Izabela Pawlowicz1, Marcin Rapacz2, Jan Bocianowski3
1
In sti tute of Plant Ge net ics, Pol ish Acad emy of Sci ences, Strzeszyñska 34, 60-479 Poznañ, Po land, 2Ag ri cul -tural Uni ver sity of Kracow, Pod³u¿na 3, 30-239 Cra cow, Po land, 3De part ment of Math e mat i cal and Sta tis ti cal
Meth ods, The Uni ver sity of Ag ri cul ture, Wojska Polskiego 28, 60-637 Poznañ, Poland
IDENTIFICATION OF AFLP MARKERS LINKED WITH LOW-TEMPERATURE RESISTANCE IN INTROGRESSIONS
TRANSFERRED FROM FESTUCA ARUNDINACEA TO LOLIUM MULTIFLORUM
ABSTRACT
BC3-152/53 pop u la tion of L. multiflorum plants com pris ing sin gle introgression of F. arundinacea ge nome had higher win ter har di ness than con trol L. multiflorum plants. AFLP anal y sis were per formed re sult ing in gen er a -tion of 19 mark ers linked with freez ing re sis tance, 7 linked with win ter har di ness and 2 mark ers cor re lated with both traits. It in di cates that Fes tu ca introgression could make the im pact on Lolium stress re sis tance.
Key words: AFLP mark ers, freez ing re sis tance, F. arundinacea, L. multiflorum, win ter har di ness
INTRODUCTION
Low tem per a ture is one of the most im por tant fac tors that limit the growth and geo graph ical in ci dence of many grass spe cies. In over-win ter ing plants from tem per ate re gions, ex pos ing them to cer tain pe ri ods of subzero tem per a -ture in creases their tol er ance to lat ter freez ing. Cold or frost ac cli ma tion is the adap tive pro cess in volv ing a num ber of bio chem i cal and phys i o log i cal changes (Levitt 1980, Guy et al. 1985). It also in cludes the ex pres sion of cold-reg u lated (COR) genes, which prod ucts are nec es sary for pro tec tion against freez ing stress (Thomashow 1999). Win ter har di ness is the plant abil ity to sur vive in low tem per a ture dur ing win ter pe riod. This is a ge no type spe cific trait com posed of sev eral com po nents whose im pact var ies with the cli ma tic con di tions (Larsen 1994). Com po nents of win ter har di ness in clude ver nal iza tion re quire ment, photoperiod re sponse, ice en case ment, wa terlog ging, des ic -ca tion, cli ma tic ad ap ta tion, plant con di tion in au tumn, -car bo hy drate star va tion, snow molds, low temperature fungi resistance, and freezing resistance (Larsen 1994).
Communicated by Andrzej Anio³
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 58 2008
In Eu rope pe ren nial grass land oc cupy more ag ri cul tural ar eas than any other crop. LoliumFes tu ca com plex is com posed of two ge nus hav ing com ple men -tary char ac ters. Within Lolium ge nus two spe cies: L. perenne and L. multiflorum are eco nom i cally the most im por tant for age grasses in tem per ate re gions, with high pro duc tiv ity and qual ity but rather poor per sis tency. The ge -nus Fes tu ca con sists of spe cies geo graph i cally di verse and better adapted to a wide range of eco log i cal con di tions. Lolium and Fes tu ca spe cies hy brid ize readily, their ho mol o gous chro mo somes pair and re com bine at high fre quency dur ing mei o sis (Jauhar 1975, King et al. 1999, Naganowska et al. 2001, Kosmala et al. 2006).Fer tile hy brids are com monly uti lized to im prove Lolium cultivars abiotic stress re sis tance, with main te nance of their good for age qual -ity, by trans fer of lim ited num ber of Fes tu ca genes (e.g. Humphreys et al. 1989, Humphreys et al. 2005, King et al. 1998, Zwierzykowski et al. 1998a, b, 1999).
Fes tu ca chro mo some seg ments introgressed into the re cip i ent Lolium spe -cies can be iden ti fied by genomic in situ hy bridi sa tion (GISH) (Humphreys and Paðakinskienë 1996, Humphreys et al. 1997, Kosmala et al. 2006, 2007). By a com bined ap proach of GISH tech nique and am pli fied frag ment length poly mor phism (AFLP) it is pos si ble to ‘tag’ genes re spon si ble for de sir able ag -ro nomic traits in breed ing p-rogrammes (Armstead et al. 2001, King et al. 1998, 2002, Humphreys et al. 2005). AFLP tech nique is a ro bust and highly ef fec tive method for DNA fin ger print ing that pro vides a large num ber of re li able and re -pro duc ible ge netic mark ers that can be used as an al ter na tive to mor pho log i cal trait anal y sis. Its ap pli ca tion re quires no prior se quence knowl edge thus is con -ve nient for spe cies in which the ge netic in for ma tion is still lim ited, like for age grasses.
This pa per dem on strates AFLP anal y sis of dip loid L. multiflorum pop u la tion of introgressive forms with sin gle F. arundinacea seg ment. Pentaploid hy brids be tween hexaploid F. arundinacea cv. Kord and dip loid L. multiflorum cv. Tur were back crossed into L. multiflorum (2x). Ob tained BC3-152/53 pop u la tion was char ac ter ized by freez ing re sis tance and win ter har di ness. The ob jec tive of the re search was gen er a tion of mark ers linked with these both traits.
MATERIAL AND METHODS Plant ma te rial
The pentaploid F1 hy brids of F. arundinacea (2n = 6x = 42) × L. multiflorum (2n = 4x = 28) used as the male par ent were back crossed two times into dip loid L. multiflorum cv. Tur used as the fe male par ent. The introgressions from F. arundinacea ge nome were iden ti fied in ob tained prog e nies through GISH. BC2-152/37/73 plant hav ing sin gle F. arundinacea seg ment (Kosmala et al. 2003, 2007) was back crossed again into L. multiflorum to pro duce BC3-152/53 pop u la tion con tain ing 80 plants.
Test for freez ing tol er ance
Eighty plants of the BC3152/53 pop u la tion were screened for their sen si bil -ity to freez ing tem per a tures. Test was per formed in con trolled con di tions with the method de scribed by Rapacz et al. (2004).
Each in di vid ual was di vided into 3 equal-sized clones and es tab lished in sand: peat (1:1) mix ture in the op ti mal growth con di tions (25°C, 10/14 h day/night photoperiod, 200 mmol×m-2×s photosynthetic pho ton flux den sity (PPFD, Philips AGRO so dium light source, Philips Light ning NV, Turnhout, Bel gium)). For 7 day pre-hard en ing well-rooted plants were trans ferred to the en vi ron men tal cham ber with fol low ing con di tions: 12°C, 10/14 h day/night photoperiod, 200 mmol×m-2×s-1 PPFD. Then plants were cold ac cli mated in sub-zero tem per a tures (2oC, 10/14 h day/night photoperiod, 200 mmol×m-2 ×s-1 PPFD). Three weeks later they were put to the stress con di tions (-2°C, 10/14 h day/night photoperiod, 200 mmol×m-2×s-1 PPFD) for 1 day. Af ter that time tem per a ture was low ered with the cool ing rate 1°C/h. Plants were ex posed in -7, -11 and -14°C re spec tively for 8 hours. Next, they were again trans ferred to 2°C, 10/14 h day/night photoperiod, 200 mmol×m-2×s-1 PPFD. De frost ing plant till ers were cut down to height of 2 – 3 cm. Then, tem per a ture was raised to 12°C let ting plants re-growth. Re gen er a tion of plants was es ti mated us ing 0 – 9 vi sual score (Larsen 1978) where 0 rep re sented dead plants with out any signs of re-growth, whereas 9 rep re sented plants with out vi sual signs of dam ages. Mean scores for 3 rep li cates of plant were cal cu lated. Sta tis ti cal sig nif i cance for dif fer ences in freez ing re sis tance be tween plants was de ter mined us ing Duncan’s mul ti ple range test at P = 0.05.
Test for win ter har di ness
Field test for win ter har di ness for 80 plants of the BC3-152/53 pop u la tion were per formed dur ing win ter pe riod 2005/2006 in Szelejewo Plant Breed ing. Win ter sur vival and spring regrowth were scored vi su ally us ing a scale of 0-9, where 0 rep re sented plants with no vis i ble green till ers and 9 plants with out vis i ble win ter dam age. Es ti ma tion was con ducted on 16th No vem ber 2006 and 15th March 2006. The mean re sults for 3 clones of each plant from pop u la tion and con trol L. multiflorum cv. Tur were cal cu lated. The sta tis ti cal sig nif i cance for dif fer ences be tween ge no types was de ter mined us ing Duncan’s mul ti ple range test at P = 0.05.
AFLP anal y sis
Genomic DNA was ex tracted by CTAB method (Murray and Thomp son 1980) and pu ri fied by phe nol ex trac tion. AFLP fin ger prints were gen er ated us -ing pro to col of Vos et al. (1995) with mod i fi ca tions by Kosmala (2004). AFLP adapt ers con sisted of a core se quence and an en zyme-spe cific se quence (EcoRI or Tru9I), whereas AFLP prim ers in cluded a core se quence, spe cific se quence and a se lec tive ex ten sion with one (preamplification) or tree (sec ond am pli fi
-ca tion) se lec tive nu cleo tides. These prim ers were used pre vi ously for map ping introgressions of win ter har di ness and drought tol er ance in L. multiflorum introgressive lines ob tained from back cross es of triploid and pentaploid L. multiflorum × F. pratensis hy brids (Skibiñska et al. 2002).
Poly mor phic DNA bands of F. arundinacea pres ent in genomes of introgressive ge no types were scored as a dom i nant (pres ent: ab sent). Link age of AFLP mark ers with freez ing re sis tance or win ter har di ness was es ti mated us ing anal y sis of re gres sion (Hastie and Tibshirani 1990).
RESULTS
The di ver sity of freez ing sen si bil ity and win ter har di ness in BC3-152/53 plants was il lus trated by his to grams (Fig. 1a, b). Freez ing tol er ance of 11 in di -vid u als was sig nif i cantly (P < 0.05) higher (1.78-3.22) than the mean value of
Fig. 1. Dis tri bu tion of freez ing re sis tance (A) and win ter har di ness (B) in BC3-152/53 pop u la tion. Both val ues was as sessed on a scale of 0-9 (Larsen 1978), where 0 rep re sented dead plants with out any signs
of re-growth and 9 plants with out vi sual signs of dam ages. Con trol (L. multiflorum cv. Tur) was marked as Lm Tur — histogram (A).
L. multiflorum (1.72). Win ter har di ness of 13 plants was better (P < 0.05) than con trol L. multiflorum.
AFLP stud ies were per formed on three groups of plants. BC3-152/53-A group con tained 10 freez ing-sen si tive and 10 freez ing-tol er ant in di vid u als. To BC3-152/53-B 10 plants win ter hardy and 10 with low har di ness af ter win ter were se lected. The third group (BC3152/53C) con sisted with 10 plants hav -ing both high freez -ing re sis tance and win ter har di ness val ues and 10 plants with the low est freez ing re sis tance and win ter har di ness val ues. Forty primer com bi na tions were used for the iden ti fi ca tion of introgressions trans ferred from F. arundinacea to L. multiflorum ge nome. The to tal num ber of AFLP mark ers ob tained was 28. Nineteen of them were linked with freez ing re sis -tance (BC3-152/53-A), 7 with win ter har di ness (BC3-152/53-B) and 2 mark ers were si mul ta neously cor re lated with both ana lysed traits (BC3-152/53-C). Their ap prox i mate size (in base pairs) ranged from 130 to 700. Primer pairs giv ing the high est num ber of poly mor phic bands de rived from F. arundinacea in re spec tive ana lysed groups of plants were fol low ing: BC3152/53A -E43M47 (23), BC3-152/53-B - E32M33 (26), BC3-152/53-C - E43M52 (21).
DISCUSSION
Spe cies from Fes tu ca ge nus are the ex cel lent source of low-tem per a ture stress tol er ance. Among them F. pratensis is the most freez ing-re sis tant and win ter hardy cul ti vated in Eu rope. Ad di tion ally its dip loid na ture causes that introgression of stress re sis tance pro ceeds rel a tively eas ily. Trans fer of freez -ing tol er ance from F. pratensis to L. perenne was re ported pre vi ously (e.g. Can ter 2000, Grønnerød et al. 2004).
Fig. 2. AFLP fin ger print ob tained by DNA am pli fi ca tion with E43M52 primer com bi na tion, sep a rated in 6% de na tur ing acrylamide gel and visu al ised with sil ver stain ing. In di vid ual ge no types
from BC3-152/53 pop u la tion are marked with num bers, and length marker with M let ter.
F. arundinacea is allohexaploid spe cies com pris ing three subgenomes, one in her ited from freez ing tol er ant F. pratensis and two from drought tol er ant F. glaucescens (Humphreys et al. 1995). It gives two ap proaches for trans fer of stress re sis tance genes to Lolium: in di rectly from the subgenomes (Humphreys and Thomas 1993, Humphreys and Paðakinskienë 1996, Humphreys et al. 1997) or di rectly from one or the other of its pro gen i tors (Zwierzykowski et al. 1999, Mor gan et al. 2001, Humphreys et al. 2005, Kosmala et al. 2006). The first backcross-breed ing pro gram aimed at the trans fer of win ter har di ness and freez ing tol er ance from F. arundinacea into L. multiflorum us ing pentaploid hy brids be tween F. arundinacea (6x) and L. multiflorum (4x) was re ported by Kosmala et al. (2006). The cur rent study, is the sec ond backcross-breed ing pro gram with the uti li za tion of these hy brid per formed on dif fer ent L. multiflorum cultivars.
Among BC3-152/53 pop u la tion 13.8% and 16.25% in di vid u als showed higher than pa ter nal L. multiflorum plants val ues of freez ing re sis tance and win ter har di ness, re spec tively. In Kosmala et al. (2006) re search only 7.2% of the tested ge no types showed more win ter har di ness then the L. multiflorum con trol. Si mul ta neously all these plants were more freez ing tol er ant than Lolium and the level of re sis tance var ied sig nif i cantly (from 0.5 to 1.5). In our ex per i ment AFLP re search re sulted in only two AFLP-mark ers linked with both traits gen er a tion. Al though freez ing tol er ance is the most im por tant com -po nent of win ter har di ness the fi nal con tri bu tion of freez ing tol er ance in win ter har di ness is very vari able and strongly de pends on the year and lo cal con di tions (Larsen 1994). More over, it was no ticed that cor re la tion be tween freez -ing tol er ance and win ter har di ness var ies clearly among spe cies. In wheat (Sutka et al. 1986) and oil seed rape (Rife and Salgado 1996) frost re sis tance is in gen eral well cor re lated with win ter har di ness. But there are spe cies, like al falfa, where this cor re la tion is rather low, de pend ing of the year of ob ser va -tions (Brouwer et al. 2000). In our ex per i ment we ob tained only two mark ers con nected with both traits. In our ex per i ment con ducted in Szelejewo Plant Breed ing me te o rol ogy data were mon i tored. Win ters in 2005 and 2006 were rather warm, with low num ber of day with the neg a tive tem per a tures op er at ing to the plant. It is sup posed that un der such mild con di tions, freez ing tol er ance could not be a fac tor de ter min ing win ter har di ness of the plants. It can not be also ex cluded that F. arundinacea be longs to the spe cies char ac ter iz ing with week genetically-based relationship between frost resistance and winter hardiness but it needs a further experimental verification.
CONCLUSIONS
To re ca pit u late, it was shown for the sec ond time that uti li za tion of F. arundinacea in backcross breed ing ap proaches takes the op por tu nity to trans -fer genes for low-tem per a ture re sis tance to L. multiflorum. In BC3152/53 pop -u la tion introgressions were in di cated by AFLP anal y sis. With the as sis tance of
prim ers used in the pres ent ex per i ment it was pos si ble to dis tin guish chro mo some re gions sep a rately re spon si ble for win ter har di ness and freez ing tol er -ance. Fes tu ca introgression could make the im pact on Lolium win ter har di ness and freez ing re sis tance as some in di vid u als from BC3-152/53 pop u la tion were char ac ter ized by higher stress tol er ance than pa ter nal L. multiflorum plants.
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