J. Lima-Brito1
*
, A. Carvalho1, C. Matos1, Pat Heslop-Har ri son2 and H. Guedes-Pinto1
1 De part ment of Ge net ics and Bio tech nol ogy, CGB/ICETA, Uni ver sity of Tras-os-Mon tes and Alto Douro, 5000-911 Vila Real, Por tu gal., 2 De part ment of Bi ol ogy,
Uni ver sity of Leicester, Leicester LE1 7RH, United Kingdom
To whom ed i to rial cor re spon dence should be ad dressed, e-mail: jbrito@utad.pt
rRNA GENE EXPRESSION AND LOCATION IN TRITICALE ASSAYED BY SILVER STAINING AND IN SITU HYBRIDISATION TECHNIQUES
ABSTRACT
In durum wheat × rye hy brids and the de rived amphiploid triticale, AABBRR, the ex pres sion of the 1R rRNA genes is largely sup pressed. Alloauto-octoploid triticales, AABBRRRR, al lows the eval u a tion if rye NOR in ac ti va tion can be over come by the in crease of rye ge nome num ber.
In the pres ent work, we used sil ver stain ing and n situ hy bridi sa tion tech niques in or der to study the nu cle o lar ac tiv ity and to lo cal ize the rRNA genes in hexaploid and alloauto-octoploid triticales.
The use of rye genomic DNA as probe al lowed the iden ti fi ca tion of the rye chro mo somes pres ent in both hexaploid and octoploid triticales (14 and 28, re spec tively). The si mul ta neous use of the ri bo somal se quence en ables the lo cal isa -tion of 18S-25S rDNA on the sat el lite chro mo somes of both triticales. On hexaploid triticale we de tected six rDNA sites (four on wheat chro mo some pairs 1B and 6B and two on rye chro mo some pair 1R), whereas on alloauto-octoploid triticale eight rDNA sites (four on wheat-pairs 1B and 6B and four on rye chro mo some pairs 1R) were ob served.
As ex pected, the max i mum num ber of ac tive AgNORs per metaphase cell was co in ci dent with the max i mum num -ber of nu cle oli per interphase nu cleus con firm ing that all and only the NORs func tion ally ac tive dur ing interphase are stained by sil ver at the next mi totic metaphase.
Com par i son of the nu cle o lar ac tiv ity be tween hexaploid and octoploid triticales ana lysed here in di cates that the in -crease in 1R chro mo somes from two to four does not change the sup pres sion of rye nu cle o lar ac tiv ity. This sup ports the sug ges tion that genomic in ter ac tions are un der strong ge netic con trol.
Key words: amphiploids, in situ hy bridi sa tion, nu cle o lar dom i nance, rDNA, sil ver stain ing, triticale.
INTRODUCTION
One of the most stud ied genomic in ter ac tion sys tems is the one con cern ing the dom i -nance of one pro gen i tor in the ex pres sion of ri bo somal loci – nu cle o lar dom i -nance or amphiplasty (Navashin, 1928). The phe nom e non has been ob served in many interspecific hy brids and their de riv a tives in plants and an i mals (see Lacadena et al. 1988 and Pikaard, 2000 for a re view). In ce re als, nu cle o lar dom i nance is a com mon fea -ture in both intra- and intergeneric hy brids (see Lacadena et al. 1988).
Ex pres sion of the ri bo somal RNA genes pro duces the nu cle oli at interphase and, where the genes are in ter ca lary on the chro mo somes, leads to sec ond ary con stric tions at Communicated by Piotr Masojæ
P L A N T B R E E D I N G A N D S E E D S C I E N C E
Volume 51 2005
the nu cle o lar or ga niz ing re gions (NORs) of metaphase chro mo somes, form ing the sat -el lites.
rRNA gene lo ca tion and ex pres sion can be as sayed by a com bi na tion of meth ods in clud ing mea sure ment of nu cle o lar vol ume and num ber (Mar tini and Flavell 1985), sil -ver stain ing (Moreno et al. 1990; Neves et al. 1997, Lima-Brito et al. 1998), and in situ hy bridi sa tion (see Jiang and Gill 1994; Leitch and Heslop-Har ri son 1992).
Anal y sis of rRNA gene ex pres sion by sil ver stain ing (Hubell 1985; Stack et al. 1991) is a pow er ful method to score ac tive rDNA sites, while in situ hy bridi sa tion shows all sites of rRNA genes.
Within the Triticeae, the to tal num ber of rDNA loci per spe cie var ies. Rye (Secale cereale L., 2n=2x=14, ge nome con sti tu tion RR), have a sin gle pair of chro mo somes with NORs, which are both nor mally ex pressed.
In bread wheat (Triticum aestivum L., 2n=6x=42, AABBDD), rRNA genes have been found on chro mo somes 1BS and 6BS (ma jor sites), 5DS, 1AS, 7DL, 1BL, and 3DS (see Jiang and Gill 1994). Loci on the two sat el lite chro mo some pairs, 1B and 6B, are large and strongly ex pressed, and the con tri bu tion of other loci to cel lu lar rRNA is small (Mar tini and Flavell, 1985).
Af ter the study of sev eral hy brids, a hi er ar chy of ex pres sion of the rRNA genes was es tab lished (see Lacadena et al., 1988). In wheat x rye F1 hy brids and in triticale, nu cle o lar dom i nance of wheat is ob served, re sult ing in an al most to tal in ac ti va tion of rye or i -gin rRNA genes (Cermeño et al. 1984; Lacadena et al. 1988; Thomas and Kaltsikes, 1993; Neves et al. 1995, 1997). Con se quently, in the large ma jor ity of cells, rye NOR ac tiv ity is un de tect able by the sil ver stain ing tech nique which re veals interphase nu cle -oli and stains metaphase NORs that where tran scribed dur ing the pre vi ous interphase (Hubel, 1985).
In bread wheat x Aegilops umbellulata (2n=2x=14, UU) hy brids and ad di tion lines, rRNA gene ac tiv ity of chro mo somes 1U and 5U from A. umbellulata is dom i nant and the 1B and 6B rDNA is sup pressed (Mar tini et al. 1982, Lacadena et al. 1988). Nu cle o -lar dom i nance was also found in bar ley x rye hy brids (Ramsay and Dyer 1983), in wheat-bar ley ad di tion lines (Santos et al. 1984), and in triticale x tritordeum hy brids (Lima-Brito et al., 1998).
In the pres ent work, we aimed to study the nu cle o lar ac tiv ity in both hexaploid and alloauto-octoploid triticale and to lo cal ize the rRNA genes. For those pur poses we used sil ver stain ing and in situ hy bridi sa tion tech niques, re spec tively.
MATERIAL AND METHODS
The fol low ing spe cies were used in the study: ten plants of re gional rye ‘Vila Pouca’ (2n=14, RR), six plants of durum wheat ‘Candial’, (2n=28, AABB); five plants of triticale ‘Douro’ (2n=42, AABBRR), reselected in De part ment of Ge net ics and Bio -tech nol ogy/Uni ver sity of Trás-os-Mon tes and Alto Douro; and ten plants of alloauto-octoploid triticale, (2n=56, AABBRRRR) ob tained by Guedes-Pinto (1988) from crosses be tween triticale ‘Corgo’ (2n=42, AABBRR) and rye ‘Vila Pouca’ (2n=14, RR).
For root tip chro mo some prep a ra tions, seeds were ger mi nated on moist fil ter pa per for 48 h at 25 °C. The 1.5-2cm ex cised root-tips were trans ferred to ice wa ter for 24-30h
at 0 °C and then fixed in eth a nolace tic acid (3:1). Spread prep a ra tions were made as de -scribed by Schwarzacher and Heslop-Har ri son, (2000). Fixed root-tips were par tially di gested with cellulase and pectinase be fore squash ing in 45% ace tic acid. Cover slips were re moved af ter freez ing with dry ice and slides air-dried.
To study nu cle o lar ac tiv ity by scor ing the num ber of sil ver stained nu cle o lar or ga niz -ing re gions (Ag-NORs) in metaphase cells and the num ber of nu cle oli in interphase cells, we used the salt-ny lon sil ver stain ing tech nique of Stack et al. (1991) with small mod i fi ca tions as de scribed by Lima-Brito et al. (1998).
For in situ hy bridi sa tion tech nique, we fol lowed the roottip spread prep a ra tion pro to -col and the probe de tec tion method de scribed by Schwarzacher and Heslop-Har ri son (2000). The probes used in hy bridi sa tion were: to tal genomic DNA from Secale cereale ‘Petkus’ la belled with digoxigenin and the ri bo somal se quence pTa 71 (Gerlach and Bedbrook, 1979) la belled with TRITC (tetramethyl rhodamine isothiocyanate). Triticum aestivum ‘Chi nese Spring’ DNA was used as block ing DNA to re duce cross hy bridi sa tion. The slides were counter stained with DAPI (4’-6’ diamidino-2-phenylindole).
Then the slides were mounted in CitifluorGlyc erol and ana lysed on a Zeiss mi cro -scope. Pho to graphs were taken on Fuji 400 col our print film, digit ised to Photo CD and con verted to grey scale in Adobe Photoshop with con trast op ti mi sa tion af fect ing the whole im age equally.
RESULTS
Ta ble 1 sum ma rizes the num ber of metaphase cells with dif fer ent num bers of AgNORs, and the fre quency of interphase cells with dif fer ent num bers of nu cle oli, af -ter sil ver stain ing. The max i mum num ber of nu cle oli seen reg u larly at in-terphase is of greater sig nif i cance than the dis tri bu tion of num bers since nu cle oli usu ally fuse dur ing interphase. There fore, sta tis ti cal anal y sis of the dis tri bu tions or max i mum num bers of interphase nu cle oli may re flect fu sion fre quen cies as well as the num bers of ac tive NORs.
In dip loid rye ‘Vila Pouca’ all mi totic metaphase cells showed two Ag-NORs (Ta ble 1) and the max i mum num ber of nu cle oli ob served in interphase cells was also two. In durum wheat ‘Candial’, all mi totic metaphase cells showed four Ag-NORs (Ta ble 1), the same as the max i mum num ber of nu cle oli ob served in interphase cells. Af ter sil ver stain ing, 88% of metaphase cells of hexaploid triticale ‘Douro’ showed four Ag-NORs (Ta ble 1 and Fig. 1a). Five or six nu cle oli were de tected in only six interphase nu clei out of 2272 scored (Fig. 1b) giv ing good ev i dence for the sup pres sion of the 1R rDNA loci. The same sup pres sion could be de tected in the alloauto-octoploid triticale in which some 89% of the metaphase cells ana lysed had four Ag-NORs (Ta ble 1 and Fig. 1c). How ever, a very small per cent age (7%) of metaphase cells showed more than four AgNORs. The num ber of nu cle oli var ies be tween one and eight (Fig. 1d), and the cor re spon dence be tween the max i mum num ber of AgNORs and nu cle oli was also ob -served.
Af ter in situ hy bridi sa tion us ing to tal genomic DNA from rye and the ri bo somal se -quence pTa 71 it was pos si ble to iden tify clearly all the rye chro mo somes (14 in 6x-triticale and 28 in the alloauto-octoploid triticale) pres ent in metaphase cells as well
1 el ba T s ll ec e s a h pr et ni f o yc ne u qe rf d n a sll ec e s a h p at e m ni s R O N-g A f o re b m u N . de i d ut s se nil e ht ni il oe l c u n f o sr e b m u n t n e re ffi d hti w la ir et a M ci m o ne G n oit uti ts n oc hti w sll ec e sa h pa te m f o . o N s R O N-g A f o . s o n t ne re ffi d s re b m u n t ne re ffi d hti w sll ec e sa h pr et ni f o yc ne u qe r F ) %( il oe lc u n f o es a h pr et ni f o . o N de vr es b o sll ec 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 'a c u o P ali V' e y R R R -5 2 1 -0 8 2 5. 9 1 8 4. -0 0 5 3 'l ai d na C' ta e h w m ur u D B B A A -0 9 -9 1 0 8. 2 5 5 6. 1 2 3 4. 6 2 1. -0 5 4 3 ' or u o D' el ac iti r T-x 6 R R B B A A -5 0 6 2 1 -9 2 4. 1 4 1 8. 7 3 1 4. 1 1 9 0. 0 8 1. 0 9 0. -2 7 2 2 el ac iti r T di ol p ot c o-ot u A oll A R R R R B B A A 1 7 9 9 1 3 1 1 -2 2 1 0 0. 0 4 4 7. 5 3 0 3. 1 1 4 7. 0 4 1. 0 4 0. 0 2 0. 0 2 0. 4 2 7 6 1 d n u of e n o n se ta ci d ni ‘ - ‘ A B D . 1 . gi F -) A( sll ec e sa h pr et ni ni il oe l c u n f o d na ) s w o r ra , s R O N-g A( sll ec e sa h pa te m ni A N Dr e vi t ca hti w s ni e t or p s ni at s e u qi n hc et g n i ni at s re vli s e h T -) B( . de ss er p p us er a R O N R 1 e m o s o m or hc ni g i r o-e yr e h T . B 6 d na B 1 se m o s o m or hc t ae h w m or f s R O N-g A r u of g n i w o hs ’ or u o D‘ el ac iti rt e di ol pa xe H i r o-t ae h w m or f s R O N-g A r u of g n i w o hs el ac iti rt di ol p ot c o-ot ua oll A -) C( .il oe l c u n r u of ot e n o m or f n oi t ai ra v a g n i w o hs ’ or u o D‘ el ac iti rt -x 6 f o sll ec e sa h pr et nI -.il oe l c u n e vi f d na e er ht hti w l le c es a h pr et ni o wt el ac iti rt -x 8 f o sll ec e sa h pr et nI – ) D( . de ss er p p us er a s R O N R 1 r u of e h T . B 6 d na B 1 se m o s o m or hc ni g
as to local ise the ma jor sat el lite NOR chro mo somes (6 in hexaploid triticale and 8 in the octoploid triticale), as can be seen in Fig. 2a and Fig. 2b.
DISCUSSION
The re sults showed good cor re la tion be tween the max i mum num ber of ac tive Ag-NORs per metaphase cell and the max i mum num ber of nu cle oli per interphase nu cleus, con firm ing that all and only the NORs func tion ally ac tive dur ing interphase are stained by sil ver at the next mi totic metaphase (see Me dina et al. 1986).
Pre vi ous re ports showed that as ploidy level in creases and, con se quently, higher po ten tially ac tive Ag-NORs are pres ent, both the num ber of nu cle oli and Ag-NORs per nu cleus shows a wider range (see Lacadena et al., 1984; Rangel-Figueiredo et al., 1985). Our data also re veals that wider spec trum of oc cur rence of nu cle oli per cell (Ta ble 1) that can be ex plained by the pos si bil ity of nu cle oli fus ing into interphase nu clei.
The NOR in rye was found to be local ised in the SAT chro mo some 1R through in situ hy bridi sa tion ex per i ments (see Appels et al., 1980) and re con firmed through sil ver stain ing and C-band ing (see Lacadena et al., 1984). Our re sults re veals that in dip loid rye the num ber of AgNORs and nu cle oli per cell were sim i lar to those ex -pected (see Rangel-Figueiredo et al., 1985).
In durum wheat, dom i nance of NORs on chro mo somes 1B and 6B over the mi -nor rDNA sites was de tected which con firms ear lier re sults pres ent by other au thors (see Viegas and Mello-Sampayo, 1975; Lacadena et al., 1988; Lima-Brito et al., 1998).
The nu cle o lar ac tiv ity of the chro mo some 1R seems to be in ac tive in a wheat back ground (Viegas and Mello-Sampayo, 1975; Thomas and Kaltsikes, 1983, Cermeño et al., 1984; Rangel-Figueiredo et al., 1985, Lacadena et al., 1988; Vieira
A B
Fig. 2. Root tips metaphases af ter counter stained with DAPI and in situ hy bridi sa tion tech nique per formed with to tal genomic DNA from rye (light grey) and ri bo somal se quence pTa71 (ar rows). (A) – 6x-triticale ‘Douro’ show ing six rDNA sites, two on the rye chro mo some pair 1R and four on wheat chro mo some pairs 1B and 6B; (B) – Alloauto-octoploid triticale pre sent ing eight rDNA sites, four on the rye-or i gin chro mo some 1R and other
et al., 1990a; Neves et al., 1997; Lima-Brito et al., 1998). The in ac ti va tion of rye NORs also oc curs in bar ley x rye (Ramsay and Dyer 1983) and H. chilense x rye (Thomas and Pickering 1985; Pohler and Schrader 1988) hy brids, wheatbar ley ad -di tion lines (Santos et al., 1984), monosomic ad -di tions of H. chilense to rye (Linde-Laursen et al., 1993), triticale x tritordeum F1 hy brids (Lima-Brito et al., 1998), and other com bi na tions in the Triticeae (see Lacadena et al., 1988).
In the triticale cultivar ‘Douro’ stud ied here, most of the metaphase cells showed four Ag-NORs giv ing good ev i dence for the sup pres sion of the 1R rDNA loci. It was also de tected a very few cells with five or six AgNORs and nu cle oli. How -ever, there is no di rect ev i dence in which chro mo some the ex tra NOR is ac tive.
Af ter in situ hy bridi sa tion, six sites of 18S-25S rDNA were de tected in hexaploid triticale AABBRR: four from wheat chro mo some pairs 1B and 6B and two from rye chro mo some pair 1R (see Fig. 2a). In the alloauto-octoploid triticale, AABBRRRR, two ad di tional sites of 18S25S rDNA were seen on rye chro mo -some pair 1R.
The anal y sis of the data ob tained by sil ver stain ing both in AABBRR and AABBRRRR triticales showed that the Ag-NORs and nu cle oli pat tern are sim i lar: four NORs and four nu cle oli be ing the high est sig nif i cant num ber ob served. A few cells with six Ag-NORs were ob served in both triticales and more nu cle oli per cell are rare al though they have been ob served.
In both cases, the max i mum num ber of NORs and nu cle oli per cell are in ac cor -dance with the to tal max i mum num ber of nu cle o lar re gions pres ent, six in hexaploid triticale and eight in octoploid triticale. How ever, it is clear that sup pres -sion of the rye nu cle o lar ac tiv ity took place in both triticales, al though a few rye NORs may be in com pletely sup pressed.
Com par i son of the nu cle o lar ac tiv ity be tween hexaploid and octoploid triticale ana lysed here in di cates that the in crease in 1R chro mo somes from two to four does not change the sup pres sion of rye nu cle o lar ac tiv ity. This sup ports the sug ges tion that genomic in ter ac tions are un der strong ge netic con trol.
We now know that genes lo cated on the long arm of rye chro mo some 1R and on chro mo some 2R in flu ence the ex pres sion of 1R in triticale (Vieira et al., 1990a, Neves et al. 1997).
Sev eral fac tors in flu ence the ex pres sion of rDNA genes in clud ing methylation (e.g. Heslop-Har ri son 1990; Vieira et al. 1990b; Neves et al., 1995; Castilho et al., 1999) or length of sub-re peats in the intergenic spacer DNA of the rDNA (Sardana et al. 1992).
CONCLUSION
In situ hy bridi sa tion tech nique us ing the ri bo somal se quence pTa71 al lowed the un equiv o cal lo cal isa tion of the rRNA genes on the wheat chro mo some pairs 1B and 6B and on the rye chro mo some pair 1R in both 6x- and 8- triticales ana lysed in this work. The study of nu cle o lar ac tiv ity of these triticales, per formed by sil ver stain ing tech nique, re vealed the sup pres sion of chro mo some 1R in most of the mi -totic metaphase and interphase cells scored.
ACKNOWLEDGMENTS
This work was par tially sup ported by FCT; pro ject POCTI/AGR/35107/99. REFERENCES
Appels, R., Gerlach, W.L., Den nis, E.S., Swift, H. and Pea cock, W.J. 1980. Mo lec u lar and chro mo somal or -ga ni za tion of DNA se quences cod ing for the ri bo somal RNAs in ce re als. Chromosoma, 78: 293-311. Castilho, A., Neves, N., Rufini-Castiglione, M., Viegas, W. and Heslop-Har ri son, J.S. 1999.
5-Methylcytosine dis tri bu tion and ge nome or ga ni za tion in triticale be fore and af ter treat ment with 5-azacytidine. J. Cell Sci., 112: 4397-4404.
Cermeno, M.C., Orellana, J., Santos, J.L., and Lacadena, J.R. 1984. Nu cle o lar or ga nizer ac tiv ity in wheat, rye and de riv a tives ana lysed by a sil ver stain ing pro ce dure. Chromosoma, 89: 370-376.
Gerlach, W.L. and Bedbrook, J.R. 1979. Clon ing and char ac ter iza tion of ri bo somal RNA genes from wheat and bar ley. Nucl. Ac ids Res., 7: 1869-1885.
Guedes-Pinto, H. 1988. 8x triticale with four Secale ge nome. Tag.- Ber. Akad. Landwirtsch. Wiss, DDR, Berlin, 266, S. 307-325.
Heslop-Har ri son, J.S. 1990. Gene ex pres sion and pa ren tal dom i nance in hy brid plants. De vel op ment (Suppl.): 21-28.
Hubell, H.R. 1985. Sil ver stain ing as an in di ca tor of ac tive ri bo somal genes. Stain Technol., 60: 285-294. Jiang, J. and Gill, B.S. 1994. New 18S26S ri bo somal RNA gene loci: chro mo somal land marks for the evo
-lu tion of polyploid wheats. Chromosoma, 103: 179-185.
Lacadena, J.R., Cermeño, M.C., Orellana, J. And Santos, J.L. 1984. Ev i dence for wheatrye nu cle o lar com -pe ti tion (amphiplasty) in triticale by siver stain ing pro ce dure. Theor. Appl. Genet., 67: 207-213. Lacadena, J.R., Cermeno, M.C., Orellana, J., and Santos, J.L. 1988. Nu cle o lar com pe ti tion in Triticeae.
Kew Chro mo some Con fer ence III: 151-165.
Leitch, I.J., and Heslop-Har ri son, J.S. 1992. Phys i cal map ping of the 18S-5.8S-26S rRNA genes in bar ley by in situ hy bridi sa tion. Ge nome, 33: 1013-1018.
LimaBrito, J., GuedesPinto, H. and HeslopHar ri son, J.S. 1998. The ac tiv ity of nu cle o lar or ga niz ing chro -mo somes in multigeneric F1 hy brids in volv ing wheat, triticale, and tritordeum. Ge nome, 41: 763-768. LindeLaursen, I, Schrader, O., and Zerneke, F. 1993. Chro mo somal con sti tu tion of rye (Secale cereale)
-Hordeum chilense ad di tion lines. Hereditas, 119: 21-29.
Mar tini, G., and Flavell, R.B. 1985. The con trol of nu cle o lus vol ume in wheat, a ge netic study at three de -vel op men tal stages. He red ity, 54: 111-120.
Mar tini, G., Dell, M. O’, and Flavell, R.B. 1982. Par tial in ac ti va tion of wheat nu cle o lus or gan is ers by the nu cle o lus or gan iser chro mo somes from Aegilops umbellulata. Chromosoma, 84: 687-700.
Me dina, F.J., Solanilla, E.L., SanchezPina, M.A., FernandezGomez, M.E., and Risueno, M.C. 1986. Cy -to log i cal ap proach -to the nu cle o lar func tions de tected by sil ver stain ing. Chromosoma, 94: 259-260. Moreno, F.J., Rodrigo, R.M., and Gar cia-Herdugo, G. 1990. Ag-NOR pro teins and rDNA transcriptional
ac tiv ity in plant cells. J. Histochem. Cytochem., 38: 1879-1887.
Navashin, M.S., 1928. Amphiplasty; a new caryological phe nom e non. Z. indukt. Abstamm.-u Vererbl. Suppl. 2: 1148.
Neves, N., HeslopHar ri son, J.S., and Viegas, W. 1995. rRNA gene ac tiv ity and con trol of ex pres sion me di -ated by methylation and im print ing dur ing em bryo de vel op ment in wheat x rye hy brids. Theor. Appl. Genet., 91: 529-533.
Neves, N., Castilho, A., Silva, M., Heslop-Har ri son, J.S., and Viegas, W. 1997. Nu cle o lar dom i nance in triticales: con trol by un linked genes. Chr. Res., 5: 125-131.
Pikaard, G.S. 2000. Nu cle o lar dom i nance: uniparental gene si lenc ing on a multi-megabase scale in ge netic hy brids. Plant Mo lec u lar Bi ol ogy, 43: 163-177.
Pohler, W., and Schrader, O. 1988. Chro mo some pair ing in hy brids of Hordeum chilense with Secale
cereale and triticale. Tag.-Ber. Akad. Landwirtsch.-Wiss. DDR, Berlin, 266: 189-198.
Ramsay, G., and Dyer, A.F. 1983. Nu cle o lar or gan iser sup pres sion in bar ley x rye hy brids. Kew Chro mo -some Con fer ence II, P.E. Brandham & M.D. Bennett eds, Lon don, UK: George Allen & Unwin, 361. Rangel-Figueiredo, T., Guedes-Pinto, H. and Mello-Sampayo, T. 1985. Nu cle o lar or ga nizer ac tiv ity in
triticales of dif fer ent ge nome cosntitution. In: Ge net ics and Breed ing of Triticale, Eucarpia meet ing, Clermont-Ferrand (France), INRA, 65-74.
Santos, J.L., Lacadena, J.R., Cermeno, M.C., and Orellana, J. 1984. Nu cle o lar or gan iser ac tiv ity in wheat-bar ley ad di tion lines. He red ity, 52: 425-429.
Sardana, R., Dell, M.O’, and Flavell, R.B. 1992. Cor re la tion be tween the size of the intergenic reg u la tory re gion, the sta tus of cytocine methylation of rRNA genes and nu cle o lar ex pres sion in wheat. Mol. Gen. Genet., 236: 155-162.
Schwarzacher, T. and Heslop-Har ri son, J.S. 2000. Pratical in situ hy bridi sa tion.BIOS Sci en tific Pub lish ers, Ox ford, U.K
Stack, S., Herickhoff, L., Sherman, J., and An der son, L. 1991. Stain ing plant cells with sil ver. I. The saltny -lon tech nique. Biotechnic. & His to chem is try, 1: 69-78.
Thomas, J.B. and Kaltsikes, P.J. 1983. Ef fect of chro mo some 1B and 6B on nu cle o lus for ma tion in hexaploid triticale. Can. J. Genet. Cytol., 25:292-297.
Thomas, H.M., and Pickering, R.A. 1985. Com par i sons of the hy brids Hordeum chilense x H. vulgare,
Hordeum chilense x Secale cereale and the amphiploid of Hordeum chilense x H. vulgare. Theor.
Appl. Genet., 69: 519-522.
Viegas, W., and Mello-Sampayo, T. 1975. Nu cle o lar or ga ni za tion in the ge nus Triticum. Broteria, 44: 121-133.
Vieira, R., Queiroz, A., Morais, L., Barao, A., Mello-Sampayo, T., and Viegas, W. 1990a. Ge netic con trol of 1R nu cle o lus or ga nizer re gion ex pres sion in the pres ence of wheat genomes. Ge nome, 33: 713-718. Vieira, R., Queiroz, A., Morais, L., Barao, A., Mello-Sampayo, T., and Viegas, W. 1990b. 1R chro mo some
