Ana Carvalho1, Henrique Guedes-Pinto1, J.S. Heslop-Har ri son2 and José Lima-Brito1*
1
De part ment of Ge net ics and Bio tech nol ogy, CGB/ICETA-UTAD, Uni ver sity of Trás-os-Mon tes and Alto Douro, Ap. 1013, 500-911 Vila Real, Por tu gal, 2 De part ment of Bi ol ogy,
Uni ver sity of Leicester, Leicester, LE1 7RH, United Kingdom, England
(*)
- Cor re spond ing au thor e-mail: jbrito@utad.pt Fax: ++351-259-350629 Phone: ++351-259-350507
MEIOTIC BEHAVIOUR AND CHROMOSOME IDENTIFICATION IN THE PORTUGUESE CULTIVAR OF TRITICALE ‘DOURO’
USING IN SITU HYBRIDISATION
ABSTRACT
The wide hy bridi sa tion has been ap plied to ce re als breed ing al low ing to the pro duc tion of new amphiploids. Triticale (x Triticosecale Wittmack) was the first syn thetic amphiploid of the Triticeae tribe and per formed well in com par i son with its par ent’s wheat and rye.
In this work we aimed to ana lyse the mei otic be hav iour and to iden tify the chro mo somes pres ent in both mi totic and mei otic metaphase cells of the por tu guese cultivar ‘Douro’ (2n=42, AABBRR) us ing in situ hy bridi sa tion tech nique with genomic DNA from rye and re pet i tive DNA se quences as probes.
Triticale ‘Douro’ pre sented a high level of ho mol o gous pair ing. The mean num ber of bi va lents was 20.14 and the av er age of ring bi va lents was higher than rod bi va lents. The to tal fre quency of univalents was re duced (1.72), pre sent ing the wheat univalents a higher fre quency com par a tively to the rye univalents. No multivalent con fig u ra -tions were seen, con firm ing the high mei otic reg u lar ity and sta bil ity of triticale ‘Douro’. The si mul ta neous use of pSc119.2 and pSc250 re pet i tive DNA probes on mi totic and mei otic chro mo somes al lowed the iden ti fi ca tion of all rye chro mo somes. The pSc119.2 probe iden ti fied some wheat chro mo somes, namely from B genome, and the chromosome pairs 4A and 5A.
Key words: chro mo some iden ti fi ca tion, in situ hy bridi sa tion, mei otic pair ing, re pet i tive DNA clones, Secale cereale, triticale.
INTRODUCTION
The wide hy bridi sa tion has been used to trans fer use ful char ac ters from wild spe -cies to crops, wid en ing their ge netic ba sis, and to pro duce new crops like triticale (see Gupta and Priyadarshan, 1982).
Triticale (x Triticosecale Wittmack), the first syn thetic amphiploid pro duced in
Triticeae tribe, with the aim of com bin ing the high pro duc tiv ity and qual ity of
wheat par ent and the rus tic ity of rye, per forms well in com par i son with its par ents in some parts of the world (Guedes-Pinto et al., 1991). This amphiploid has been used mainly as grain for an i mal feed ing (see Varughese et al., 1996, Myer, 2002),
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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
for graz ing, for age, for age/grain dual pur pose and si lage (Pfeif fer, 1992; Royo et
al., 1996; Carnide et al., 1996).
‘Douro’ is a re cent por tu guese cultivar of hexaploid triticale reselected at Uni -ver sity of Trás-os-Mon tes and Alto Douro and reg is tered in Por tu guese Na tional Cat a logue of Va ri et ies since 1996. It pres ents high pro duc tiv ity and qual ity when com pared with other va ri et ies used as con trol in tri als local ised at dif fer ent lo ca -tions of North of Por tu gal.
Pre vi ous re ports in triticale cor re lated its high pro duc tiv ity with its mei otic sta -bil ity. Ker nel shriv el ling and uni va len cy have been con sid ered the most im por tant prob lems of new es tab lished triticales (see Jouve and Soler, 1996). Both ef fects have been cor re lated in part with the pres ence of the large ter mi nal heterochromatin blocks on rye chro mo somes (Thomas and Kaltsikes, 1976; Gustafson and Bennett, 1982). The ab sence of telomeric heterochromatin in rye chro mo somes is as so ci ated with an im prove ment in mei otic sta bil ity in triticale (Kaltsikes and Gustafson, 1982).
Cband ing and more re cently flu o res cence in situ hy bridi sa tion (FISH) tech -niques have been the most use ful meth ods for iden ti fy ing chro mo somes in rye, wheat and triticale mi totic metaphase cells (see Lukaszewski and Gustafson, 1987; Schwarzacher et al., 1989; Cuadrado and Jouve, 1994).
FISH tech nique us ing re pet i tive DNA se quences was per formed to ana lyse the re pet i tive DNA com po si tion in the telomeres of rye chro mo somes in rye and triticale mi totic cells (Cuadrado and Jouve, 1996).
Cuadrado et al (1997) used the same strat egy in mei otic metaphase I cells of ph1b mu tant wheat x rye hy brids which al lowed the iden ti fi ca tion of sev eral chro mo -somes of wheat and rye.
Genomic in situ hy bridi sa tion (GISH) tech nique uses the to tal genomic DNA as probe and con sti tutes a pow er ful method to study the chro mo some pair ing in dif fer -ent spe cies be long ing to Triticeae tribe (King et al., 1994; Miller et al., 1994).
In this work, we used the in situ hy bridi sa tion tech nique, with to tal genomic DNA from rye and re pet i tive DNA se quences as probes, in or der to ana lyse the mei otic pair ing and to iden tify the pa ren tal chro mo somes on mei otic and mi totic metaphase cells of triticale ‘Douro’.
MATERIAL AND METHODS
Plant ma te rial
Root tips and an thers from 6x-triticale ‘Douro’ (2n=6x=42, AABBRR) were used. The spread prep a ra tion of the eth a nol:ace tic acid (3:1) fixed ma te rial es sen -tially fol lowed the meth ods de scribed by Lima-Brito et al. (1996) and Fernández-Cal vin et al. (1995).
Cover slips were re moved af ter freez ing with dry ice and the slides air-dried. Prep a ra tions were used im me di ately or store at –20°C for sev eral months be fore use. The chro mo some pair ing was ana lysed on 100 pol len-mother-cells (PMCs) at metaphase I.
Probes prep a ra tion, in situ hy bridi sa tion and reprobe
In situ hy bridi sa tion was per formed on mei otic chro mo some spreads with to tal
genomic DNA from Secale cereale L. and with the re pet i tive DNA se quence pTa71 (Eco RI frag ment of 8,9 kb con tain ing 18S-5.8S-25S rRNA genes and the intergeneric se quences) (see Gerlack and Bedbrook, 1979). Both were la belled by nick trans la tion with bi o tin-16-dUTP (Roche Mo lec u lar Biochemicals) , and digoxigenin-11-dUTP (Roche), re spec tively, and used as probes. Unlabelled to tal genomic DNA from durum wheat ‘Candial’ was sheared to frag ments about 250 bp, by autoclaving, and used as block ing DNA at 80 times the amount of the DNA genomic probe.
These mei otic chro mo some spreads were later reprobed (see Schwarzacher and HeslopHar ri son (2000) reprobe method) and used, in ad di tion to the mi totic chro -mo some spreads, for in situ hy bridi sa tion with the fol low ing two re pet i tive DNA probes: pSc119.2 (Bedbrook et al., 1980; McIntrye et al., 1988), with a 120 bp tan -dem re peated se quence unit, and pSc250 with a re peated se quence of 476 bp (see Vershinin et al., 1995 for a re view). Both probes were la belled by PCR, with digoxigenin-11-dUTP and bi o tin-16-dUTP, re spec tively.
The in situ hy bridi sa tion tech nique and probes de tec tion were per formed fol low ing FernándezCal vin et al. (1995) and LimaBrito et al. (1996) pro to cols, for an -thers and root-tips, re spec tively. The hy bridi sa tion mix ture con tained 50% formamide, 10% (v/v) of 20xSSC, 10% (w/v) dex tran sul phate and 0.05% salmon sperm DNA. To this mix ture were added 10 ng/µl of genomic DNA probe, 800 ng of block ing DNA and 3ng/µl of the rDNA clone pTa71. The other re pet i tive DNA probes (pSc119.2 and pSc250), used on mi totic and reprobed mei otic prep a ra tions, were added to the mix ture at a con cen tra tion of 2,5 ng/µl.
The an ti bod ies used for the de tec tion step were sheep antidigoxigenin con ju -gated to rhodamin (Roche), di luted in 4xSSC, for digoxigenin-la belled probes and streptavidin con ju gated with fluorescein (FITC; Roche), di luted in 4xSSC, for bi o -tin-la belled probes.
The slides were mounted with Vecta Shield with DAPI (4’,6-diamino-2-phenilindole, Vec tor Lab o ra to ries) and ob served on an AxioPlan 2.0 Im ag ing (Zeiss) epifluorescence mi cro scope equipped with ap pro pri ate light fil ters. The im ages were ob tained by an AxioCam (Zeiss) with the AxionVision soft ware, af ter tri ple ex po sure with three fil ters sets, and pre pared for print ing with Adobe Photoshop 6.0 soft ware.
RESULTS
Mei otic chro mo some be hav iour
Af ter in situ hy bridi sa tion per formed on mei otic chro mo some spreads of PMCs, at metaphase I, with to tal genomic DNA from rye and the ri bo somal se quence, as probes, (Fig. 1), it was pos si ble to dis crim i nate the pa ren tal ge nome or i gin of the hexaploid triticale (2n=42, AABBRR).
The fre quen cies of the dif fer ent mei otic con fig u ra tions ob served in hexaploid triticale ‘Douro’ are sum ma rized on Ta ble 1.
Some of the PMCs ana lysed (35%), dem on strated the ex pected ho mol o gous chro mo some pair ing of 21 bi va lents (four teen bi va lents from wheat and seven bi -va lents from rye). The most part of PMCs (46%) pre sented 20 bi -va lents, re sult ing in a to tal mean num ber of 20.14 (Ta ble 1). The to tal num ber of ring bi va lent con fig -u ra tions (14.74) was higher than rod bi va lent con fig -u ra tions (5.4), show ing the rye open bi va lents, a high fre quency (3.54) when com pared to wheat rod bi va lents. The to tal num ber of univalents was re duced, show ing the wheat univalents the higher av er age (Ta ble 1). The to tal per cent age of chro mo some arm as so ci a tions was 83,05%.
No multivalent con fig u ra tions were seen con firm ing the mei otic sta bil ity and reg u lar be hav iour of this hexaploid triticale cultivar.
Chro mo some iden ti fi ca tion
The use of re pet i tive DNA se quences as probes on mi totic metaphase chro mo -some spreads (Fig. 2) and af ter reprobe of the mei otic metaphase I prep a ra tions (Fig. 3), al lowed the iden ti fi ca tion of all rye chro mo somes and some wheat chro -mo somes by com par i son with the ideo grams and karyo types pre sented by Mukai Ta ble 1 Av er age chro mo some con fig u ra tions and mean per cent age of chro mo some arm as so ci a tions
ob served on 100 PMCs from the por tu guese triticale ‘Douro’. (Range in brack ets)
Univalents Bivalents
XTA/PMC % Chromosome
Arm. Assoc.
Rye Wheat Rods Rings Total
Rye Wheat Rye Wheat
0.66 1.06 3.54 1.86 3.13 11.61 20.14 31.75 83.05
(0-4) (0-2) (1-7) (0-5) (0-6) (8-14) (18-21) (28-35)
Fig .1. PMC at metaphase I of triticale ‘Douro’ (2n=6x=42, AABBRR) af ter counter stained with DAPI and in
situ hy bridi sa tion. A) – Seven rye bi va lent con fig u ra tions (five rods and two rings) de tected by the genomic
DNA rye probe (white). The 14 wheat bi va lents (one rod and thir teen rings) are counter stained with DAPI (grey). B) – Six rDNA sites (ar rows) were de tected by the rDNA pTa71 probe. Two of them were local ised on
and Nakahara (1993), Cuadrado and Jouve (1994), Cuadrado et al. (1995,1997), Alkhimova et al. (1999) and Schwarzacher and Heslop-Har ri son (2000).
All rye chro mo somes were iden ti fied by in situ hy bridi sa tion with both pSc119.2 and pSc250 (Figs. 2 and 3). The karyotype mainly used for this pur pose was that of Schwarzacher and Heslop-Har ri son (2000). The wheat chro mo somes, mainly that of B ge nome and the two chro mo some pairs 4A and 5A, were iden ti fied by the pSc119.2 probe (Figs. 2 and 3), fol low ing Mukai and Nakahara (1993) as well as Cuadrado et al. (1994) karyo types.
Fig ure 3 pres ents two PMCs af ter in situ hy bridi sa tion with genomic rye probe and the ri bo somal se quence. In Fig. 3-A it could be seen seven rye bi va lents (five rods and two rings), 13 wheat bi va lents and two univalents of wheat-or i gin. Fig ure 3-C pres ents one PMC with five rye bi va lents, 13 wheat bi va lents and six univalents (four of rye or i gin and two of wheat or i gin). In both cases, the ri bo somal se quence al lowed the iden ti fi ca tion of the 1R, 1B and 6B sat el lite chro mo some pairs.
The si mul ta neously hy bridi sa tion with pSc119.2 and pSc250 on these PMCs, al -lowed the iden ti fi ca tion of all rye bi va lent con fig u ra tions and of the un paired rye chro mo somes (Fig. 3-B and 3-D). Some wheat chro mo somes from B ge nome were iden ti fied as well as the two chro mo some pairs 4A and 5A (Figs. 3-B and 3-D). The unlabelled bi va lents and univalents pre sented in Figs. 3-B and 3-D cor re spond ing to wheat chro mo somes from A ge nome.
Fig. 2. Root tip metaphase af ter counter stained with DAPI and in situ hy bridi sa tion. A) – Probed with
pSc250 (white sig nals on telomeric chro mo some ends); B) – Probed pSc119.2 (white band ing
pat tern); C) – Si mul ta neous hy bridi sa tion with pSc119.2 and pSc250 probes al lowed
the iden ti fi ca tion of all rye chro mo somes
Fig. 3. Two PMCs at metaphase I af ter counter stained with DAPI and in situ hy bridi sa tion with genomic
DNA from rye (white) and pTa71 (ar rows), as probes (A and C), that had been later reprobed with pSc119.2 (light grey) and
pSc250 (white), si mul ta neously (B and D).
DISCUSSION
Mei otic chro mo some pair ing
Pre vi ous re ports showed the use ful ness of the in situ hy bridi sa tion tech nique us -ing both to tal genomic DNA and re pet i tive DNA se quences, as probes, for ge nome dis crim i na tion and chro mo some iden ti fi ca tion (King et al., 1994; Miller et al., 1994; Cuadrado et al., 1997).
The same was ver i fied in our work af ter in situ hy bridi sa tion per formed on 100 PMCs of ‘Douro’ triticale us ing si mul ta neous to tal genomic DNA from S. cereale and the ri bo somal se quence, pTa71 (see Fig. 1 and Figs. 3-A and 3-C).
In 6x-triticale ‘Douro’, homoeologous par ing was not de tected, prob a bly due to the ac tive pres ence of the sup pres sor homoeologous pair ing sys tem (Ph) on wheat com po nent. Most of the PMCs ana lysed showed a high level of ho mol o gous pair -ing since 81% of the PMCs pre sented a range of 20-21 bi va lents (see Ta ble 1).
The oc cur rence of a av er age num ber of bi va lents lower than 21 can be ex plained by the pres ence of un paired rye and wheat chro mo somes, spe cially from the A ge -nome (see Figs. 3-B and 3-D). How ever, the uni va lent fre quency was re duced and the av er age num ber of wheat univalents was higher than rye univalents (Ta ble 1). Thomas and Kaltsikes (1976) re ported that the fail ure of mei otic pair ing takes place pre dom i nantly in rye chro mo somes lead ing to a pre pon der ance of rye univalents in triticales. Our re sults are con tra dic tory to this ev i dence and agreed with those re ported by Merker (1973).
Bernardo et al. (1990) also re ported that chro mo some in sta bil ity of hexaploid triticales was due to the pres ence of un paired rye chro mo somes and open bi va lents at metaphase I. This fact can ex plain the par tial ste ril ity, aneuploidy and re duced pro duc tion, pres ent even in ad vanced lines (Tsuchiya, 1969; Merker, 1971). Nev er the less, Müntzing (1979) early re ported that does not ex ist a strict cor re la tion be -tween the above re ferred char ac ters and pro duc tion.
In our work, the av er age num ber of ring bi va lents was higher than rod bi va lents (see Ta ble 1), con firm ing the above re ferred high mei otic sta bil ity. These re sults are not un ex pected since ‘Douro’ has been im proved af ter sev eral years of breed ing programme.
The mean per cent age of chro mo some arm as so ci a tions was 83.05%, re veal ing a high de gree of pair ing.
Chro mo some iden ti fi ca tion
FISH tech nique us ing re pet i tive DNA se quences was suc cess ful on chro mo some iden ti fi ca tion as re ported by Mukai and Nakahara (1993), Cuadrado and Jouve (1994), Cuadrado et al. (1995,1997), Alkhimova et al. (1999) and Schwarzacher and Heslop-Har ri son (2000).
In our work the si mul ta neous use of pSc119.2 and pSc250, al lowed the iden ti fi -ca tion of all rye chro mo somes and some of the wheat com po nent, both on mi totic and mei otic metaphase cells (see Figs. 2 and 3).
The in situ hy bridi sa tion us ing to tal genomic DNA from rye and pTa71, only iden ti fied the three pairs of sat el lite chro mo somes: 1R, 1B and 6B (see Figs. 3-A and 3-C).
The si mul ta neous use of pSc119.2 and pSc250 probes un able the com plete iden ti fi ca tion of A and B genomes from wheat. The pSc250 probe is spe cific for rye ge -nome and does not have homology with wheat (Vershinin et al., 1995; 1996). But when we used it, si mul ta neously with pSc119.2 (Figs. 2 and 3B and 3D), was pos -si ble to iden tify all rye chro mo somes, some of the B ge nome chro mo somes and the pairs 4A and 5A from wheat (Mukai and Nakahara, 1993; Cuadrado et al., 1997; Schwarzacher and Heslop-Har ri son, 2000).
The band ing pat tern re vealed by the pSc119.2 probe on A and B wheat genomes, spe cially on B chro mo somes, seems to be very con served on wheat (Cuadrado et
al., 1997).
The chro mo some iden ti fi ca tion given by pSc250 and pSc119.2 con firmed the ex is tence of ho mol o gous pair ing, dis cussed above.
The wheat univalents de tected were mostly from A ge nome as it ap peared unlabelled af ter FISH with pSc119.2 (see Fig. 3B and 3D). The rye univalents iden -ti fied were mostly the chro mo some pairs 2R and 3R (see Fig. 3B).
FISH with highly re pet i tive probes, in clud ing pSc119.2, and pTa71, was per formed by Cuadrado et al. (1997) for chro mo some pair ing anal y sis on a ph1b com -mon wheat x rye hy brid plant. Those au thors achieved a suc cess ful iden ti fi ca tion of homoeologous chro mo some pair ing be tween rye-rye, wheat-rye and wheat-wheat. They also es ti mated the pair ing fre quency of the chro mo some arms and at trib uted the lower val ues to the fol low ing rye arms: 2RS and 5RS. This could be re lated with the pres ence of the two un paired chro mo somes 2R (univalents) in most part of the PMCs.
Cuadrado et al. (1997) sug gested that rye par ent (‘Centeio do Alto’) might have ef fec tive ge netic fac tors on pair ing fre quency, due to its re duced amount of C-heterochromatin com pared to other rye cultivars.
Rye chro mo somes are eas ily iden ti fied by its char ac ter is tic large blocks of telomeric heterochromatin (Sarma and Natarajan, 1973) whereas the wheat chro -mo somes have it on in ter sti tial re gions (Lukaszewski and Gustafson, 1987; Bernardo et al., 1990). This is sue fa cil i tate the iden ti fi ca tion of rye and wheat com -ple ments on triticale, but it could not be dis carded the pres ence of poly mor phism in rye band ing pat tern be tween cultivars. Some au thors sug gested con tro ver sial the o -ries about the in flu ence of the large blocks of telomeric heterochromatin in the grain shriv el ling, for ma tion of ab er rant nu clei on en do sperm and grain shriv el ling (Bennett, 1977; Gustafson and Bennett, 1982; Gupta and Priyadarshan, 1982). They even con nected these fea tures with the con tri bu tion of 4R, 5R and 6R chro -mo somes that have a no ta ble telomeric heterochromatin (Bennett, 1977).
The rye chro mo somes de lay at anaphase was re lated with seg ments of later rep li -ca tion that cor re spond ing to telomeric heterochromatin re gions (Bennett, 1977). None the less, Varghese and Lelley (1983) ver i fied that nei ther the to tal heterochromatin nor the heterochromatin of each ge nome have cor re la tion with the mei otic reg u lar ity or grain shriv el ling.
The 6x-triticale ‘Douro’ showed strong telomeric blocks of heterochromatin on rye mi totic and mei otic chro mo somes (Figs. 1, 2 and 3). It also pre sented some dif fer ences on the hy bridi sa tion pat tern of these re gions when com pared to the karyo -types and ideo grams ana lysed (Cuadrado and Jouve, 1994; Alkhimova, 1999;
Schwarzacher and HeslopHar ri son, 2000). Nev er the less, we ana lysed and com -pared karyo types from a dip loid rye spe cie and we are study ing triticale.
More over, the rye cultivar used in the pro duc tion of the 6x-triticale ‘Douro’ is un known.
We ob tained some dif fer ences on the hy bridi sa tion pat tern of rye chro mo somes com par a tively to the karyotype re ported by Cuadrado and Jouve (1994) for 6x-triticale. This fact can be ex plained by dif fer ent or i gins of rye used in these triticales.
Cuadrado and Jouve (1994) re ported that there was no hy bridi sa tion sites of pSc119.2 probe on chro mo some pairs 2R and 3R. Our data re vealed hy bridi sa tion sig nals of pSc119.2 in all rye chro mo some pairs of triticale ‘Douro’ (see Figs. 2 and 3).
CONCLUSIONS
In situ hy bridi sa tion tech nique was a pow er ful tool for the study of mei otic be
-hav iour and chro mo some iden ti fi ca tion of 6x-triticale cultivar ana lysed here. This cultivar, named ‘Douro’, dem on strated a high mei otic sta bil ity, reg u lar ity and uni for mity that al ready led it to be reg is tered in the Por tu guese Na tional Cat a -logue of Va ri et ies.
ACKNOWLEDGEMENTS
This work was sup ported by FCT - pro ject POCTI/35107/AGR/1999. REFERENCES
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