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Physical Mapping of the 5S rRNA MultigeneFamily inCommon WheatY. Mukai, T. R. Endo, and B. S. Gill

Insitu hybridization in conjunction with deletion mapping was used to physically mapthe 5S rRNA multigene family in Triticum aestivum L. cv. 'Chinese Spring.' Twelve5S rRNA loci were mapped on chromosomes of homoeologous group 1 (arms 1AS1BS, an d 1DS) an d group 5 (arms 5AS, 5BS, and 5DS). The 5S rRNA loci were mapped(fraction of the distance from the centromere) at positions 0.77, 0.96, 0.76, 0.63, an0.64 on arms 1AS, 1DS, 5AS, 5BS, an d 5DS, respectively. Th e 5S rRNA locus on BSwa s mapped at position ca. 0.5 of the satellite length. Deletions were used to furthemap the 5S rRNA loci to chromosome bands 1AS12, 1BS32, 1DS22, 5AS22, 5BS22and 5DS22. The 5S rRNA loci were not associated with major C-bands on arms 1BS1DS, and 5BS. The mapped 5S rRNA loci on the chromosomes of homoeologougroup 1 are designated 5S-Rma-A 1, 5S-Rrna-B 1,and 5S-Rrna-D 1, and the homoeologous group 5 set are designated 5S-Rma-A2, 5S-Rrna-B2, and 5S-Rrna-D2. The 5rRNA locus in 1AS is a new finding InChinese Spring wheat.

From the Department of Plant Pathology, Kansas StateUniversity, Manhattan. Dr. Mukai is now at Osaka KyoikuUniversity, Osaka, Japan, and Dr. Endo is now at NaraUniversity, Nara, Japan. Research supported in partby USDA-CRGO grant 86-CRCR-1-2222 and USDA-CSRSspecial research grant "Wheat Genetics Resource Cen-ter at Kansas State University." Dr. Endo was sup-ported by a grant from the Japan Society for the Pro-motion of Science under the U.S.-Japan CooperativeScience Program. The work was completed while Dr.Endo and Dr. Mukai were visiting scientists at KansasState University under the auspices of the Wheat Ge -netics Resource Centerand the U.S.-Japan CooperativeScience Program. Contribution No. 90-175-J from theWheat Genetics Resource Center and Kansas Agricul-tural Experiment Station, Kansas State University,Manhattan. Address reprint requests to Dr. B. S.Gill,Department of Plant Pathology, Throckmorton Hall,Kansas State University, Manhattan, KS 66506-5502.Journal of Heredity 1990,81:290-295; 0022-1503/90/$2.00

The structure, organization, and evolutionof the 5S rRNA (ribosomal ribose nucleicacid) multigene family have been studiedin detail in the Triticeae.'T7 's The 5S rRNAmultigenes exist in two sizes: the long unit(ca. 500 bp), consisting of a 120-bp codingand 380-bp spacer region, and a short unit(ca. 400 bp), consisting of a coding regionof the same length and a smaller spacerregion (ca. 280 bp). 9 L8 In most cases, bothshort and long units are found in homo-geneous tandem arrays of repeating unitsin homoeologous groups 1 and 5, respec-tively. In wheat 5S rRNA loci have beenassigned to chromosomes B, ID, 5A, 5B,and tentatively 5D. 5.'8There is limited information on thephysical location of the 5S rRNA loci. Ap-pels et al., 2 using in situ hybridization (ISH),mapped 5S rRNA loci distal to the sec-ondary constrictions in chromosomes 1Bof wheat and IR of rye and in a nonnu-cleolar chromosome in barley. Because oftechnical limitations, the physical locationof 5S loci on other chromosomes of wheatand rye was no t determined. More re-cently, Reddy and Appels'7 mapped an ad-ditional 5S rRNA locus in chromosome 5Rof rye.Using a different approach, Kota andDvorak" took advantage of a spontaneousdeletion of chromosome 5BS and mappeda 5S rRNA locus close to the prominentinterstitial band. From this data and those

of Appels et al.,2 Dvorak et al.5 speculatethat 5S multigene families may be locatedin the intercalary heterochromatin bandon the short arms of chromosomes 5A, 5B1B (in the satellite), and D.In these studies, ISH or deletion stockwere used in combination with Southernblotting. Both methods are individually insufficient for the detailed physical localization of a gene on a chromosome, especially with reference to band positionin a karyotype. When the two approacheare combined, more accurate location oa gene in a specific chromosome banshould be possible.Rayburn and Gill'6 reported a biotin-labeling technique for the mapping of DNsequences in plant chromosomes by meanof ISH. This technique has several advantages, including rapid detection and accurate localization of hybridization siteon chromosomes. The biotin-labelingtechnique also permits the location of single-copy DNA sequences in plant chromosomes, suggesting that the techniqumay be as sensitive as the isotope-labelintechnique for the detection of hybridization sites.'In addition to the sensitive ISH methoda number of deletion stocks have becomavailable since the discovery by Endo 7 oa genetic system that allows the isolatioof a virtually unlimited array of deletiostocks in common wheat. In this system

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deletions are recovered in backcrossprogenies of monosomic addition plantswith an Aegilops cylindrica chromosomecrossed as a female with normal wheat.The deletions occur in gametes/zygotesthat lack the alien chromosome, andhomozygous deletion plants can be re-covered in a pure wheat background bymeans of selfing. The homozygous dele-tion stocks are usually viable because ofthe polyploid nature of wheat. Moreover,the location of breakpoints in most of thedeletion chromosomes can be determinedby means of banding analysis. 6 We reportthe physical mapping of 5S rRNA loci inchromosomes of wheat, using ISH in con-junction with an array of chromosomedeletion stocks.Materials and MethodsEuploid, ditelosomic (DT), and deletionstocks of Triticum aestivum cv. 'ChineseSpring' were used. We obtained seed ofDT stocks from Dr. E. R. Sears. The dele-tion stocks were derived from the back-crossed progeny of the monosomic addi-tion of an A. cylindrica chromosome toChinese Spring.7 The deletions we usedlacked portions of the short arms of chro-mosomes I B, ID, 5A, 5B, and 5D; they aredescribed in Table I (see also Figure 1).We identified the chromosome, arm, re-gion, and band number location of 5S rRNAloci using DT and deletion stocks. We cal-culated the position of ISH sites on telo-somic chromosomes as a fraction of thetotal telosome length (FL) from the cen-tromere. We calculated the FL position ofbreakpoints in deletion chromosomes fromat least 10 C-banded chromosomes of boththe deletion chromosome and the normalhomologue, using the equation S/L dele-tion chromosome over S/L normal homo-logue, multiplied by 1, where S = shortarm and L = long arm.For cytological preparations, we ger-minated seeds in a moist dish for 3 daysat 230C.Th e root tips were preteated at 0"Cfor 16 to 20 h an d fixed in 3:1 alcohol:glacial acetic acid for I or 2 days. We thenstained the root tips in acetocarmine forseveral hours and then scraped out themeristems and squashed them in 45% ace-tic acid. Slides were stored at -70"C untilused. For C-banding, we used the proce-dure of Endo.6

We obtained the rye 5S DNA probe(pScT7) from R.Appels.'8 It contains a Se-calecereole5SDNA inserted into the BamHIsite of pUC8. Probe DNA was labeled bymeans of nick-translation (Bethesda Re-

search Laboratories, Gaithersburg, Mary-land) with biotin-16-dUTP (BoehringerMannheim, Indianapolis, Indiana) accord-ing to the supplier's instructions.Th e ISH protocol followed that of Ray-burn and Gill'6 with some modifications.Slides were denatured in 70% formamide/2 x SSC at 70C for 2 min and dehydratedin an ethanol series at -20 0C. The hybrid-ization mixture consisted of 50% form-amide, 2x SSC, 10% dextran sulfate, salm-on sperm carrier DNA at 500 #g/ml, andbiotin-labeled pScT7 at 4 Mg/ml. Th e la-beled pScT7 DNA was denatured at 100Cfor 10 min. Hybridization was performedfor 6 h in a humidity chamber at 37C.Posthybridization washes and detection ofhybridization sites were as described byRayburn and Gill.'6Photographs were taken with an Olym-pu s NC SPlanApo 100 (oil) objective, usingKodak technical Pan film 2415.ResultsCytological Characterization ofDeletion StocksWe analyzed the deletion stocks by meansof C-banding to determine the band lo-cation and FL positions of the breakpoints(Figure 1 and Table 1). Th e C-bandinganalysis indicated that each deletion ex-cept one listed in Table 1 resulted from asingle breakpoint, with a concomitant lossof the distal segment. We propose a formaldesignation that provides information ontype of aberration (del = deletion); nameof chromosome, arm, region, and band thatsuffered the deletion; and FL chromatindeleted. Details of the chromosome no-menclature system have been publishedelsewhere.'Physical Mapping of 5S rRNA Loci byISHISH analysis with the rye 5S clone consis-tently indicated 12 sites on metaphasespreads of chromosomes from root tip cellsof Chinese Spring wheat (Figure 2) . In mostcells, the signal was most intense in onlyfive pairs of chromosomes, although all 12chromosomes showed a moderate tostrong signal (Figure 2). Twelve sites ofhybridization also occurred in the inter-phase nucleus (Figure 3). Depending onthe orientation of a chromosome, we ob-served either one signal covering bothchromatids or two distinct sites of hybrid-ization on each chromatid.We identified all chromosomes by meansof DT analysis. Chromosome 1B showed a5S rRNA locus in the satellite region of the

Table 1. Deletion stocks used In mapping 5SrRNA loci by In situ hybridization n Trfficumaestivum cv . 'Chinese Spring'Deletionstock Formal designation, Breakpoint bIBS-4b Del(lB)(S32:0.26) 0.74 0.04IDS-2 Del(ID)(S12:0.35) 0.65 0.055AS-2 Del(5A)(S22:0.03) 0.97 0.045AS-3 DeI(5A)(S22:0.26) 0.74 + 0.045BS-I Del(5B)(S22:0.33) 0.67 0.045BS-2 Del(5B)(S22:0.51) 0.49 0.035DS-I Del(5D)(S22:0.31) 0.69 + .05

a Different deletions with breakpoints in the same banar e distinguished by indicating the fraction of thdeleted arm. Colon indicates a single break withconcomitant loss of the distal fragment.Fraction of the length of the normal short arm.

short arm (Figure 4). This chromosomwas further identified by arm ratio and thmorphology of the satellite (Figure 4) . Thchromosome pair with a weak signal neathe terminal end was identified as IASChromosome pair ID had a terminal signain the short arm. The remaining three pairof chromosomes with subterminal sites ohybridization on short arms were 5A, 5Band 5D. Signal intensity was greatest ochromosomes 1B and 5B; intermediate ochromosomes 5A, 5D, and ID; and weakeson chromosome IA.We measured the FL position of the SrRNA locus on the cytologically identifable telosomic chromosomes (Figure 4)The FL location of 5S rRNA loci was 0.7 0.04 in AS, 0.96 0.07 in IDS, 0.76 0.05 in 5A, 0.63 + 0.04 in 5B, and 0.640.05 in 5D. In the IBS arm, the 5S rRNAlocus can be mapped at approximately thmidposition (0.5) of the satellite lengthThis can be deduced from del(lB)(S32with a breakpoint at 0.36 of the satellitlength that lacks the 5S rRNA locus. Because the ISH site in an intact satellite appears to be located at a midposition, whiccannot be less than the 0.36 position,can be assumed to be at about 0.5 positioof the satellite length.5S rRNA Location In DeletionChromosomes by ISH AnalysisThe 5S rRNA loci were allocated to specifibands by means of ISH analysis of deletiostocks (Figures I and 5).

Chromosome IA. No deletions weravailable. The 5S rRNA locus is in banS12 at FL 0.77.Chromosome B. We analyzed one deletion stock (Table 1). The deletion hadbreakpoint in band S32 distal to the proximal C-band S31 of the satellite and lacke

the 5S rRNA locus. Th e breakpoint i

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del(lB)(S32) was calculated at 0.36 of thesatellite length and 0.74 of the arm length(secondary constriction was not includedin the measurements).Chromosome ID. One deletion was ana-lyzed (Table 1). Del(ID)(S12) with abreakpoint at position 0.65 just proximalto the prominent C-band on IDS(21)lacked the 5S rRNA locus. The 5S rRNA isin band S22 at 0.96.Chromosome 5A. We analyzed two dele-

tions (Table 1). Del(5A)(S22) with abreakpoint at 0.97 showed a 5S rRNA locus(data not shown). Del(5A)(S22) with abreakpoint at 0.74 lacked a 5S rRNA locus.Th e FL position was determined to be at0.76 using telosomic analysis. The data in-dicate that the 5S gene is located in bandS22.

Chromosome 5B. Del(5B)(S22:0.51),which lacked half the 5BS arm, did nothave a 5S rRNA locus. Del(5B)(S22:0.33),with a breakpoint toward the distal end ofband S22, retained the 5S rRNA locus. Theintensity of the label was reduced consid-erably, suggesting a breakage in the 5S lo-cus itself. The FL value (0.63) from dele-tion analysis is close to the FL value (0.67)calculated from telosomic mapping.Chromosome 5D. Del(5D)(S22), with abreakpoint at 0.69 of the S22 band, re-tained the 5S locus. As band S21 (C-band)is located at 0.56, this places the 5S rRNAlocus in S22 at 0.64.DiscussionThis is the first report of physical mappingof the 5S rRNA multigene family in thethree sets of chromosomes of homoeolo-gous groups 1 and 5 in T. aestivum. Fol-lowing the rules of wheat loci nomencla-ture, homoeologous group I loci aredesignated as 5S-Rrna-A1, 5S-Rrna-B1, and5S-Rrna-D1, and homoeologous group 5as

Figure 1. C-banding and in situ hybridization anal-ysis of deletion stocks in Chinese Spring wheat. Eachset of chromosomes includes anormal C-banded chro-mosome on the left (arrow indicates the position ofthe deletion breakpoint), followed by the C-bandeddeletion chromosome, the acetocarmine-stalned dele-tion chromosome, and the deletion chromosome afterin situ hybridization with 5S DNA probe pScT7. Dele-tions analyzed (left, top to bottom): del(IB)(S32).del(ID)(S12), del(SA)(S22:0.03), and del(5A)(S22:0.26). Note the morphology of the IBL arms of thedel(lB)(S32) chromosome, which showed a translo-cation of an unidentified chromatin at he terminal end.Del(5A)(S22:0.03) showed a 5S rRNA locus and wasindistinguishable from labeled 5D chromosomes in thesame cell; it is not shown. Right, top to bottom: del(5B)(S220.33), del(5D)(S22:0.51), and del(5D)(S22).5. sites are visible in del(58)(S22:0.33) and del(5D)(S22) chromosomes. Other deletions lacked 5SrRNA loci.

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Figure 2. Left: An acetocarmine-stained metaphase spread of double ditelosomic 5D S of Chinese Spring wheat, showing 40 normal chromosomes and a 5DS telosomipair (arrows). Right: The same cell shown after in situ hybridization with biotin-labeled 5S rye DNA probe (pScT7), showing 12 distinct sites of hybridization. Note thlabeled 5DS telosome pair and the satellited chromosome IB pair.

5S-Rrna-A2, 5S-Rrna-B2, and 5S-Rrna-D2.Previously, Appels et al., 2 using an iodinat-ed 5S RNA probe in conjunction with ISH,demonstrated a 5S rRNA locus in the sat-ellite region of chromosome B. Later, 5SrRNA loci were assigned to the short armsof chromosomes B, ID, 5A, 5B, and ten-tatively 5D from variation in restrictionfragments of different aneuploids ofChinese Spring wheat.5 Evidence for thepresence of the 5S rRNA locus in DS wasobtained from the missing 410-bp Taqlfragment in nullisomic IDS plants. The lo-cus of 5S rRNA in 5AS was indicated frommissing Haelll and Sstl fragments (0.5 to1.5 kb) in DT5AL plants, and on 5BS frommissing 0.5-kb Taql fragments in DT5BLplants. A 5S rRNA locus was indicated on5DS on the basis of the reduced hybrid-ization to a high-molecular-weight Taqlsmear and the absence of a minor 1.l-kbband in DT5DL plants. Apparently, no 5SrRNA locus wa s detected in IAS despitethe use of a number of restriction enzymeson nullisomic IAS plants.5 Our data con-firmed the presence of a 5S rRNA locus inthe 5DS chromosome arm and detected a5S rRNA locus in the IAS arm.

The detection of a 5S rRNA locus in theIA chromosome in hexaploid ChineseSpring wheat wa s not unexpected. Dvoraket al. 5 reported 5S rRNA loci on chromo-somes IA and 5A in diploid and tetraploidwheats. Chromosome IA contained a short360-bp 5S rRNA unit, as detected in Mboldigest. Failure to observe this size unit inChinese Spring wheat led to the conclu-sion that IA in Chinese Spring has lost the5S rRNA locus. ISH results clearly show

that A in Chinese Spring contains a 5SrRNA locus. Chromosome IA in ChineseSpring probably contains the same 360 bpunit that Dvorak et al. detected in diploidand tetraploid wheats. The 5S rRNA locusprobably was not detected in their exper-iments because of the low copy number(as deduced from the intensity of ISH) andthe masking by the abundant 410-bp and500-bp 5S rRNA families.Dvorak et al.5 obtained evidence for two5S rRNA loci in Lophopyrum elongatum,in all diploid species of Triticum, and inAegilops, with the exception of A. spel-toides (syn. T. speltoides). A. speltoides

showed only a long unit (5S-Rrna-2) locusIn polyploid wheats, the 5S-Rrna-B1 locuswas observed in T. turgidum but not in Ttimopheevii, indicating an apparently closephylogenetic relationship between Aspeltoidesand T. timopheevii. However, thenumber of 5S rRNA loci in T. timopheeviremains an open question in view of ouresults in regard to the detection of the5S-Rrna-AI locus in Chinese Spring wheaand the fact that an Mbol fragment of unknown origin was observed in T. timopheevii.5Among eukaryotic organisms, 5S rRNAgenes are part of the 18SS-28S rRNA arrays

Figure 3. An interphase nucleus of Chinese Spring wheat after in situ hybridization with the 5S rye prob(pScT7), showing 12 sites of hybridization. Note the relatively diffused sites (arrows) that appear to be associatewith the nucleolar body.

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in yeast and certain fungi.3 In all other eu-karyotes, both 5S and 1SS-28S families areindependent arrays that are on the samechromosomes or are on separate chro-mosomes. In wheat, NOR (nucleolus or-ganizing region) loci that contain 18S-28SrRNA families have been found on groups1, 5, an d 6 chromosomes. There is appar-ent linkage between 5S and 18S-28S rRNAloci on chromosomes IA, 5A, B, and 5D,as short arms of these chromosomes con-tain both loci. Although in chromosomeIBS the I8S-28S rDNA array is separatedfrom the 5 rDNA array by a stretch ofDNA ca. 35 million bp long (see below),in interphase nuclei a pair of 5S loci weremore diffused and apparently were asso-ciated with a nucleolar body (Figure 3).This 5S-Rrna-BI locus, in association withthe NOR locus on IBS, may be involvedin the organization of the nucleolus. Incorn, even though they are on differentchromosomes, 55S and NOR loci may as-sociate with a nucleolar body during cer-tain stages of cell division.' 5Except for chromosome A, chromo-some arms that carry 55S rRNA loci alsocarry interstitial C-bands. Dvorak et al. 5speculated that C-bands may be the sitesof 55SRNA loci. Ou r data do not supportthis hypothesis. In BS, del(lB)(S32) con-tained the C-band, bu t the 55 locus wasdeleted. However, in certain clear C-bandpreparations, a faint C-band that lies be-tween bands 531 and S33 may be the siteof the 5S locus. In 1DS, the major C-band(S21) lies at FL 0.70, whereas the 5S rRNAlocus is at 0.96. In SAS, the major C-bandis at FL 0.72 and the 5S rRNA locus is veryclose to it at FL 0.76. In 5BS, del(5B)(S22:0.33) with a breakpoint at FL 0.67 lackedthe prominent C-band but retained the 5Slocus at FL 0.63. Thus, deletion analysisand the measurement of relative positionsof C-bands and 55S rRNA loci do not indi-cate any association between majorC-bands and 5 rRNA loci.Based on a total haploid DNA contentof wheat of 16 x 109 bp 4.12and a total lengthof the haploid chromosome complementof 248 Am' 0 and assuming constant DNAdensity along the chromosome length, 1,M of chromosome length contains an es-timated 70 million bp of DNA. From dele-tion analysis and arm location of the genesas determined by ISH, 55 DNA sequencescan be delimited to chromosome lengthsof about I m for most of the loci we ana-lyzed. Thus, for the IBS arm, the 5S rRNAlocus lies at 0.5 length of the satellite (totalsatellite length of I m) or within an es-timates 35-million-bp stretch of DNA. As

Fllre 5. Asterisks indicate the location of 5SrRNA loci on specific bands of the short arms of group I agroup 5 chromosomes. Deletion breakpoints and the position of the 55SRNA locus are also indicated. Deletichromosomes, shown to the right of each arm, show only the 5S rRNA locus, if detected.

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most recombination in the IBS arm is re-stricted to the distal part of the satellite,tightly linked markers may lie within a dis-tance of 1million bp from a gene of inter-est, and this is well within the resolvingpower of pulsed-field gel electrophore-sis. 8 ' 9 Moreover, using an appropriatecombination of deletions, DNA probes forspecific chromosomal segments can beobtained through the use of methods suchas the phenol emulsion reassociationtechnique. 4The deletions we used were producedin the progeny of plants that were mono-somic for a gametocidal chromosome ofA. cylindrica. Banding an d ISH results in-dicated that most deletions resulted froma single break, with a concomitant loss ofa terminal chromosome segment. This isan important observation. First, it rulesou t the occurrence of complex deletion/duplication events produced as a resultof the breakage-fusion-bridge cycle, asreported by McClintock in corn.' 3 Thosedeletions would be unsuitable in geneticmapping experiments. Second, deletionsoccur in tissues (presumably either in ga-metes before fertilization or in zygotes im-mediately after fertilization), where theyare immediately healed and/or occasion-ally result in translocations. Ou r limitedexperience indicates that these deletionsare cytogenetically stable. Third, becausedeletions are simple and can occur any-where in the genome and in the progenyof plants that lack the alien chromosome,they provide an elegant deletion analysissystem in wheat. As the genome of wheatis unusually large and complex, deletionsprovide an opportunity for cytogenetic andmolecular delineation of agronomicallyimportant traits to small chromosome seg-ments, possibly leading to molecular clon-ing of previously intractable genes ofwheat.Th e combination of the biotin-labeling

technique with deletion stocks has beenshown to be an efficient approach for thephysical mapping of 5S rRNA loci in sixpairs of homoeologous groups 1 and 5chromosomes in Chinese Spring wheat.Th e location of the 5S rRNA locus in 5Dwa s confirmed, and a new location of 5SrRNA locus in the 1A chromosome ofChinese Spring wheat was demonstrated.Biotin-labeling is a sensitive technique forthe accurate physical mapping of 5S rRNAan d may be the method of choice for ISHanalysis. ISH is an especially powerfultechnique for gene localization in caseswhere restriction fragment variation is rareor absent. Other attractive applications in-clude the analysis of the location an d geneorder of DNA sequences on chromosomesand cytogenetic identification of aneu-ploid stocks, which are now based on anumber of methods, such as cytologicalidentification of chromosomes, geneticsegregations, and gene dosage effects.2 0

References1. Ambros PP, Matzke MA. and Matzke AIM, 1986.Detection of a 7 kb unique sequence (T-DNA) in plantchromosomes by in situ hybridization. Chromosoma34:11-18.2.Appels R,Gerlach WL, Dennis ES, Swift H, nd Pea-cock WJ, 1980. Molecular and chromosomal organi-zation of DNA sequences coding for the ribosomal RNAsin cereals. Chromosoma 78:293-311.3. Appels Ran d Honeycut RL, 1986. rDNA: evolutionover abillion years. In: DN A Systematics: plants (DuttaSK, ed). Boca Raton: CRC Press; 82-135.4.Bennett MD, 1972. Nuclear DN A content an d mini-mum generation time In herbaceous plants. Proc RSo cLondon Se r B 181:109-135.5. Dvorak J,Zhang H-B. Kota RS, and Lassner M, 1989.Organization and evolution of the 5S ribosomal RNAgene family in wheat and related species. Genome 32 :1003-1016.6. Endo TR, 1986. Complete identification of commonwheat chromosomes by means of a C-banding tech-nique. Jpn J Genet 61:89-93.7. Endo TR, 1988. Induction of chromosomal structuralchanges by achromosome of Aegilops cyindricaL. incommon wheat. J Hered 79:366-370.

8.Canal MW, Young ND, and Tanksley SD , 1989. Pulsedfield gel electrophoresis and physical mapping of argeDNA fragments in the Tm-2a region of chromosome 9in tomato. Mol Gen Genet 215:395-400.9. Gerlach WL and Dyer TA, 1980. Sequence organization of the repeating units in the nucleus of wheawhich contain 5S rRNA genes. Nucleic Acids Re s 84851-4865.10. Gill BS, 1987. Chromosome banding methods, standard chromosome band nomenclature, and applications in cytogenetic analysis. In: Wheat and wheat improvement, 2nd ed. (Heyne EGC,d). MadisonWisconsin: Am. Soc. Agron.; 243-254.11.Kota RS and Dvorak J, 1986. Mapping of a chromosome pairing gene and 5S rRNA genes in TnricumoestivumL. by a spontaneous deletion in chromosomearm 5Bp. Ca n J Genet Cytol 28:266-271.12. May CE and Appels R.1987. The molecular geneticof wheat: toward an understanding of 16 billion baspairs of DNA. In: Wheat and wheat improvement, 2nded. (Heyne EG , ed). Madison, Wisconsin: Am. SocAgron.; 165-198.13. McClintock B, 1942. The stability of broken endof chromosomes in Zea mays. Genetics 26:234-281.14. Nussbaum RL, Lesko JG. Lewis RA , Ledbetter SAand Ledbetter DH , 1987. Isolation of anonymous DNAsequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation. Proc Natl Acad Sci US A84:6521-6525.15. Phillips RL, McMullen MD, and Rubenstein, I, 1988Ribosomal DNA in maize. In: Chromosome structureand function (Gustafson JP and Appels R,eds). NewYork: Plenum; 201-214.16. Rayburn AL and Gill BS, 1985. Use of biotin-labeledprobes to map specific DNA sequences on wheat chromosomes. J Hered 76:78-81.17. Reddy Pand Appels R,1989. A second locus fothe 5S multigene family in Secale L.: sequence divergence in two lineages of the family. Genome 32:456467.18. Scoles GJ, Gill BS, Xin Z-Y, Clarke BC , McIntyre CLChapman C,and Appels R, 1988. Frequent duplicatioand deletion events in the 5S RNA genes and the associated spacer regions of the Trlticeae. Plant Syst Evo160:105-122.19. Snape JW, Flavell RB , O'Dell M, Hughes W G, andPayne PI, 1985. Intrachromosomal mapping of the nucleolar organizer region relative to three marker locon chromosome I Bof wheat (Triticum aestivum). TheoAppl Genet 69:263-270.20 . Young ND, Miller JC, and Tanksley SD, 1987. Rapichromosomal assignment of multiple genomic clonein tomato using primary trisomics. Nucleic Acids Re15:9339-9348.

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