physical and genetic mapping of chromosome 9s in maize ... fileusing mutations with terminal...

Copyright 0 1996 by the Genetics Society of America

Physical and Genetic Mapping of Chromosome 9s in Maize Using Mutations With Terminal Deficiencies

Shiaoman Chao,*” Jack M. Gardiner,*.‘ Susan Melia-Hancock* and Edward H. Coe, Jr.*>+

*Department of Agronomy, University of Missouri, Columbia, Missouri 6521 1 and USDA-ARS, Plant Genetics Research Unit, Columbia, Missouri 6521 1

Manuscript received December 8, 1995 Accepted for publication May 11, 1996

ABSTRACT Deletion mapping was employed to determine the physical order of five morphological variants, pydl,

yg2, wdl, u28 and u31, with respect to restriction fragment length polymorphism (RFLP) markers located at the distal end of chromosome 9s in maize. The genetic materials used were a series of terminal- deficiency mutants, newly derived with MCCLINTOCK’S original stocks developed in the 1940s, via breakage-fusion-bridge cycles. A combined physical map and genetic map has been constructed based on data gathered from both genetic complementation tests and RFLP analysis. The location of u3l in relation to RFLP markers was further determined by interval mapping. The physical distance between the healed telomeric end and the most distal RFLP marker in two terminal-deficiency lines was established by using pulsed field gel electrophoresis and verified by BaZ31 digestion. The results from this study set a foundation for studies on the mechanism of healing of broken chromosome ends in higher plants.

C YTOGENETIC materials containing chromosomal alterations, such as translocations, inversions, and

deficiencies, derived by various physical means, unques- tionably have made a tremendous contribution to our understanding of chromosome behavior in plants (BURNHAM 1962). In maize, such materials often have been used for gene mapping and linkage group assign- ment dating back to the 1930s (BURNHAM 1930; MCCLINTOCK 1931). Terminal deficiencies generated by nondisjunction in B-A translocations have been employed widely to map genes to chromosome arms (BECKETI 1991) and, in one notable study, to derive segmental location of genes and of endosperm size fac- tors on the long arm of chromosome 10 (LIN 1982).

One process by which randomly generated terminal deficiencies can be derived is the breakage-fusion- bridge cycle. During meiosis, plants heterozygous for a paracentric inversion will give rise to a dicentric bridge following a crossover within the inverted segment, defi- cient for the terminal portion. Random breakage at this bridge configuration leads to formation of chromosomes with further deficiencies or duplications. The fate of the chromosomes with broken ends has been examined, and it was found that when a chromatid is broken at meiotic anaphase, fusion will occur between two sister halves of this chromatid and a bridge will reform during the following mitotic anaphase (MCCLIN-

Curresponding author; Edward H. Coe, Jr., USDA-ARS, Curtis Hall, University of Missouri, Columbia, MO 65211. E-mail: [email protected]

Taipei, Taiwan.

IA 52242.

’ Present address: Institute of Botany, Academia Sinica, Nankang,

* Present address: Biology Department, University of Iowa, Iowa City,

Genetics 143 1785-1794 (August, 1996)

TOCK 1938). This process is referred to as the breakage- fusion-bridge cycle. It has also been demonstrated that this cycle will continue in all subsequent gametophytic and endosperm mitoses following its origin at a meiotic anaphase (MCCLINTOCK 1939, 1941a). However, this cycle will cease when the broken chromosome enters into the zygote. The broken end heals permanently as no further fusion and breakage are found in sporophytic mitoses or any other tissues of the later genera- tions. This behavior applies to breakage events derived from crossing over in an inversion, from radiation, or from crossing over in an inverted duplication ( MCCLIN- TOCK 1944). Terminal-deficiency mutants involving the short arm of chromosome 9 of maize, derived from the duplication-generated breakage-fusion-bridge cycle, were used by MCCLINTOCK (1944) to determine the physical order of variants for pale-yellow ( p y d l ) , yellow- green ( y g 2 ) , and white ( w d l ) seedlings. These variants proved to involve one gene ( y g 2 ) and deficiencies of two extents, in which w d l uncovered yg2 and p y d l , yet p y d l did not uncover yg2, observations that otherwise contradict an “allelic series” interpretation.

With the recent rapid development of restriction fragment length polymorphism (RFLP) technology, terminal-deficiency mutants with deletions ranging from small segments to whole arms have been used to con- struct RFLP-based physical maps in maize (WEBER and HELENTJARIS 1989) and wheat (WERNER et al. 1992a; HOHMANN et al. 1994; O~IHARA et al. 1994), and to position the centromere in tomato (VAN WORDRAGEN et al. 1994). In this report, we present the results from using a series of newly derived terminal-deficiency materials like those described by MCCLINTOCK (1941a, 1944) to establish the physical and genetic order of five mor-

1786 S. Chao et al.

FEMALE PARENT MALE PARENT dup wx Sh C C Sh wx /df wx Sh x rearrg. c sh Wx /df wx Sh

9s c Sh wx

c sh Wx 11 V

0 Sh wx Sh wx i 0 (poor pollen transmission) (crossover occurring

between reverse repeats)

BRIDGE, BREAKAGE, FUSION CYCLE

1 random breakpoint I

\ fCShwx Sh wx n " . OR -

A n

c sh Wx " c sh Wx (variegated endosperm;

stabilizes in embryo)

selfing

CShwx " - ..

c - " c

colored endosperm white seedling (wd)

OR colored endosperm

pale-yellow seedling (pyd)

colored endosperm green seedling

phological markers in relation to RFLP markers located at the distal end of chromosome 9s in maize. Addition- ally, we have used pulsed field gel electrophoresis and BaB1 digestion to verify the physical distance from the most distal RFLP marker to healed telomeres in two terminal-deficiency lines.

MATERIALS AND METHODS

Generation of terminaldeficiency lines and complementation tests The genetic stock used in this study was from the genetic cross, dup wx Sh C C Sh wx/ df wx Sh X df wx Sh/ rearranged c sh Wx, provided by Dr. PAUL CHOMET. Crossovers occurring within the duplicated segments will result in formation of a dicentric bridge during meiosis, and progeny having healed chromosomes with terminal deficiencies can be recovered subsequently (Figure 1). Seeds were selected with variegated-colored endosperms, which are indicators of broken chromosomes as a result of continuation of the breakage- fusion-bridge cycle. Among plants from variegated kernels, a portion are expected to contain a terminal deficiency of appropriate size. The plants were both selfed and crossed to a tester, yg2 c sh wx. As depicted in Figure 2, the extent of terminal deficiency will be revealed based on segregation pat- tern and ratio among testcross progeny and selfed progeny, as the physical order of pydl relative to yg2 and wdl is known (MCCLINTOCK 1944). Individual phenotype was scored by growing progeny in a sandbench. Plants segregating for white seedlings among selfed progeny will show yellow-green seedlings in testcross progeny. In contrast, only green seedlings

colorless endosperm green seedling

FIGURE 1.-Genetic croses used to derive terminaldeficiency limes. Formation of a dicentric bridge fol- lows displaced crossing over b e tween sister chromatids of a reverse duplication chromosome, leading to breakagefusion-bridge cycles, random breakage of the bridge, and oftencorresponding broken chromosomes in the two sperms. In postfertilization divisions of the endosperm, breakagefusion-bridge cycles continue and display color variegation, signaling that a broken chromosome may also be present, stabilized, in the embryo. Self-polli- nation of deficiency-bearing plants may give white or paleyellow segregants showing linkage with C (colored endosperm).

will be found in testcross progeny of mutations whose deletion of a terminal chromosome segment does not include yg2. Skewed segregation ratios may indicate that a longer terminal segment has been deleted, and such chromosomes are not transmitted well through pollen.

Complementation tests were carried out by crossing all the terminal-deficiency lines to testers containing the seedling variants in this region: pydl (pale-yellow) reference deletion, wdl (white) reference deletion, yg2 (yellow-green), v28 (virescent), and v 3 l (virescent). The v28 mutation, originally isolated and designated ~ ~ - 2 7 by M. G. NEUFFER, was placed on the short arm of chromosome 9 by M. G. NEUFFER and D. ENGLAND (unpublished data); experiments reported in this paper show that the v28 locus is lost in the deficiency defined by each of the wdl isolates. The v31 mutation, originally isolated and designated v V 2 8 by M. G. NEUFTER, was placed on the short arm of chromosome 9 by M. G. NEUFFER and D. ENGLAND (unpublished data) and was mapped between shl and wxl by 0. E. NELSON (personal communication, 1995); experiments reported in this paper identify that the v31 locus is not contained within the deficiency defined by any of the wdl isolates. RFLP analysis: For each of the wd or pyd lines, genomic

DNA was prepared from all three genotypes, ie., wd or pyd homozygotes (wd/wd or pyd/pyd), green heterozygotes (+/ wd or +/pyd , in the colored class from selfs: see Figure l ) , and green homozygotes (+/+, in the colorless class from selfs), from 2-week old seedlings harvested from the sandbench. The materials were ground to a powder using mortar and pestle in the presence of liquid nitrogen. Genomic DNAs were isolated using the CTAB procedure described previously

Physical Map of Maize Chromosome 9s 1787

pyd breakpoint distal to yg.? wd breakpoint proximal to yg2

\ ,YgZCShwx " b FShwx " OR c

Yg2 c sh Wx Yg2 cshWx "

Z c s h wx testcross to yg "

yg2csh wx "

,YgZCShwx ~

, C Shwx - yg2csh wx " yg2 c sh wx "

" " ,. 50% 1 50%

colored endosperm green seedling

colored endosperm yellow-green seedling

Yg2 c shWx ~ Yg2 c shWx

yg2 c sh wx yg2 c sh wx "

" ,. 50% n 50%

FIGURE 2.-Genetic crosses of derivatives bearing terminal deficiencies (Figure 1) to determine whether the deficiency includes the locus of yg2. Crosses on yg2 may show complementation or not, revealing the extent of the newly derived terminal deficiencies. In this study, as in the studies of MCCLINTOCK, each of the derivatives that showed pale yellow seedlings on selfing (Figure 1) complemented yg2, and each one that showed white seedlings did not.

colorless endosperm colorless endosperm green seedling green seedling

(GARDINER et al. 1993) with minor modifications. Southern transfer was done using Amersham's Hybond N+ membrane in alkaline solution, and hybridization was carried out in CHURCH and GILBERT buffer (CHURCH and GILBERT 1984). Absence of a fragment hybridizing to probes for loci along the map was used to identify the extent of deficiency in the lines.

Interval mapping of u31 was performed using a F2 population segregating for u31. The rationale of interval mapping is based on the fact that the closest adjacent markers should most often stay as homozygotes when the target gene becomes homozygous (LANDER and BOTSTEIN 1987; HOISINGTON and COE 1990). The method allows us to score FWLP patterns among individuals homozygous for the target gene from a segregating population to determine the nearest RFLP markers flanking the target gene. Approximately 100 F2 seeds were planted in a flat and plants were grown in a growth chamber. Genomic DNA was prepared for each of the 21 individual v3lu31 homozygous seedlings following the method of CONE (1989). Green plants were bulked for genomic DNA isolation. Polymorphic DNA markers were identified by screening genomic DNA from A619, the line used to produce the F2 population; from bulked green plants; and from bulked virescent plants with six restriction enzymes. DNA markers detecting polymorphisms between A619 and the virescent bulk were hybridized to DNA of individual virescent plants. Segregation data were recorded to place u31 in relation to the cosegregat- ing FWLP markers.

High molecular weight (HMW) DNA isolation, pulsed field gel electrophoresis and Southern hybridization: HMW DNA from terminaldeficiency lines was isolated from 2-week old seedlings according to the method reported by CHEUNG and CALX (1990) except that the enzymes used to generate proto- plasts were 2% cellulase (Onozuka RS) and 0.1% Pectolyase E3. After digesting the DNA with rare-cutting enzymes, NotI, MluI and ASCI, large DNA fragments were separated on a 1% agarose gel in 0.5X TBE buffer using a CHEF gel unit (Bio- Rad) at 14". The gel was electrophoresed for 24 hr at 200 V with pulse time ramping from 60 to 90 sec. DNA fragments ranging from 50 kb to 2 Mb can be well separated under these conditions. Southern transfers were performed as described before, except that before transferring the gel was depuri- nated in 0.25 N HCl for 30 min following 1 min of short wavelength UV (254 nm) irradiation. A hybridization probe, pAtT4, containing 400 bp of Arabidopsis telomere repeats, was provided by Dr. E. J. RICHARDS (RICHARDS and AUSUBEL 1988).

But31 digestion: Approximately 5 micrograms of HMW DNA in an agarose block were digested with Bat31 (New En- gland Biolabs, 1000 units/ml). The enzyme was diluted 20- fold before use, and 1 pl of the diluted enzyme was used in a reaction mix of 250 p1 (enzyme concentration was 0.2 units/ ml). A series of time courses was employed to vary the extent of digestion. The BuE31 reactions were stopped by transferring the agarose block into 20 mM EGTA solution. The DNA was then processed for restriction enzyme digestions.

RESULTS

Complementation tests of fyd and wd lines: From the process outlined in Figures 1 and 2 we have recovered lines segregating pale-yellow seedlings or white seedlings. We have only included lines giving expected segregation ratios, as viable homozygous terminaldeficiency individuals are necessary for these studies. Com- plementation tests were made with the @dl and wdl reference sources (Table 1 ) . Ten lines complementing wdl but not pydl (denoted as pyd lines) and six lines not complementing wdl (denoted as wd lines) were identified, and complementation of yg2, u28 or 1131 was further tested. The results show that all the pyd lines complement all three mutant variants we have tested, while all the wd lines complement u31 but fail to complement yg2and u28 (Table 1). Since none of the terminaldeficiency lines uncovered only yg2 or u28, the physical location of u28 in relation to yg2 and wdl cannot be inferred from this study. Thus, the known physical order among the five types we have analyzed is pydl (yg2, u28, wdl )-u3l-centromere (Figure 3).

RFLP analysis of fyd and wd lines: As indicated previously by MCCLINTOCK'S (1944) data both pydl and wdl are distal to the C locus, and when the terminal- deficiency chromosome carries the C locus, crossing over between C and genes distal to C is expected to be reduced. As a result, the majority of the colored seeds among selfed progeny will give rise to either mutant seedlings with genotype wd/wd or pyd/pyd, or green seedlings with heterozygous genotype + / w d or +/pyd,

1788 S. Chao et a1.

TABLE 1

Complementation tests establishing that newly derived pyd and wd lines show complementation and noncomplementation patterns consistent with those found by MCCLINTOCK (1944)

Lines pydl-ref bn 19.0 7 umclO9 wdl-ref Y@ u28 u31

pydl-G2 Pale yellow Absent + Green Green Green Pyd 1 -G3 Pale yellow Absent + Green Green Green @dl-G4 Pale yellow Absent + Green Green Green pydl-G5 Pale yellow Absent + Green Green Green Pyd I -G6 Pale yellow Absent + Green Green Green pydl-G7 Pale yellow Absent + Green Green Green Pydl-GS Pale yellow Absent + Green Green Green @dl-GY Pale yellow Absent + Green Green Green pydl-GlO Pale yellow ND + Green Green Green B d l - G I 1 Pale yellow ND + Green Green Green wd 1 -G2 Absent + White Yelgreen Virescent Green wdl-G3 Absent Absent White Yelgreen Virescent Green wdl-G4 Absent + White Yelgreen Virescent Green wd 1 -G5 Absent Absent White Yelgreen Virescent Green wdl-GG ND ND White Yelgreen Virescent Green wdl-G7 Absent Absent White Yel-green Virescent Green

In all cases tested, the phenotype observed was in accordance to that expected for a terminal deletion of the pyd type (breakpoint distal to yg2) or wd type (breakpoint proximal to yg2). Additionally, u28 and u31 tests reveal that u31 is proximal to pyd and wd breakpoints, while 1128 is proximal to pyd and distal to each of the wd breakpoints. RFLP tests show that bn19.07 is distal to the tested fqd and wd breakpoints, while umcl09 is proximal to each of the pyd and to some but not all of the wd breakpoints. ND, not done; +, present.-

whereas colorless seeds are mostly homozygous green genotype +/+ (Figure 1). In this way, we were able to compare RFLP patterns among three genotypes for each line (an example is shown in Figure 4). RFLP analysis was performed on five of six wd lines and eight of 10 pyd lines. Markers located at the distal end of chromosome 9s were examined. The results showed that bnZ9.07 was absent from all of the pyd lines and wd lines analyzed (data not shown), while umclO9 was present in all the Pyd lines and two of the wd lines (lines G2 and G4), but absent from three wd lines (lines G3, G5 and G7) (Figure 4). These data enabled us to inte- grate four morphological traits, # d l , yg2, v28 and wdl, into the RFLP map (Figure 3). However, the order between bn19.07 and pydl is not resolved, as there is no terminal-deficiency line with breakpoints between pydl and bnZ9.07.

The location of v31 in relation to RFLP markers was determined using homozygous v31 v31 individuals from an F2 population. The segregation data are shown in Table 2, and the location of v31 is placed in the region containing asgl9, asg82 and npi266, establishing that v31 is proximal to pydl, wdl, yg2, and u28. The physical and genetic map for both morphological markers and RFLP markers at the distal end of chromosome 9s is shown in Figure 3.

Analysis of the physical distance between distal RFLP markers and the 9s telomere: Previous results, includ- ing ours (J. GARDINER, E. COE and S. CHAO, unpub lished data), have indicated that plant telomeres contain simple repeat sequences (CCCTAAA) , organized in a tandemly repeated fashion. When pAtT4, an Arabi-

dopsis telomere repeat probe, is used as a hybridization probe to HMW DNAs of wd lines digested with several methylation-sensitive restriction enzymes, a number of individual restriction fragments are observed, arising from hybridization of the telomere repeat to the termini of the 10 maize chromosomes. Also observable are high levels of polymorphism between the various pyd and wd lines, with wdl-G? and wdl-G5 being typical examples (Figure 5A). This is due in part to the fact that 19 of the 20 chromosomal termini are potentially heterozygous for any given end (unlike the 9s termini, which are homozygous).

Further examination of the wdl-G3 and wdl-G5 lines revealed them to be of particular interest. As noted earlier, these two wd lines had received breaks more proximal than wdl-G2, wdl-G4, and wdl-refand had lost the umclO9 locus but retained the phplOOO5 locus. This defines the location of the breakpoint in these two lines between umclO9 and phplOOO5. Reprobing the South- ern blot used to generate Figure 5A with phplOOO5 allowed the corresponding NotI and ASCI restriction fragments to be identified for this probe in wdl-G3 and wdl-G5. No restriction fragment was visible for MZuI, presumably because phplOOO5 hybridized to a fragment that was smaller than 50 kb that had run off the gel. The two autoradiographs (Figure 5, A and B) were carefully aligned using the blot edges and sample wells as matching points. Using this approach, it was determined that the 500-kb NotI and the 250-kb ASCI fragment of wdl- G3 and the 250-kb ASCI fragment of wdl-G5 identified by phplOOO5 comigrate with fragments identified by the telomere repeat. However, the 500-kb NotI fragment

Physical Map of Maize Chromosome 9s

physical and genetic map

1789

6

6 "

-- umcll3a 1 6

csu95a -- umclO9 \

"shl " bzl

-- bn19.07a

3 -- PhP70005

11

1 : ; a w l 9 6 asg82

10 6 -- umclO5a, npi266

5 -- ~ ~ ~ 9 4 6

6

$ :: csu43 sus 1

1 2/'bn18.17

1- bn17.50 ' urnc95

14

" rPa8 9 1 csudl 1

4 npi425d

? csu59 -:csu145a

9 A asgu

bn15.09 r ibnll4.28 asg 12

asg59b

npi97b 1 csu54b 10

95

A agrrl18b

-- bn19.07a

-- umclO9

-- phplOOO5

-- csu95a

-- umcl13a -- shl

" bzl -- asgl9 " asg82

- - umc 105a, npi266

95

-- bn19.07a I -- agrrl 18b

I" I Ysz, PYd7

I I wd7

-- umclO9

-- csu95a -- umcl13a -- shl -- bzl -- asgl9 -- asg82 I V31 -- npi266 '

-- phplOOO5

identified by phplOOO5 in wdl-G5 did not comigrate with a telomere repeat-hybridizing band. Taken together, this suggested that the 250-kb AscI fragment in both wdl-G3 and wdf-G5 and the 500-kb Not1 fragment in wdl-G3 might represent the terminal restriction fragments for these enzymes. An alternative interpretation of the comigration of the phplOOO5 and telomere repeat-hybridizing bands is that the bands are coinciden- tally of similar size but physically unrelated.

To differentiate between the two alternatives, a series of Bul31 exonuclease digestions were done to determine if the comigrating 250-kb ASCI fragments seen with phplOOO5 and the telomere repeat are both preferen- tially sensitive to Bul31 exonuclease. This would be expected if both phpZOOO5 and the telomere repeat hy- bridize to the same terminal restriction fragment. Bal31 is routinely used to identify telomeric restriction fragments, since chromosomal termini in HMW DNA (2- 5 Mb) are hypersensitive to mild Baa1 exonuclease treatment before restriction endonuclease digestion

FIGURE 3.-Chromosome 9University of Missouri RFLP map (August 15, 1995) and the combined physical and genetic map of chromosome 9s developed from this study. Ambiguous orders are shown by dotted lines. (Note: only the order is shown on the combined map; the distance is not in scale).

(RICHARDS and AUSUBEL 1988). A series of timed BuB1 digestions of wdl-G5 followed by AscI digestion shows that both of the 250-kb ASCI fragments identified by ph~~OOU5 and the telomere repeat are BuB1 sensitive. After 10 rnin of But31 treatment the 250-kb hybridization band corresponding to phplOOO5 decreases, while the heterodisperse smear below gradually increases, and after 40 min of Bul31 digestion the 250-kb band is lost (Figure 6A). Reprobing this blot with the telomere repeat shows that after 5 min of Bal31 digestion the intensity of the 250-kb hybridization band is becoming heterodisperse, until after 40 min no individual band is observable. It is not unexpected that hybridization of the telomere repeat probe is slightly more sensitive to Bal31 digestion since phplOOO5 may be as much as 250 kb centromere-proximal to the terminal telomere repeats on the same restriction fragment. As a control to monitor nonspecific exonuclease digestion, the blot was probed a third time with npi266, a probe that maps interstitially on chromosome 9s and should be unaf-

1790 S . Chao et al.

pydl lines

62 63 64 GS G6 67 G8 G9 GI0

FIGURE 4.-Genomic DNAs of segregants from wd and pyd lines, digested with Hind111 and probed for presence or absence of the umcl09 locus, demonstrating that some of the zud mutants have lost this locus. The DNA concentration for pydI/pydl of line G5 was low, and the hybridization signal was barely visible. The lane marked * is a wd line with assumed genotype + / d l . Note absence of the 4.3kb urn109 band in wd163, G5, and G7, showing that the deficiency breakpoint has occurred between urn109 and the centromere.

fected by mild BuB1 digestion conditions (Figure 6C). of the HMW DNA in these samples, therefore, has re- After 40 min of BuB1 digestion, the intensity of the mained intact and is unaffected by the mild BuBl diges- npi266 hybridizing band remains unchanged. The bulk tion conditions used. These data, showing specific BuB1

TABLE 2

Segregation data for u32 u32 homozygotes scored for RFLP markers on chromosome 9s

u31/u31 F2 individuals

RFLP markers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

umclO9 B H B B B H B H B B B H H H B H H B B H H phplOOO5 B H B B B H B B B B B H H H B H H B B B H csu95a B H B B B B B B B B B B B H B B H B B B H umcl13a B H B B B B B B B B B B B H B B H B B B H sh 1 B H B B B B B B B ' . B B B B H B B B B B B H bZl B B B B B B B B B B B B B B B B B B B B H

asg82 B B B B B B B B B B B B B B B B B B B B B npi266 B B B B B B B B B B B B B B B B B B B B B

asgl9 B B B B B B B B B B B B B B B B B B B B B

From an F2 population segregating for v31, v31/v31 individuals were subjected to RFLP analysis. RFLP markers are shown in their previously known map order from distal to centromere proximal. B signifies homozygosity for the allele from the u31 parent and H signifies heterozygosity (in this sample, no homozygotes, A, were obtained for the other allele). Note the complete homozygosity (B) of the v31 parent allele in the asgl9-asg82-npi266 region, indicating that v31 lies in this region.


A wd-G3 wd-GS A

8254

*

365- 285- 2 2 5 -

B wd-G3 Wd-G5

kb 825 785

450 365

Mlu I Asc I Not

restriction map

I Mlu I Asc I

wd-G3 : , 250kb 1

Not I Asc I ' k breakpoint

250kb y wd-G5 : I 14

Not I Asc I Not I

+ phplOOO5

FIGURE 5.-Correspondence of telomere repeat-probed fragments with fragments probed by phpl0005. HMW DNAs of wdl-G3 and G5 were digested with NotI, MluI and ASCI and probed with pAtT4 (A) and phpl0005 (B). The restriction map shows the terminal restriction site for ASCI in wdl-G3 and G5 (see text). The terminal NotI restriction site is shown for wdl-G3 and GS; note that wdl-G5 has a NotI site that was lost in the chromosome breakage event that produced wdl-G3.

sensitivity for the 250-kb ASCI fragment detected by both the telomere repeat and phplOOO5, demonstrate that they both reside on the same 250-kb ASCI restriction fragment, that phplOOO5 is close to that restriction site rather than to the telomere, and that the distance between phplOOO5 and the healed end in wdl-G5 is no more than 250 kb.

The finding that the 500-kb Not1 restriction fragment identified by php10005 in wdl-G5 does not comigrate with a band identified by the telomere repeat probe

450 - 365 - 285 - 225 -

B - 4-

Asc I digest P + - kb 0' 5' 10' 20' 40' 2 u

1,125- 1,020-j 945

!Ha] 680

C c1

Asc I digest 20 + - kb 0 5' 10' 20' 40' q 8

FIGURE 6.-Concurrent end digestion of fragments carrying phpl0005 and telomere repeats. HMW DNA of wdl-G5 was treated with Bat31 for different times followed by ASCI digestion. The last track is HMW DNA of line G5 double-digested with NotI and ASCI. The filter was probed with phpl0005 (A), pAtT4 (B) , and npi266 (C). A demon- strates that the 250-kb fragment probed by phpl0005 is sensitive to BaB1 digestion, as expected for a terminal restriction fragment. Note that the bulk of the HMW DNA is intact even after 40 min of Bal31 digestion, evidenced by unaltered hybridization for the internally located site npi266.

1792 S. Chao et al.

was surprising and warranted further investigation. Considering the results previously obtained, indicating that phplOOO5 is within 250 kb of the healed end of chromosome 9s in wdl-G5, and the fact that phplOOO5 did not comigrate with a telomere repeat-hybridizing band in NotI-digested wdl-G5 DNA, we conclude that wdl-G5 has a NotI site distal to the terminal ASCI site (Figure 5B). Upon digestion with NotI, the telomere will be separated from the fragment carrying phplOOO5. The comigration of phpl0005 and the telomere repeat bands in wdl-G3, with the 500-kb NotI and 250-kb ASCI fragments, is interpreted to mean that wdl-G3 has lost the terminal NotI site that has been retained in wdl- G5. The difference between wdl-G5 and wdl-G3 with respect to this terminal NotI site is likely to be due to a breakpoint difference, since the healed chromosome 9s in both of these lines was derived from the same genomic origin. Furthermore, the terminal NotI site in wdl-G5 must be very close to the breakpoint of wdl-G3 because the 500-kb NotI fragment identified by phplOOO5 in wdl-G3 and wdl-G5 is similar in size. To examine this possibility, HMW DNA from wdl-G5 was double digested with NotI and ASCI, and a single 250-kb fragment, identified by phplOOO5, migrated the same distance as in the single ASCI digest (Figure 6A). This suggests that the terminal NotI site in wdl-G5 is very near the healed end containing the telomere repeats since there is no observable difference between the single- and doubledigested wdl-G5 HMW DNA. Small dif- ferences of 10-20 kb between the single- and doubledigested DNA would be very difficult to resolve under the pulsed field gel electrophoresis conditions used in these experiments. To further substantiate this idea, HMW DNA from both wdl-G3 and wdl-G5 was treated with BaZ31 exonuclease and followed by NotI digestion and probing with phplOOO5 (data not shown). The results from this experiment indicate that the 500-kb band in both wdl-G3 and wdl-G5 is sensitive to Bal31 and that this band in each line disappeared at about the same rate over the time course of Bal31 digestion. Therefore, it is evident that the physical structure of chromosome 9s in wdl-G3 and wdl-G5 is very similar except for a NotI site near the telomere in wdl-G5.

DISCUSSION

The methods available to generate terminal-deficiency lines are not limited to ionizing radiation. In wheat the gametocidal chromosome originating from certain alien species, if introduced into cultivated wheat species, can cause extensive chromosome breakages at random places (ENDO 1990). Maize transposable ele- ments, such as Ds or Mu, have also been demonstrated to be capable of causing chromosome breakage leading to terminal or interstitial deletions (MCCLINTOCK 1951; TAYLOR and WALBOT 1985). Recently, a set of deletion lines involving chromosome 9S, induced by Mu, has

been recovered (ROBERTSON and STINARD 1987; ROE ERTSON et al. 1994). Based on cytogenetic evidence, the latter authors have demonstrated that, among lines that were noncomplementing to wdl, two had terminal deficiencies while others may carry internal deletions of various lengths covering the region containing prdl, yg2 and wdl . FWLP analysis should provide information on the extent of deletions found among these materials. More importantly, since it appears that Mu-induced chromosome deletions are confined to smaller regions, lines carrying various lengths of internal deletions may be useful to resolve the ambiguities we have found in this study, namely the location of v28 and the relative order of bn19.07 to pydl.

At the present time the use of terminaldeficiency stocks for physical map construction has made little impact on the advancement of fine-structure genome analysis for diploid plant species. One of the main obsta- cles is poor transmission of chromosomes with longer deletions through pollen, resulting in low recovery of homozygous deficiencies. Moreover, during the process of producing deletion lines through either physical or biological means, more complex rearrangements can be induced, such as small interstitial deletions, which can complicate the construction of a fine structure physical map. Thus, more detailed and laborious cytological work, or other analyses independent of the physical mapping per se, are required before deletion lines can be considered definitive tools. Nonetheless, the use of terminal-deficiency lines has already provided useful information to correlate the genetic map with the physical map in wheat. From our present study, the materials have allowed us to incorporate RFLP markers with morphological markers quickly, and they are consistent with and extend conventional mapping results (Figure 7). In addition, the materials provide us with useful re- sources to investigate the telomere structure at the broken ends. The NotI, wdl-G5 terminal restriction fragment of -500 kb or the 250-kb ASCI fragment from G3 or G5 could be retrieved using phpl0005 as a verifying probe to derive clones carrying the 9s “healed” telomere.

Investigating breakage-fusion-bridge events in the 1940s, MCCLINTOCK was the first to observe permanent healing of broken chromosome ends in the sporophytic tissues. The recent molecular characterization of telomeres has demonstrated that there are tandemly repeated simple sequences at the ends of chromosomes. The sequence is very similar from simple eukaryotes such as yeast to complex organisms, such as humans and higher plants (PARDUE 1994). Evidence from humans (WILKIE et al. 1990) and Tetrahymena (Yu and BLACKBURN 1991) has suggested that the healing of broken ends is a result of direct addition of Grich telomere repeats onto the broken ends and that telomerase plays an essential role in this process. Other healing mecha- nisms that do not require telomerase have also been


9s

agrr 1 1 86

bn19.07a

PYd 1

php 7 0005 Asd csu95a

FIGURE 7.-Portion of chromosome 9, showing locations of the breakpoints for the wd isolates in this study, defined by analyses of the presence us. absence of the umcl09 locus, associations of the new (healed) telomeres with HMW restriction fragments, physical distances from the telomeres to restriction sites, and end digestions with Bat31 endonuclease.

reported, one mediated via recombination with existing wild-type telomeres found in yeast, Saccharomyces cermi- siae (WANG and ZAKIAN 1990), and the other through transposition of a telomere-specific retrotransposon to maintain the length of chromosome ends found in Dro- sophila (MASON and BIESSMANN 1995). In higher plants, thus far, the results gathered from in situ hybridization have indicated that de nouo synthesis of telomere repeats may have been involved in chromosome healing of the broken ends of barley and wheat chromosomes (WANG et al. 1992; WERNER et al. 1992b; TSUJIMOTO 1993). By examining wheat materials carrying chromosomes with newly broken ends induced by the gametocidal chromosome system, TSUJIMOTO (1993) further indicated that the addition of telomere sequences to the broken end took place soon after chromosome breakage occurred, while restoration of telomeres to full length may take more than one generation. It is interesting to note from these data that a broken end is considered healed as soon as a few telomere repeats are added on, and that the length of telomere repeats seems not important for the telomere to function. These data have also strongly implied that direct addition of telomere repeats to broken ends may be used for chromosome healing in higher plants. So far, the reported chromosome breakage induced by means of either the breakage-fusion- bridge cycle in maize or the gametocidal chromosome system in wheat have all been found to take place during meiosis or gamete formation. After fertilization the

broken chromosome enters the zygote, and ends are healed before the first mitosis. Thus, a recombination- based mechanism would be unlikely to play any role in healing of broken ends in plants. However, the actual process is still waiting to be studied and the terminal- deficiency lines we have generated in this study should be valuable for such work. Since direct isolation of telomere sequences specific to 9s can be complicated by telomeres present at the other 19 ends, the strategy we employed using pulsed field gel electrophoresis to establish the physical distance from the most distal RFLP marker to the telomere repeats followed by veri- fication with Bat31 digestion is valid. However, more research needs to be done to identify end fragments from other deletion lines, so that we will be able to understand if there is a preferential site required for healing as found in other organisms. In humans, by examining a series of terminal deletions at the a-globin gene, it was found that during de nouo healing a small complementary region, mainly Grich, was required for the RNA component of telomerase to initiate and prime healing (FLINT et al. 1994).

Tne phenomenon is very intriguing as to why broken chromosome ends in the sporophytic tissues can heal, whereas in the gametophytic and endosperm tissues they cannot. An exception to this is ring-shaped chromosomes, which can go through breakage and fusion cycles in sporophytic tissues without showing any signs of healing (MCCLINTOCK 1941b). With the advanced development of molecular genetics techniques, the dif- ferences may be possible to resolve in the future.

The experiments were conceived and the deletions were produced and tested by J.M.G.; additional tests were completed by E.H.C., and RFLP analyses and BaB1 digestions were designed and carried out by S.C. We thank J. BIRCHLER, M. MCMULLEN, and D. S. ROBERTSON for helpful criticisms and advice on the manuscript. This work was supported in part by a grant from the Midwest Plant Biotechnology Research Consortium with matching funds provided by Cargill Seeds, Agrigenetics (Mycogen), United Agriseeds (DowElanco), Funks Seeds (CIBA Geigy), and Asgrow Seeds (Upjohn); and in part by the National Research Initiative Competitive Grants Office, US. Depart- ment of Agriculture. This is a contribution from the Missouri Agricul- tural Experiment Station, Journal Series Number 12428. Mention of a particular product or brand name does not constitute an endorse- ment or recommendation by the University of Missouri or the US. Department of Agriculture and is provided solely for informational purposes.

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Communicating editor: W. F. SHERIDAN

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