centri-fuging at 12,000g for 10 min; the pellet was removed. DNA was pre-cipitated from the supernatant by addition of 2 volumes of ethanol,followed immediately by centrifugation. The DNA so isolated was re-suspended in 100 mL of 10 mM Tris, pH 8.0, and stored at 48C. Ran-dom amplified polymorphic DNA (RAPD) analysis and microsatelliteDNA analysis were performed according to Williams et al. (1990) andBell and Ecker (1994), respectively. We monitored the inheritance of24 RAPD markers and 11 microsatellite markers. The x2 test for inde-pendence was performed using Microsoft Excel 5.0. For DNA gelblots, 1 mg of DNA was digested with 10 units of the appropriate re-striction enzyme per microgram of DNA, electrophoresed on an0.8% agarose gel, blotted on Biodyne-B (Pall, Ann Arbor, MI) nylonmembrane, and hybridized at 608C in 2 3 SSC (1 3 SSC is 0.15 MNaCl and 0.015 M sodium citrate). Washes were in 0.2 3 SSC at608C. Radiolabeled probe was prepared by the random priming poly-merase reaction with the Prime-it kit from Amersham.

Analysis of DNA similarity was performed by using the WU-BLAST2 program (using the AATDB server, which was terminated, orthe server at EMBL, http://dove.embl-heidelberg.de/cgi/blast2). Incertain cases, the TAIR NCBI-BLAST 2 was used, modifying thequery parameters as follows: nucleic mismatch, 24; nucleic match,5; gap opening, 10; and gap extension, 10. The use of WU-BLAST orof the modified NCBI-BLAST was required to recognize more distantrelationships between DNA sequences. For the K7 and K9 loci, sim-ilarity searches were performed using the DNA sequences depositedin GenBank. For the L6 locus, a sequence from nucleotides 37,000 to40,000 of bacterial artificial chromosome F22K20 was used. Proteinsimilarity searches were performed using the gapped NCBI BLAST2.0 server at NCBI. Gene predictions were performed on the Net-Gene2 server (http://www.cbs.dtu.dk/services/NetGene2/) (Hebsgaardet al., 1996). Pairwise BLAST comparisons were performed on theDIYB BLAST server (http://www.proweb.org/Tools/new-blast.html).

To detect the occurrence of possible mutations, we amplified theputative promoter of the RAP2.1 gene as an 800-bp fragment by thepolymerase chain reaction (PCR) with a proofreading thermostablepolymerase mix (Klentaq-LA; Clontech, Palo Alto, CA) from the al-lotetraploid in which the RAP2.1 gene was silenced and from the Ar-abidopsis parent. Two PCR products per genotype, each derived

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from an independent PCR reaction, were sequenced in both orienta-tions by using the amplification primers.

Cytology

Flower buds were fixed for 24 hr in 1:3 (v/v) glacial acetic acid/100%ethanol and stored in 70% ethanol at 48C. Anthers were dissected,placed in a drop of acetocarmine (2% carmine in 50% acetic acid) or49,6-diamidino-2-phenylindole (0.3 mg/mL) on a glass slide, andsquashed with an iron needle. A glass cover slip was applied, theslide was heated gently over a flame, and pressure was applied toburst and spread the pollen mother cells. A Nikon Microphot wasused to view and photograph the chromosomes. Images were digi-tized and adjusted for contrast in Photoshop (Adobe, Menlo Park,CA). Meiotic abnormalities were scored in blind tests by observingthe following number of meioses per genotype (metaphase I andanaphase I, respectively): Sue-1, 6, 33; Sue-2, 51, 9; 612, 10, 12;Care-1, 45, 8; 49-2B, 34, 12; 605A, 39, 18; and 605B, 30, 19. In non-blinded tests, we further scored 30 metaphase I and 30 anaphase I ofA. thaliana 612. Pollen viability was estimated after staining with 1%acetocarmine and counting at least 100 cells per genotype.

Gene Expression Analysis

Amplified fragment length polymorphism (AFLP)–cDNA was per-formed with mRNA purified from comparable organs from the paren-tal and F2 allotetraploid plants of comparable developmental age(from rosette leaves, before production of a visible inflorescence, orfrom flower buds). Because the F1 plants grew at different rates, theywere not included in the expression analyses. RNA was purified from100 mg of tissue (mature rosette leaves and inflorescence top, in-cluding buds and flowers) by using Trizol reagent according to themanufacturer’s instructions (Gibco Life Technologies) and subjectedto oligo(dT) affinity purification by using the magnetic separation kitof Promega. The mRNA was subjected to AFLP-cDNA analysis(Bachem et al., 1996). In a first round of experiments, leaf and flowerAFLP-cDNAs were made from diploid Ler, tetraploid Ler (612), two F2

allotetraploids, C. arenosa, and, as control, from a 1:1 mix of tetra-ploid Ler and C. arenosa cDNAs. In a second round of experiments,leaf and flower AFLP-cDNAs were prepared from duplicate mRNApreparations from the genotypes used above but using four F3 al-lotetraploids. In this second analysis, as a control, we also examinedthe AFLP-cDNA profiles of two different tetraploid A. thaliana individ-uals. The expression patterns were identical, suggesting that genesilencing is rare within inbred A. thaliana. Differential products wereeluted from the gel by boiling in water for 2 min, reamplified, andcloned using the Invitrogen TA-cloning kit. Several clones were se-quenced for each product by using the dye-terminator kit of AppliedBiosystems (Palo Alto, CA). The three silenced genes reported herewere isolated from the first round of AFLP-cDNA experiments. Often,the AFLP gel band yielded different cloned cDNAs. To determinewhich sequence represented the silenced cDNA, sequence-specificprimers were designed from the Primer3 Web site (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and used in reversetranscription (RT)–PCR analysis. For RT-PCR, new mRNA prepara-tions were made both from the plants originally used for the AFLP-cDNA analysis and from other allotetraploid individuals. RT was per-formed on 10 to 25% of each mRNA preparation and used randomhexamers as primers. Of the RT reaction product, 5% was used for

RT-PCR. The RT-PCR primers were L6-L, 59-TCTCCAGCAAAT-GATGAACAA-39; L6-R, 59-CCGGAGAGTCTCAATTTGGT-39; K7-A,59-TGGAGAGGCTTATGGACG-39; K7-B, 59-GCTCTTCTTAATGTG-TTG-39; K9-L, 59-TTGTTTTTGTTTTTGATAAGAACTCTG-39; and K9-R,59-CACTCGCTAGCTTCTCATGG-39. The K7 primers were specificfor C. arenosa DNA, the K9 primers were specific for A. thaliana DNA,and the L6 primers preferentially amplified the C. arenosa DNA.

IPCR Isolation of DNA Flanking the AFLP-cDNA

To isolate the DNA flanking the AFLP-cDNA, we used the AFLP-cDNA sequence of K7 and L6 and the available cDNA sequence ofRAP2.1 (K9). We designed outward-facing primer pairs for inversePCR (IPCR) (Ochman et al., 1988), using the Web-based Primer3program. The primer sequences were as follows: for K7, K7-AL (59-TCTCCTTTGCCTATTTAAAGGCTGT-39) and K7-AR (59-TCCAAC-GTCCATAAGCCTCTCC-39); for K9, K9-AL (59-CCGGTAATAAAA-CCCGACTTGAATC-39) and K9-AR (59-TGGTTAGGCACTTCTTCT-TGAGGTG-39); and for L6, L6-AL (59-GGAGGACCGCGGCAAT-AAG-39) and L6-AR (59-TTCATCATTTGCTGGAGAATGAAA-39). Ge-nomic DNAs from A. thaliana and C. arenosa were digested in sepa-rate reactions with six restriction enzymes (AseI, SphI, HindIII, EcoRI,SpeI, and XbaI). Each digested DNA was ligated (2 ng/10 mL reactionvolume), and 200 pg was used in long-distance PCR (20-mL reactionvolumes) with an annealing step at 658C (Henikoff and Comai,1998a). Reactions that produced PCR products of desirable sizewere used for cloning with the Invitrogen Topo-TA kit. In the case ofK7, control reaction with native genomic DNA showed that ligation ofa circular fragment is not a requirement for amplification (see Re-sults). The correct flanking fragments were identified by matching theknown sequences adjacent to the IPCR primers. The sequences ofK7, K9, L6, and their respective IPCR products have been depositedin GenBank (see Figure 7 for the accession numbers). The putativepromoter of RAP2.1 was amplified from the DNA of A. thaliana 612 andfrom the silenced hybrid 623-K1 (Figure 6, individual 11) by using prim-ers K9-AR (see above) and primer K9-PL (59-TGGGTATTCAGC-CCATTTTAAACC-39). Two independently amplified PCR products pergenotype were sequenced in both directions with the amplificationprimers.

ACKNOWLEDGMENTS

We acknowledge the assistance of Jiang Aimin with the molecular typ-ing of the allotetraploids, Chi-Min Fu with the embryo developmentstudies, and Jorja Henikoff with BLAST search strategy. This work wassupported in part by U.S. Department of Agriculture–National Re-search Initiative Competitive Grants Program Grant No. 97-35301-4429 to L.C. and by a National Institutes of Health grant to B.B.

Received April 19, 2000; accepted June 28, 2000.

REFERENCES

Allard, R.W. (1960). Principles of Plant Breeding. (New York: JohnWiley).

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1566 The Plant Cell

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Phenotypic Instability and Rapid Gene Silencing in Newly Formed … · 1997); novel homeotic phenotypes in Digitalis (Schwanitz, 1957), Gilia (Grant, 1956), and cotton (Meyer, 1970);