tools and methodologies for cytogenetic studies of plant chromosomes

ISSN 0095-4527, Cytology and Genetics, 2008, Vol. 42, No. 3, pp. 189–203. © Allerton Press, Inc., 2008.

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1

HISTORICAL PERSPECTIVES

Prior to the 1920s, cytological studies were carriedout on biological tissues that were embedded in paraf-fin, sectioned, and stained (Wilson, 1925; Darlington,1937). The methods that were in vogue were not suffi-ciently refined to permit the detection of such grossmorphological features as centromeres, secondary con-strictions, and satellites of chromosomes. They merelyfacilitated the determination of chromosome numbersand permitted detection of approximate size differencesamong the chromosomes in somatic and meiotic cellsof many eukaryotic species. For example, in the genus

Triticum

, Sakamura (1918) showed that the somaticcells of the species

monoccocum, turgidum

, and

aesti-vum

possessed 14, 28, and 42 similar-sized chromo-somes, respectively.

During the 1920s and 1930s, innovations wereintroduced which facilitated cytological and karyotypicanalyses. In 1921, Belling described a technique forstudying meiosis in plant species that involved thesquashing of anthers. This method permitted the sepa-ration of PMCs and facilitated the spreading of theirchromosomes. In 1929, Kagawa, working with

Triti-cum

and

Aegilops

species, demonstrated that treatmentwith chloral hydrate before fixing and staining of thecells shortened the chromosomes. This made it easier toseparate them and study their gross morphological fea-tures: centromeres, secondary constrictions, and satel-lites. Pretreatment with other agents such as alpha bro-monaphthalene (Schmuck and Kostoff, 1935), colchi-cine (O’Mara, 1939), paradichlorobenzene (Meyers,1945) and cold water (Hill and Myers, 1945) also per-mitted identification of these chromosomal substruc-

1

The text was submitted by the authors in English.

tures. By the early 1940s, the squashing technique, con-comitant with appropriate modifications and prtreat-ment, completely replaced the method of microtomesectioning of tissues in chromosome studies usingsomatic and meiotic tissues of most species (Hillart,1938; Aase, 1935; O’Mara, 1939).

The squash technique, with appropriate modifica-tions, was also used successfully in chromosome stud-ies of insects, amphibians, and other animals (White,1954), excluding mammals (Hsu, 1979). Mammaliancytology had to await the innovations of hypotonicsolutions (Hsu, 1952), in vitro culturing of tissues andcells (Hsu and Pomerat, 1953), and their colchicine pre-treatment (Hsu and Pomerat, 1953) in order to obtaingood spreads of somatic chromosome complements.Using the innovative procedures, the chromosomenumber in man was first correctly determined to be2

n

= 46 by both Tjio and Levan, and Ford and Hammer-ton, in 1956. In the ensuing years this technique, com-bined with the use of phytohemagglutinin (Nowell1960) to stimulate cell divisions, was applied to karyo-typing euploids, aneuploids, and individuals with chro-mosomal abnormalities in numerous mammalian spe-cies (see Hsu and Benirschke 1967–1977). The varioustechniques that have been used to this day for karyotyp-ing hundreds of species using gross chromosomal mor-phological features are detailed in La Cour (1947), Dar-lington and La Cour (1960), Sharma and Sharma(1965), and Haskell and Wills (1968).

STANDARD SOMATIC KARYOTYPESIN PLANT SPECIES

In plant species, such as maize (Rhoades andMcClintock, 1935), tomato (Rick and Barton, 1954;

Tools and Methodologies for Cytogenetic Studiesof Plant Chromosomes

1

G. Fedak

a

and N.-S. Kim

b

a

Eastern Cereals and Oilseeds Research Centre Agriculture and Agri-Food Canada 960 Carling Avenue Ottawa,Ontario, K1A 0C6 Canada

b

Department of Molecular Biosciences Kangwon National University Chunchon, 200-701 Korea

Received February 1, 2008

Abstract

–A brief overview is presented in advances in cytogenetic methodology and the development of ane-uploid stocks since the 1920s. The methodologies range from first reports of chromosome numbers of majororganisms, the development of chromosome karyotypes, then aneuploid stocks in major crop plants. Molecu-lar inputs include chromosome banding techniques, molecular marker maps, and in situ hybridization meth-odologies. All of the new techniques have greatly increased the degree of resolution obtained from cytogeneticstudies.

DOI:

10.3103/S0095452708030067

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Rick et al., 1964) and rice (Chu, 1967; Kurata andOmura, 1978), individual chromosomes (and thereforetrisomies) could not be identified in somatic cells usingstandard straining procedures because the chromo-somes were either too small and/or similar in morphol-ogy. In most plant species, however, at least a few of thechromosomes and trisomies could be identified in stan-dard somatic karyotypes. For example, in barley(Tsuchiya, 1960) and

Ptunia axiillaris

(Reddi and Pad-maja, 1982), three of the chromosome pairs and triso-mies could be identified using standard procedures.In beets (Romagosa et al., 1986), eight out of nine chro-mosomes and trisomies could be identified in the stan-dard fashion, and in

A. strigosa

, Rajhathy (1975) wasable to distinguish all chromosomes and identify all tri-somies from standard somatic karyotypes.

Although numerous karyotypic studies have beencarried out since 1939 in several species of the genus

Triticum

, only the species

T. monococcum

(AA),

T. tur-gidum

(AABB), and

T. aestivum

(AABBDD) will bereviewed here. Studies by Camara (1943), Coucoli andSkorda (1966), Giorgi and Bozzini (1969), and Kerbyand Kuspira (1988) have shown that there are 14 similarchromosomes in the diploid complement of

T. mono-coccum

; one ST pair, two M pairs, and four SM pairs.Depending on the accession line studied, either one ortwo chromosome pairs were found to possess satellites(Camara, 1943; Riley et al., 1958; Coucoli and Skorda,1966). The karyotype of

T. turgidum

consists of twoSAT pairs, two ST pairs, seven SM pairs, and three Mpairs of chromosomes (Giorgi and Bozzini, 1969a;Kerby and Kuspira, 1988). Since the A genome has thechromosome constitution given above, the karyotype ofthe B genome in

T. turgidum

must consist of two SATpairs, one ST pair, three SM pairs, and one M pair ofhomologues. Two of these chromosome pairs possesslarge satellites (Pathak, 1940; Riley et al., 1958) thatbelong to the B genome (Okamoto, 1957). The satel-lites in the A genome in

T. turgidum

are suppressed(Riley et al., 1958). At most, four to six of the chromo-somes in the somatic complement of this species can bedistinguished using standard procedures.

Depending on the genotype studied, either one(Sears, 1954), two (Morrison, 1953), three (Pathak,1940; Camara, 1943) or four (Kagawa, 1929, Schulz-Schaeffer and Haun, 1961) satellited chromosomes areobserved in

T. aestivum.

These belong to the B and Dgenome (Sears, 1954; Schulz-Schaeffer and Haun,1961). Camara (1943), Morrison (1953), Sears (1954,1958). Schulz-Schaeffer and Haun (1961), and Gill(1987) have shown that the chromosomes in thesomatic complement of bread wheat are similar in size.For example, Gill (1987) reported that they range inlength from 8.4 um for chromosome ID to 13.8 um forchromosome 3D. The latter observations also show that(i) except for the chromosome 4A pair, all other pairs inhomoeologous groups 1, 4 and 5 are highly heterobra-chial (ST); (ii) except for the chomosome 7B pair, allother pairs in homoeologous groups 6 and 7 are M; and

(iii) chromosome pairs in homoeologous groups 2 and3, as well as chromosome pairs 4A and 7B, are SM.Even in the best somatic metaphase spreads, only a lim-ited number of chromosomes and chromosome pairscan be distinguished by standard methods.

CHROMOSOME IDENTIFICATIONDURING MEIOSIS

Standard staining procedures render nucleoli clearlyvisible at the pachytene stage and permit the detectionof chromosomes that carry NORs. Moreover, in corn(Rhoades and McClintock, 1935), tomato (Rick andBarton, 1954), and rice (Khush et al., 1984), it is possi-ble to identify each monovalent, bivalent, and multiva-lent association on the basis of its length and chro-momere pattern during pachytene. Thus, a chromo-some in triplicate in these species is easily identified byexamination of the trivalent configuration withpachytene using standard staining techniques.

Banding techniques.

In almost all species, the use-fulness of standard staining procedures, however, hasbeen limited. Although they have facilitated the ascer-tainment of chromosome numbers and gross morpho-logical features of chromosomes, they have not permit-ted an accurate and unequivocal identification of all thechromosomes, and therefore the aneuploids, of a spe-cies. An exhaustive analysis of the karyotype requiresthe use of staining procedures that can reveal each chro-mosome as a specific, unique, and constant pattern ofalternating dark and light banding regions, topologi-cally equivalent to the bands in the polytene chromo-somes in salivary gland cells of

D. melanogaster.

Darlington and La Cour (1940) demonstrated thatwith cold treatment of somatic cells of

Trillium erec-tum

, some regions of chromosomes revealed uniquepatterns by appearing thinner and less intensely stainedthan the rest of the chromosomes. The utilization of flu-orescent and other dyes, together with various modifi-cations in the pretreatment of cytological material inthe late 1960s, heralded a new era of cytogenetics. Newand reliable staining procedures were introduced, eachof which was capable of revealing a unique “bandingpattern” of the chromosomes of a given species.By 1972 the application of one or another of five majorbanding techniques (Q, G, R, C, and N) for the purposeof karyotypic analysis came into vogue. These have ledto a more precise cytogenetic and phylogenetic analysisof various eukaryotes.

Q-BANDING

Caspersson and his colleagues in 1968 were first todemonstrate that fluorescent dyes such as quinacrineand quinacrine mustard bind preferentially to certainregions of normal mitotic chromosomes of

Cricetulusgriseus, V. faba

and

T. erectum.

As a result, unique pat-terns of brightly fluorescent regions alternating withnonfluorescent (dark) regions were produced in each


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TOOLS AND METHODOLOGIES FOR CYTOGENETIC STUDIES 191

chromosome. Weisblum and de Haseth (1972) andBurkholder (1988) have shown that fluorescent dyesinteract with AT base pairs and those regions of DNAthat are sufficiently AT-rich (70–100%) fluoresce andappear as bright bands (Q bands). Q-banding permitsidentification of all the chromosomes and their homo-logues in most species. For example, in man all 23 pairsof homologous chromosomes can be distinguished onthe basis of their Q-banding patterns (Caspersson et al.,1971). In

Scilla sibirica

, all eight chromosome pairscan be identified (Caspersson et al, 1969). Q-bandingdoes not require any pretreatment and is the simplest ofall the banding methods. Compared to other bandingtechniques, it has several disadvantages; the fluorescentbands are not permanent; the technique requires the useof ultraviolet light and does not stain the ends of chro-mosomes. As a consequence, Q-banding has been usedto a limited extent and since the late 1970s (Pinkelet al., 1988) has been largely replaced by other bandingmethods. In plants, Q-banding studies have been lim-ited to a few in

Trillium, Scilla, Allium, Crepis, Lilium,Secale

, and

Vicia

(Caspersson et al., 1969; Vosa andMarchi, 1972a; Kongsuwan and Smyth, 1977; Sch-weizer, 1980; Rowland, 1981).

G-BANDING

In 1971, Drets and Shaw, Patil et al., Seabright, andSumner et al. independently developed a protocol foranimal species whereby each chromosome segmentand chromosome revealed a unique pattern of bands.Each of the protocols, by using a variety of treatmentsbefore fixing -chromosomes and staining with Giemsa,yielded a banding pattern in normal mitotic chromo-somes that was similar to the one revealed by theQ-banding technique. The dark regions are the topolog-ical equivalents of Q-bands and are called G-bands,whereas the light regions are equivalent to the nonfluo-rescent dark ones revealed with the use of fluorescentdyes (Drets and Shaw, 1971; Dutrillaux and Lejeune,1975). Application of G-banding methods to prophaseand prometaphase chromosomes in animals revealed alarger number of bands than at metaphase which per-mited more precise karyotyping and cytogenetic analy-sis (Yunis, 1981; Iannuzzi, 1990).

The basis for G-banding is currently unknown. Oneplausible explanation is that offered by Comings(1978), who postulated that prophase and metaphasechromosomes contain a basic chromomeric structurethat can be enhanced. This enhancement occurs byinducing a certain rearrangement of the fibers awayfrom the light bands toward the G-bands, possibly someextraction of light-band DNA with denatured nonhis-tone proteins, followed by the marked enhancement ofthis pattern through the ability of thiazin dyes inGiemsa to side stack on available DNA. Sumner (1982)and Burkholder (1988) have proposed alternate mecha-nisms.

Although the technique has been attempted in manyplant species, G-bands have been generated in the chro-mosomes of only a few species:

Tulipa gesneriana

(Fil-ion and Blakey, 1979),

Pinus resinosa

(Drewry, 1982),and

Vicia hajsatana

(Wang and Kao, 1988). The failureto produce G-bands in the chromosomes of most plantspecies, including those in

Triticeae

, has been attrib-uted to the increased condensation of plant chromo-somes (Greilhuber, 1977; Drewry, 1982). Anderson,et al. (1982), however, failed to show consistent differ-ences in the degree of compaction, based on measure-ments of lengths and volumes of chromosomes fromseveral plant and animal species. Wang and Kao (1988)demonstrated that improper pretreatment of plant chro-mosomes alters the organization of their chemical con-stituents and renders them unresponsive to the

G

-band-ing procedure.

REVERSE (R)-BANDING

This banding technique was developed by Dutril-laux and Lejeune in 1971. Mild denaturation by heatand subsequent staining of chromosomes with Giemsaor a fluorochrome dye revealed a banding pattern that isthe reverse of the patterns produced by the G- andQ-banding methods (Bobrow et al., 1972; Comings,1973; Dutrillaux et al., 1973). Specifically, if the chro-mosomes are stained with Giemsa, the dark bands(R bands) produced with this technique are equivalentto the light bands produced by the G-banding techniqueand vice versa (Dutrillaux and Lejeune, 1971, 1975).If a flurochrome dye such as acridine orange or olivo-mycin is used, fluorescent R-banding is the reverse ofQ-banding in that the R-bands fluoresce bright greenand the non-R-bands show a faint red color (Schweizer1976; Lin et al., 1980; Schmid and Guttenback, 1988).R-banding is particularly useful in the detection ofstructural rearrangements involving the ends of chro-mosomes in that it stains telomeres as T-bands (Dutril-laux et al., 1973).

R-bands have been detected in only a few plant spe-cies, e.g.,

S. sibirica, V. fava, Allium

spp., none of whichbelong to the

Tririceae

tribe (Schweizer, 1980; Deum-ling and Greilhuber, 1982; Loidl, 1983). Moreover,since the R-bands in these species are few in numberand faint in expression, they have not been used forkaryotyping and cytogenetic studies.

R-bands can be produced by GC-specific fluoro-chromes (Schweizer, 1976; Van de Sande et al., 1977;Holmquist et al., 1982), although the mechanism ofR-banding is unknown (Burkholder, 1988). The mech-anism proposed by Comings (1978) may also explainR-banding if the DNA and proteins in the G- andR-bands are selectively denatured under different con-ditions of pH, salt concentrations, and temperature.

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C-BANDING

Pardue and Gall (1970) and Jones (1970) indepen-dently demonstrated a procedure which with stringenttreatment of chromosomes prior to fixation and stainingwith Giemsa, stained only the regions of constitutiveheterochromatin in chromosomes of

Mus musculus.

The regions, now referred to as C-bands, were observedto be proximal to the centromeres of all the chromo-somes in this species and have since been demonstratedin chromosomes of the guinea pig (Yasmineh andYunis, 1975),

Drosophila

spp. (Gall and Atherton,1974; Brutlag et at., 1977),

Rattus rattus

(Yosida andSagai, 1975), and many other animal species. Constitu-tive heterochromatin usually appears as “satelliteDNA” when nuclear chromosomal DNA is fragmentedand centrifuged (Kit, 1961). It consists of short, highlyrepeated base pair sequences in tandem (Southern,1970; Corneo et al., 1970; Gall and Atherton, 1974;Brutlag et al., 1977) in one or more regions of all ormore chromosomes in most species. Arrighi and Hsu(1971) showed that C-bands are located next to the cen-tromeres of each chromosome, next to the secondaryconstrictions of chromosomes 1, 9, and 16 as well asthe satellites of acrocentric chromosomes in man. Withfew exceptions, constitutive heterochromatin in animalspecies reveals a consistent pattern of distribution.Therefore, C-banding in animal species does not corre-spond to a banding pattern in a strict sense. Its applica-tion in these species is limited because it does not allowprecise recognition of individual chromosomes.

Several lines of evidence indicate that the productionof C-banding is due to the extraction of non-C-bandDNA and denaturation of proteins in these regions. TheDNA in constitutive heterochromatin is resistant toextraction, remains within the chromosomes, and istherefore stainable by Giemsa (Pathak and Arrighi, 1973;Dille et al., 1987; Burkholder, 1988).

Since the initial studies in

V. faba

by Vosa and Mar-chi in 1972, chromosomes of many species of

Aegilops,Agropyron, Elymus, Hordeum, Secale

, and

Triticum

(Gill and Kimber, 1974a and 1974b; Linde-Laursen,1975; Vosa, 1976; Gerlach and Peacock, 1980; Singhand Tsuchiya, 1981b; Seal, 1982; Teoh and Hutchin-son, 1983; Endo, 1986; Morris and Gill, 1987) andother plant species (Linde-Laursen et al., 1980; Loidl,1983) have revealed C-bands. These studies show thatthere is a fundamental difference in the distribution ofconstitutive heterochromatin within chromosomes ofanimals and plants. C-bands in the chromosomes ofplants can be located at various sites including theregions they characteristically occupy in the chromo-somes of animals. Moreover, in most plant species, e.g.,

Allium carinatum

(Loidl, 1983),

Hordem

spp. (Linde-Laursen et al., 1980), and

Agropyron elongatum

(Endoand Gill, 1984a), some of the chromosomes do notreveal C-bands next to their centromeres. Thus, in manyplant species, a unique C-banding pattern occurs ineach arm of each chromosome in the genome. This

allows individual chromosomes in the somatic cells tobe identified on the basis of their patterns (loc. cit.).

Gill and Kimber (1974a) published the first reporton C-banding patterns of chromosomes of

T. aestivum.

Despite the efforts of many investigators in the interim,Endo (1986), using an improved C-banding technique,was able to unequivocally identify all 21 chromosomesin the genome of cvs. Chinese Spring and Norm 61 of

T. aestivum.

These banding patterns are currentlyaccepted as standard patterns for the chromosomes ofcommon wheat (Gill, 1987; Gill et al., 1988). Ferreret al. (1984) applied the C-banding protocol to thestudy of chromosomes in meiocytes and clearly identi-fied nine of the 21 chromosome pairs.

Except for the work of Simeone et al. (1988), theefforts of other investigators (Zurabishibili et al., 1978;Seal, 1982; Lukaszewski and Gustafson, 1983) to iden-tify the A and B genomes of

T. turgidum

on the basis oftheir C-banding patterns have been inconsistent. Sime-one et al. (1988) reported C-banding patterns for chro-mosomes of

T. turgidum

that were equivalent to thoseof their homologues in the A and B genomes of

T. aes-tivum

and therefore permitted their unequivocal identi-fication. Shang et al. (1989) used the HKG (HCI-KOH–Giemsa) method to produce banding patterns in chro-mosomes of

T. turgidum

that were in part similar totheir C-banding patterns, thus allowing identification ofsome of the chromosomes.

C-banding of the chromosomes of

T. monococcum

has been reported by both Gill and Kimber (1974a) andKuz’menko et al. (1987). The banding patterns of thechromosomes in these two investigations were partiallydissimilar. Moreover, the C-banding patterns of someof the chromosomes in these publications were differ-ent from those for the A-genome chromosomes in

T. aestivum

, thus precluding their identification. Usingthe HKG method, Shang et al. (1988 and 1989)reported banding patterns for chromosomes of

T. mono-coccum

, partially, resembled their C-banding patterns.Although the banding patterns revealed by the HKGmethod rendered some of the chromosomes distin-guishable from the others, they did not allow for theirunequivocal identification.

C-banding has been used in the identification of ane-uploids (Linde-Laursen, 1978b, 1982; Zeller et al.,1987), translocations, and other structural rearrange-ments (Gill and Kimber, 1977; Lukaszewski andGustafson, 1983; Lapitan et al., 1984) and the precisephysical mapping of genes (Kota and Dvorak, 1986;Jampates and Dvorak, 1986). C-banding analysis of

durum-timopheevi

and

durum-speltoides

hybrids byChen and Gill (1983) has supported Dvorak’s sugges-tion (1983) that chromosomes 4A and 4B should bereassigned to the B and A genomes, respectively. More-over, C-banding has clarified and further substantiatedphylogenetic conclusions based on chromosome pair-ing in interspecific and intergeneric hybrids (Gill andKimber, 1974a; Hutchinson and Miller, 1982; Chen andGill, 1983; Morris and Gill, 1987).


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N-BANDING

In 1973 Matsui and Sasaki developed a techniquethey called N-banding, which selectively stained NORsin the chromosomes of mammalian species. Funakiet al. (1975) improved this procedure and demonstratedthat N-bands were confined to the NORs of the chromo-somes of 27 eukaryotic species that they studied. Faustand Vogel (1974) and Pimpinelli et al. (1976) observedthat the bands obtained with this procedure are notNOR-specific in

D. melanogaster

or the mammalianspecies studied. Nevertheless, these non-NOR bandswere and continue to be referred to as N-bands. Usingthe method of Funaki et al. (1975), with slight modifi-cations, Gerlach (1977) and Jewell (1979), workingwith

Triticum

and

Aegilops

species, respectively,clearly demonstrated that N-bands do not necessarilycorrespond to NORs. Moreover, at least some of thechromosomes in the species analyzed had uniqueN-banding patterns, permitting their identification.Gerlach (1977) identified nine of the 21 chromosomesof common wheat on the basis of their N-banding pat-terns. Subsequently, Endo and Gill (1984a) identified16 of the 21 chromosomes of common wheat, includingfive in the A genome, using an improved N-bandingprotocol. Jewell (1979) identified all 14 chromosomesof

Aegilops variabilis

on the basis of their N-bandingpatterns. The technique has also been used to identifychromosomes in barley (Singh and Tsuchiya, 1982b),rye (Jewell, 1981; Schlegel and Gill, 1984), lentils(Mehra et al., 1986), and

Elymus

spp. (Morris and Gill,1987). N-banding has also been used to identify varioustypes of aneuploids (Singh and Tsuchiya, 1981b; Zelleret al., 1987), alien addition and substitution lines(Islam, 1980), and translocations and deletions (Jewell,1978). It should be noted that N-banding has beenattempted in

T. monococcum

by B.S. Gill (personalcommunication) and in our laboratory without success.Why this should be, since some of the A genome chro-mosomes in

T. aestivum

contain N-bands, is unknown.

Gerlach (1977), and subsequently others, noted thatmany of the N-bands occupy the same positions asC-bands, implying that the N-banding technique, likethe C-banding one, identifies constitutive heterochro-matin and that at least two classes of heterochromatinoccur in wheat, rye, and other species. This was con-firmed by Schlegel and Gill (1984) and Endo and Gill(1984a). Some heterochromatic regions in each chro-mosome stain positively using both C- and N-bandingprocedures. These regions are referred to as C + N?bands. Other such regions stain positively only withC-banding techniques. These heterochromatic seg-ments are called C + N

–

bands. Schlegel and Gill (1984)have shown that only N-bands (C + N

+

bands) possessmultiple copies of the (GAA) n (GAG) n sequenceDNA. The base pair sequences in C + N

–

bands have notbeen identified.

Gill (1987) and Gill et al. (1988) have proposedbanding nomenclatures for the chromosomes of

T. aes-tivum

cv. Chinese Spring.

Identification of NORs and Nucleoli.

Nucleolusorganizer regions (NORs) are the sites of rRNA genesin the chromosomes of animal (Ritossa and Spiegel-man, 1965; Wallace and Birnstiel, 1966; Hendersonet al., 1972, 1974) and plant species (Phillips et al.,1971; Flavell and O’Dell, 1975; Hutchinson and Miller,1982). Methods have been developed for the selectivestaining of these chromosomal regions both in animals(Goodpasture and Bloom, 1975; Howell et al., 1975;Verma and Babu, 1984) and plants (Hizume et al.,1980; Lacadena et al., 1984; Mehra et al., 1985;Cunado et al., 1986). The Ag–As (ammoniacal silver)method selectively stains those sites on chromosomeswhich correspond exactly to regions that can bedetected by in situ hybridization with rDNA probes(Howell et al., 1975; Miller et al., 1976a and 1976b).It seems that this procedure stains only the NORs thatare functionally active during the preceding interphase(Howell, 1977; Schmiady et al. 1979). There is evi-dence to suggest acidic or nonhistone proteins associ-ated with the rDNA regions are responsible for theselective staining of NORs (Howell et al., 1975; Wangand Juurlink, 1979; Howell, 1985).

In situ hybridization and its application to wheatcytogenetics.

Gall and Pardue (1996) and John et al.independently reported a procedure that facilitated thecytological detection of hybrid nucleic acid regions.This technique involves the annealing of radioactivelylabelled nucleic acid sequences to cytological (chromo-somal) preparations in situ (on slides) and subsequentdetection of the hybrid regions by autoradiography.Specific DNA sequences in the chromosomes of animaland plant species have been localized with this tech-nique. These include some highly repetitive short base-pair sequences in the chromosomes of animals (Pardueand Gall, 1970; Brutlag et al., 1977) and plants(Gerlach and Peacock, 1980; Appels and Mclntyre,1985; Ganal et al., 1988; Lapitan et al., 1989), moder-ately repeated sequences such as rRNA genes in thechromosomes of animals (Wimber and Steffensen,1970, 1973; Henderson et al., 1972) and plants (Wim-ber et al, 1974; Gerlach and Bedbrook, 1979; Masciaet al., 1981; Clark et al., 1989), and some single copygenes in animals (Harper and Saunders, 1981; Hender-son, 1982; Olsen et al., 1989) and plants (Ambros et al.,1986; Huang et al., 1988).

Gerlach and Peacock (1980) isolated a highly repet-itive DNA sequence from

T. aestivum

cv. ChineseSpring and hybridized it to cytological preparations of

T. aestivum, T. dicoccoides, T. monococcum

, and

Ae. squarrosa.

A number of heavily labelled chromo-somes were observed in the preparations of

T. aestivum

and

T. dicoccoides

, but not in those of

T. monococcum

and

Ae. squarrosa.

On the basis of these results, theauthors concluded that most of the heavily labelled

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chromosomes belong to the B genome. C-banding stud-ies by Endo (1986) and Gill (1987) and N-bandingreports by Endo and Gill (1984b) support this conclu-sion. Peacock et al. (1981) demonstrated that the DNAsequence is composed of repeated (GAA) n and (GAG)n units. Rayburn and Gill (1985) showed that the majorC-and N-bands correspond to sites which contain thissatellite sequence. In situ hybridization studies with ahighly repetitive D-genome specific DNA sequenceisolated from

Ae. squarrosa

were used by Rayburn andGill (1986) to identify D-genome chromosomes inhexaploid wheat.

At least four pairs of chromosomes (1A, 1B, 5D,and 6B) of

T. aestivum

contain NORs (Crosby, 1957;Darvey and Driscoll, 1972). If NORs are the sites ofribosomal RNA (rRNA) genes, then all these chromo-somes should be expected to possess clusters of rRNAgenes. Flavell and Smith (1974a, 1974b) and Flavelland O’Dell (1976, 1979) showed that in hexaploidwheat, a large proportion of the rRNA genes are onchromosomes IB and 6B, with only a small proportionof the genes residing on chromosomes 1A and 5D.Gerlach and Bedbrook (1979) cloned the 18S + 26SrRNAgenes of

T. aestivum

into a bacterial plasmid andshowed that the probe derived from this clone hybrid-ized to regions on chromosomes 1B and 6B. Milleret al. (1980) showed that the same rDNA probe hybrid-ized to minor NORs on chromosomes 1A and 5D inbread wheat. A similar approach with a different rDNAprobe enabled Appels et al. (1980) to confirm the loca-tion of rRNA genes on chromosomes IB, 5D, and6B only. They speculated that too low a level of rRNAgenes on chromosome 1A may have precluded theirdetection by in situ hybridization experiments. In situhybridization experiments in

T. turgidum

and

T. timopheevi with rDNA probes confirmed the assign-ment of rDNA loci to chromosomes IB and 6B (Appelsand Dvorak, 1982; Dvorak and Appels, 1982). Milleret al. (1983) showed that, in T. Urartu, a labelled rDNAprobe hybridized in situ to a region on chromosome 5Athat corresponds to the NOR. Some genotypes ofT. urartu and other diploid wheat have been shown tohave two chromosome pairs with nucleolus organizers(Gerlach et al., 1980). The second NOR must, bydeduction, be located on chromosome 1A. Frankel et al.(1988), using a synthetic tetraploid AABB and a3H-labelled rDNA probe, clearly demonstrated that theNORs of two pairs of A-genome chromosomes werelabelled after in situ hybridization. Apart from onebeing more heavily labeled than the other, the two chro-mosome pairs (1A and 5A) could not be distinguishedcytologically.

Information on the location of 5S rRNA genes inwheat species is scanty. Appels et al. (1980) localizedthe 5S rRNA gene cluster to chromosome 1B of T. aes-tivum at a site distinct from and distal to the NORregion. Kota and Dvorak (1986) mapped these genes toa single site on the p arm of chromosome 5B, using aline with a spontaneous deletion. Lassner and Dvorak

(1985) and Kota and Dvorak (1986) suggested thatchromosomes 5A and 5D may also carry the genes for5S rRNA. Scoles et al. (1987), using cloned 5S rDNAsequences, obtained unequivocal evidence for the pres-ence of 5S rRNAgenes on chromosomes 1B, 1D, and5B in T aestivum. Studies by Dvorak et al. (1989) haveshown that chromosomes 1A and 5A of T. monococcumvar. aegilopoides carry 5S rRNA genes. Moreover, theyindicate that the 5S rDNA on chromosome 1B is linkedto the Nor-B1 locus.

Production of molecular probes. Molecularprobes are derived from cloned recombinant DNA mol-ecules, e.g., plasmids such as pBR 322 with cDNAgenes. These molecules are generated using restrictionand other DNA modifying enzymes and then cloned ina proper host (Mertz and Davis 1972; Watson et al.,1983). Protocols for generating recombinant DNA mol-ecules, cloning them, excising the relevant DNA frag-ment, e.g., 5S rDNA genes from these molecules andsubsequent use in in situ hybridization and other exper-iments, are detailed in Watson et al. (1983) and Win-nacker (1987). The procedures used in the cereals aregiven by Gerlach and Bedbrook (1979) and Lawrenceand Appels (1986).

Restriction fragment length polymorphism(RFLP) for chromosome identification. RFLP mark-ers are currently being extensively used in genetic map-ping (Wyman and White 1980, Helentjaris 1987, Whiteand Lalouel, 1988) and the determination of genomehomologies among crop species (Bonierbale et al.,1988; Sharp et al., 1989). They have also been used inthe identification of the critical chromosomes in aneup-loid plants in tomoto (Young et al., 1987) and breadwheat (Gale et al., 1988).

Sharp et al. (1989) isolated specific RFLP probes foreach arm of the seven chromosomes of the homoeolo-gous genomes of Triticum. These 14 homoeologousprobes permit the identification of chromosomes ineach genome of diploid and polyploid wheats.

ANEUPLOID STOCKS

Several types of aneuploid stocks have been pro-duced in wheat that have been used for decades forstudies of physical mapping. These stocks are in theseries of 21 mulli tetras produced by Ernie Sears (Sears,1966) that have been used for assigning genes to indi-vidual chromosomes (Sears, 1954; 1966) and the 24ditelo stocks for mapping genes to individual chromo-some arms (Sears and Sears, 1978). These stocks havealso been used for a number of studies: from assigningsome of the original molecular markers to individualchromosomes in the construction of the first molecularmarker maps (Anderson et al., 1992) to assigning ESTsto physical positions on chromosome arms (Han et al).The trisomic series in barley and the ditelo series in bar-ley (Tsuchiya, 1960, Fedak et al., 1971, 1972) and tri-somic series in T. monococcum (Friebe et al., 1990) and

CYTOLOGY AND GENETICS Vol. 42 No. 3 2008


rye (Kamanoi and Jenkins, 1962) have also been usedfor physical marker mapping. Another aid in wheatcytogenetics was the publication of a standard karyo-type and nomenclature system for hexaploid wheat(Gill et al., 1995). The addition lines of Betzes barley inChinese Spring wheat (Islam, 1980) have also beenused in initial studies of assigning molecular markers tochromosomes in the initial phases of the developmentof molecular maps in barley.

The deletion stocks in wheat are another usefulcytogenetic tool. They were produced by the gameto-cidal action of a particular genotype of Ae. speltoides(Endo and Gill, 1996) that caused a series of terminaldeletions on all wheat chromosomes followed by resto-ration of the telomere function. The deletion stocksconsist of 101 lines with 119 chromosome segmentdeletions for subarm mapping. These stocks providecomplete coverage of the wheat genome, subdividing itinto 159 chromosome lines. These stocks were recentlyused to physically map 16 000 wheat ESTs (Qi et al.,2004). Such a map is finding numerous applicationssuch as a source of SNP discovery (Somers et al.,2003), microsatellite discovery (Thiel et al., 2003),comparative mapping, hastening of the speed of genediscovery in wheat, plus provision of a framework forconstructing BAC-contigs of wheat. The stocks havealso been useful on a smaller scale, for example, in thephysical mapping of ESTs upregulated in wheat fol-lowing fungal attack (Han et al., 2005) Another tool forcytogenetic analysis of genomes, cloning, and taggingof genes is the BAC libraries, which have been con-structed in diploid (Lyavetsky et al., 1999) and hexap-loid wheats.

CHROMOSOME ADDITION LINES

Another set of cytogenetic stocks that permit analy-sis of single donor chromosomes are the addition linesof maize in oat (Riera-Lizarazu et al., 2000), whichwere produced by means of oat by maize pollination(Riera-Lizarazu et al., 1999). Yet another cytogenetictool is that of chromosome sorting. Chromosome sort-ing is a technique to provide aliquots enriched in partic-ular wheat chromosomes (Kubalakova et al., 2005) thatcan be used for molecular cytogenetic studies.

Complete series of addition lines from numerousalien species have been produced on a wheat back-ground. Chromosomes from more than 20 alien specieshave been added to wheat; in some cases, a completeset of addition lines has been produced. From 18 ofthese combinations, chromosome substitution lineshave also been produced. In many cases, translocationshave been induced between the chromosomes of thecrop plant and the alien species. A compendium of suchcytogenetic stocks appears as an appendix in the pro-ceedings of international wheat genetics symposia(e.g., Proceedings of the Seventh International WheatGenetics Symposium, Cambridge E, July 13–19, 1998,pp. 1374–1387) Various molecular methods have been

devised to monitor the transfer of traits from alien spe-cies into crop plant chromosomes (Fedak, 1999).Numerous translocations have been induced betweenalien and wheat chromosomes from the alien additionlines as a means of transferring disease resistance towheat (Friebe et al., 1996).

MOLECULAR MARKER MAPS

Another class of fairly recent genetic tools aremolecular linkage maps. These are not physical chro-mosome tools but are very useful in chromosome andgenome identification and monitoring alien introgres-sions. RAPD markers were basically random basesequences that were extensively used to “ta ” genescontrolling agronomic traits (Williams et al., 1990).The next class of markers were RFLPs. Extensive mapsconsisting of 1000 RFLP markers were generated inwheat (Gale et al, 1995). These markers were character-ized as too expensive and cumbersome for applicationto marker assisted selection but played a significant rolein uncovering relationships between diverse grassgenomes (Gale and Devos, 1998). For example, theoverall colinearity between wheat, barley, maize, andrice genomes was established (Feuillet and Keller,1999) which permitted the sharing of markers acrossthe maps. Because the rice genome was fully sequenced(Goff et al., 2002), this permitted the extensive use ofrice sequences to perform fine mapping in wheatgenetic studies (Liu et al., 2006). RFLP technology,particularly the use of cDNA probes, has been and stillis a valuable method of monitoring alien chromatinintrogression into the wheat genome, mainly because ofsequence conservation and the systemic relationshipamong Triticeae. In more recent fine mapping experi-ments, it is being discovered that the collinearitybetween chromosomes of species such as wheat andrice is not as close as initially assumed (La Rota andSorrels, 2004).

Other molecular maps were developed with PCR-based markers including RAPDs (William et al., 1990),AFLPs (Vos et al., 1995), and microsatellites (SSRs)(Roder et al., 1998). High density microsatellite mapscoupled with high throughput capillary electrophoresisare the essential components of a marker-assistedbreeding program. The high density consensus map ofSomers et al. (2004) is adequate for QTL detection.High density maps are essential for map-based cloningin that they increase the probability of discoveringmarkers flanking the gene in question and reduce thenumber of BAC clones containing the gene.

FISH AND GISH ANALYSIS

Another class of methodologies that facilitate theidentification of specific genomes, individual chromo-somes, or chromosomal segments is the use of fluores-cent signals. The first procedure to use fluorescentlabels to distinguish plant chromosomes was the pro-

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cess of genomic in situ hybridization (GISH) (Shwarza-cher et al., 1989). This technique was then used to iden-tify parental genomes and genome organization, inaddition to alien genome–chromosome introgression(Jiang and Gill, 1994). The identification of the threegenomes of the hexaploid has been achieved, butproved difficult to repeat consistently (Mukai et al.,1993; Sanchez-Moran et al., 1999). A modification ofthe multicolor GISH technique (Han et al., 2003, 2004)permitted the unequivocal identification of all threewheat genomes plus the presence of alien chromo-somes and translocations. Other cytogenetic tools suchas the Afa family, a repetitive sequence cloned fromAe. squarossa (Raybum and Gill, 1986); PSc119.2-arye subtelomeric heterochromatic sequence (Bedbrooket al., 1980); and PTa71, an rDNA clone of the 18S-8.5S-26S ribosomal sequence (Gerald and Bedroock,1979) can be used singly, in combination, or in combi-nation with FISH to identify individual chromosomesof wheat or barley. For example, using a combination ofFISH, Afa and PScll9.2. Kubalakova et al. (2005) wereable to identify all chromosomes of durum wheat.

Additional tools that complement the use of FISHtechnology for cytogenetic analysis of individual chro-mosomes are 5S and 25S genes, plus BAC clones thatcan be differentially strained to identify individualchromosomes (Zhang et al., 2004). The fiber FISHtechnique, using appropriate probes, which is appliedto stretched somatic chromatin, can provide better res-olution of gene and repeated sequence arrangements onwheat and barley chromosomes (Valanik et al., 2004).

It is obvious from this review that in recent years,classic cytogenetic analysis has been replaced bymolecular methods. In recent years, however, there hasbeen a renewed interest in minichromosomes. Theseare structures that consist of a centromere and minimalproximal chromatin into which genes of interest willpotentially be integrated. The minichromosomes willnot pair with those of the regular complement, they willbehave normally at meiosis, and will provide stabletransmission of the introgressed trait. This technologyis most advanced in mamalian cells. In plant cells it isbeing developed in maize from B chromosomes (Yuet al., 2007).

It is interesting to note that Gus Wiebe, a ResearchCoordinator for USDA, had developed lines of 16 chro-mosomes of barley in the 1960s. His theory was thatremoving the telomeres from the additional chromo-somes would limit their pairing with normal chromo-somes and would thus stably carry additional traits forthe barley genome.

Another recent development is the use of ricesequence information for fine mapping in wheat, bar-ley, and maize. In many cases, the colinearity ofsequences between rice and the other species is not asprecise as originally predicted. More recently, it hasbeen suggested that the genome of Brochypodium

could be used as a model system for the study of grassgenomes (Bossolini et al., 2007).

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tools and methodologies for cytogenetic studies of plant chromosomes

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