Maria Chrz¹stek
In sti tute of Ge net ics and Plant Breed ing, Ag ri cul tural Uni ver sity, ul. Akademicka 15, 20-934 Lublin, Poland
IDENTIFICATION OF GLUTENIN AND SECALIN SUBUNITS IN ADDITION AND SUBSTITUTION LINES
OF WHEAT (TRITICUM AESTIVUM L.) USING BIOCHEMICAL MARKERS
ABSTRACT
The aim of pa per was eval u a tion of com po si tion of high mo lec u lar weight (HMW) and low mo lec u lar weight (LMW) glutenin sub units in wheat-rye ad di tion lines cv. Grana - cv. Dañkowskie Z³ote, sub sti tu tion line 1B/1R and ini tial forms, as well as, lo cal iza tion of Sec genes on chro mo somes ‘Dañkowskie Z³ote’ rye. Anal y sis were per formed us ing SDS-PAGE elec tro pho re sis.The com po si tion of HMW glutenin for all ad di tion lines and ‘Grana’ wheat was: N - 6+8 - 2+12. Com po si tion of LMW glutenin was es ti mated only for 2R and 6RL ad di tion lines, wheat, and octoploid triticale. In both ad di tion lines the sub units N-6+9-15 were iden ti fied, in wheat cv. Grana sub units 1-6+9-15 were pres ent, whereas in octoploid triticale poly mor phism was ob served (1-6+9-15 or 1-11+13-N). Rye stor age pro teins (secalins) was found in ad di tion lines: 1R, 2R and sub sti tu tion line 1B/1R. It can be con cluded that genes re spon si ble for secalin biosynthesis in rye cv. Dañkowskie Z³ote are localized on 1R and 2R chromosomes.
Key words: ad di tion lines, HMW glutenins, LMW glutenins, 1B/1R sub sti tu tion line, SDS-PAGE elec tro pho re sis,
secalins
INTRODUCTION
Bak ing value of wheat flour is de ter mined mainly by glutenins (Payne et al. 1987, Verbruggen et al. 2001), and there fore iden ti fi ca tion of genes re spon si ble for these pro -teins syn the sis is very im por tant from prac ti cal point of view.
On the base of nu mer ous stud ies, it was found that com plex loci Glu A1, Glu B1, Glu D1 lo cal ized on long arms of chro mo somes 1A, 1B and 1D re spec tively are re spon si -ble for wheat high-mo lec u lar weight glutenin biosynthesis (Law rence and Shep herd 1981, Payne et al. 1982). New tech niques for DNA mo lec u lar weight mark ers iden ti fi ca tion made pos si ble to sep a rate highmo lec u lar weight glutenins into “x” type (frac -tions of lower mo bil ity) and “y” type (frac -tions of higher mo bil ity) (Lafiandra et al. 1997). Rog ers et al. (2001) pre dicted the con se quences of the lack of some “x” or “y” sub units in al most isogenic lines of wheat cv. Sicco. In gen eral, the ab sence of those sub units neg a tively af fected the elas tic ity and ex ten si bil ity of glu ten and wors ened the Communicated by Danuta Miazga
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 52 2005
physicochemical prop er ties of the dough. High-mo lec u lar weight glutenins en coded by Glu D1 lo cus (Nieto et al. 1998), es pe cially 5+10 sub units, have the ma jor in flu ence on grain qual ity of bread wheat (Payne et al. 1987, D’Ovidio et al. 1994, Lafferty and Lelley 2001).
Robin et al. (1998) ob served the evo lu tion of loci de ter min ing biosynthesis of high-mo lec u lar weight glutenins in genomes A, B, D and G of wheat. Ap ply ing DNA mo lec u lar mark ers, the au thors found the philogenetic as so ci a tions among Triticum
aestivum, Triticum monococcum, Triticum timopheevi, Triticum turgidum, Triticum urartu and Aegilops squarrosa and they pre dicted prob a ble form ing time for par tic u lar
genomes and loci en cod ing glutenins.
While the in flu ence of HMW glutenins on flour prop er ties is es tab lished, the assotiation be tween LMW glutenin sub units and breadmaking qual ity is still re -searched. Among oth ers LMW glutenins af fect on dough ex ten si bil ity (Verbruggen et
al. 2001) and pasta-mak ing qual ity of durum wheat (Liu and Shep herd 1996).
Low-mo lec ular we ight glu ten ins are en cod ed by Glu A3, Glu B3 and Glu D3
ge nes that are lo cal ized on short arms of chro mos omes from the first ho
-moeolog ous gro up (1A, 1B, 1D) (Van Cam penh out et al. 1995).
Ge nes re spons ible for se cal in bio synt hesis were lo cal ized on 1R and 2R chro
-mos omes (Sy benga et al. 1990, 1991, Orell ana et al. 1993). Se cal ins ne gat ively
affec ted the tech nol ogi cal pa ram ete rs of whe at and tri tic ale flo ur. Sub stit uti on
1B/1R and trans loc ati ons 1BL.1RS and 1AL.1RS cau se de crea se in flo ur ba
-king qu ality (Lee et al. 1995, Gray bos ch 2001). Com preh ensi ve stu dies upon
elim ina tion or re duc ing this un favor able effect ma inly by direc ted trans loc ati
-ons and sub stit uti on of chro mos omes are con duct ed (Kum lay et al. 2003).
The aim of pres ent pa per was to eval u ate the com po si tion of HMW and LMW glutenin sub units in wheat-rye ad di tion lines cv. Grana - cv. Dañkowskie Z³ote and sub sti tu tion line 1B/1R as well as lo cal iza tion of Sec genes in ‘Dañkowskie Z³ote’ rye us ing bio chem i cal mark ers (SDS-PAGE elec tro pho re sis).
MATERIAL AND METHODS
Se ries of lines of hexaploid wheat (Triticum aestivum L.) cv. Grana with added pairs of com plete or telocentric chro mo somes of rye (Secale cereale L.) cv. Dañkowskie Z³ote (1R, 2R, 3R, 3RS, 4R, 5R, 6R, 6RL, 7R) as well as sub sti tu tion line 1B/1R of cv. Grana / cv. Dañkowskie Z³ote were the sub ject of stud ies. All lines were ob tained at In -sti tute of Ge netic and Plant Breed ing of Ag ri cul tural Uni ver sity in Lublin (Miazga and Chrz¹stek 1987, Chrz¹stek and Miazga 1988). At the same time, ini tial forms; wheat cv. Grana, rye cv. Dañkowskie Z³ote and the octoploid triticale ob tained cross ing them, were stud ied.
All ma te ri als were pre vi ously cy to log i cally tested in or der to choose sta ble forms. Num ber of chro mo somes in so matic cells and chro mo some con fig u ra tions in mei o sis were stud ied.
The com pos iti on of high mo lec ular we ight glu ten in (HMW) su bun its and low
mo lec ular we ight glu ten in (LMW) su bun its, as well as pre sence of se cal ins
were de term ined using elect rophore sis tech nique on poly acryl ami de gel in pre
-sence of so dium do dec yl sul fate (SDS-PAGE) acc ordi ng to La emmli (1970)
met hod. Anal ysis were per form ed in WIBEX elect rophore sis la bor ato ry in Po
-znañ. Ident ifi cat ion of glu ten in su bun its was per form ed on the ba sis of Pay ne
and La wrence (1983) ca tal ogue. Ten ker nels from eve ry line and init ial form
were anal yzed. In or der to sim plify high-mo lec ular we ight glu ten in su bun its
ident ifi cat ion, stan dard va riet ies of hexa ploid whe at Ju bil atka and Be gra were
ap plied. Loci of al lels en cod ing low-mo lec ular we ight glu ten in su bun its were
fo und using ane uploid li nes of ‘Chin ese Sprin g’ and ‘Pavon’ whe at.
RESULTS AND DISCUSSION
Usu ally from three to five high-mo lec u lar weight glutenin sub units oc cur in hexaploid wheat va ri et ies. Lo cus Glu A1 is re spon si ble for one sub unit, lo cus Glu B1 for one or two sub units and lo cus Glu D1 en codes usu ally two sub units. In lit er a ture data, one can find re ports on the dis cov ery of new allels of par tic u lar loci en cod ing un -iden ti fied high-mo lec u lar weight glutenin sub units (Tahir et al. 1986).
Anal y sis of high-mo lec u lar weight glutenins in stud ied cv. Grana wheat lines with added or sub sti tuted chro mo somes of rye cv. Dañkowskie Z³ote re vealed that lo cus
Glu A1 de ter mined block N (Null) in all lines and in wheat, which means the lack of
glutenin sub units (Ta ble 1). Lo cus Glu D1 en coded sub units 2+12 in all stud ied lines and wheat. Pres ence of 6+8 sub units en coded by Glu B1 lo cus was found in tested ad di tion lines and ‘Grana’ wheat. Re ceived re sults are agreed with these ob tained pre vi -ously by Brzeziñski (1993). In sub sti tu tion line 1B/1R no sub units en coded by Glu B1 lo cus was ob served. How ever, the band char ac ter is tic for rye pro teins, i.e. secalins, was clearly vis i ble on electrophoregram (Fig. 1). This band was sit u ated in place re spec tive for ‘Dañkowskie Z³ote’ rye. All sub units pres ent in ‘Grana’ wheat, i.e. N-6+8-2+12
Fig. 1. Elec tro pho retic sep a ra tion of high mo lec u lar weight glutenins and secalins in ad di tion line 1R, sub sti tu tion line 1B/1R and ini tial forms. Lanes: 1 - stan dard, 2 - ‘Dañkowskie Z³ote’ rye,
3 - ‘Grana’ wheat, 4 - octoploid triticale ‘Grana’ × ‘Dañkowskie Z³ote’, 5-9 ad di tion line 1R, 10-11 sub sti tu tion line 1B/1R, 12 - stan dard
and one secalin band char ac ter is tic for rye, were ob served in ad di tion line 1R. Also on electrophoregram of ad di tion line 2R, be sides glutenin sub units re spec tive for ‘Grana’ wheat, two bands con firm ing the pres ence of rye pro teins were found (Fig. 2). No secalin bands were found in other lines. From anal y ses it fol lows that genes con trol ling secalin syn the sis oc curred on 1R and 2R chro mo somes of ‘Dañkowskie Z³ote’ rye. Many au thors found that 1R chro mo some had three loci en cod ing rye stor age pro teins. Lo cus Sec 3 lo cal ized on long arm of this chro mo some is re spon si ble for highmo lec u -lar weight secalin syn the sis ho mol o gous with wheat high-mo lec u -lar weight glutenins (Orellana et al. 1993). Com plex lo cus Sec 1 de ter min ing syn the sis of type w and 40 K
g secalins oc curred on short arm of 1R chro mo some in a re gion of sat el lite (Sybengaet al. 1990). Pro teins en coded by Sec 1 are ho mol o gous with wheat gliadins. Link age be
-tween loci Sec 3 and Sec 1 are well known (Carillo et al. 1990, Sybenga et al. 1990). The third lo cus (Sec 4) is sit u ated be tween Sec 1 and Sec 3, on short arm of 1R chro mo -some (Benito et al. 1990) and is re spon si ble for w-secalin syn the sis.
Gene Sec 2 that is re spon si ble for syn the sis of type 75 Kg secalins hav ing no an a -logues in other ce re als was iden ti fied on 2R chro mo some (Sybenga et al. 1991, Murray et al. 2001). The lat est stud ies (Malyshev et al. 1998) re vealed the pres ence of an other gene (Sec 5) de ter min ing secalin syn the sis that is also lo cal ized on short arm of chro mo some 2R. Genes re spon si ble for syn the sis of 75 K g-secalin type were also found on 6R chro mo some in Secale montanum (Shevry et al. 1985).
Prob a bly, dur ing evo lu tion pro cess of Secale cereale as a con se quence of translocation, genes en cod ing secalins were trans ferred from chro mo some 6R
Secale montanum into chro mo some 2R Secale cereale. New al leles of pre vi ously
re ported genes are iso lated and char ac ter ised (De Bustos and Jouve 2003).
Fig. 2. Elec tro pho retic sep a ra tion of high mo lec u lar weight glutenins and secalins in wheat and ad di tion lines. Lanes: 1 – stan dard, 2 – ‘Grana’ wheat, 3 -1R, 4 - 2R, 5 - 3R, 6 - 3RS, 7 - 4R, 8 - 5R, 9 - 6R,
Within stud ied ad di tion lines ‘Grana’ – ‘Dañkowskie Z³ote’ and sub sti tu tion line 1B/1R, no poly mor phism re fer ring to the com po si tion of high-mo lec u lar weight glutenin sub units was observed.
While genes en cod ing high-mo lec u lar weight glutenins are rel a tively easy to iden tify, lo cal iza tion genes con trol ling low-mo lec u lar weight glutenin syn the sis is dif fi cult, be cause of the fact that they are strongly linked with genes con trol ling biosynthesis of gliadins sit u ated on the same chro mo somes (Dubcovsky et al. 1997). Fur ther dif fi cul ties re sult that the low-mo lec u lar weight glutenins are close to some gliadins, al bu mins and glob u lins, which makes electrograms dif fi cult to in -ter pret. Even hy poth e ses that some low-mo lec u lar weight glutenins are mod i fied gliadins en coded by Gli genes, have arisen (Metakovsky et al. 1997). Ac cord ing to Payne and Cor field (1979), low-mo lec u lar weight glutenin sub units B and C are sim i lar to type g gliadins in re spect to mo lec u lar weight, and D sub unit to type w
gliadins. De spite of these dif fi cul ties, nu mer ous stud ies are car ried out aim ing to learn ge netic con di tions of low-mo lec u lar weight glutenin syn the sis. Ap pli ca tion of the new est tech niques for DNA mo lec u lar weight mark ers iden ti fi ca tion made pos si ble to lo cal ize genes en cod ing lowmo lec u lar weight glutenins and poly mor -phism oc cur ring among them (D’Ovidio 1992, Van Campenhout et al. 1995). Low-mo lec u lar weight glutenins are en coded by genes Glu A3, Glu B3 and Glu D3 lo cal ized on short arms of the first homoeologous group chro mo somes (1A, 1B, 1D). Sreeramulu et al. (1997) re ported the iden ti fi ca tion of new sub units of low-mo lec u lar weight glutenins be ing un der con trol of Glu D4 and Glu D5 genes that are found on 1D and 7D chro mo somes in hexaploid In dian wheat va ri et ies.
The at tempt to pre lim i nary eval u a tion of the ‘Grana’ wheat lines with added and sub sti tuted ‘Dañkowskie Z³ote’ rye chro mo somes with re spect to low-mo lec u lar weight glutenin com po si tion ap ply ing aneuploid wheat lines (‘Chi nese Spring’ and
Ta ble 1 Com po si tion of high mo lec u lar weight glutenin sub units in ad di tion lines ‘Grana’ - ‘Dañkowskie Z³ote’,
1B/1R sub sti tu tion line, ini tial form and stan dard wheat va ri et ies
Chromosomes and varieties Composition of subunits controlled by loci Presence ofsecalin
Glu-A1 Glu-B1 Glu-D1
1R N 6+8 2+12 + 2R N 6+8 2+12 + 3R N 6+8 2+12 3RS N 6+8 2+12 4R N 6+8 2+12 5R N 6+8 2+12 6R N 6+8 2+12 6RL N 6+8 2+12 7R N 6+8 2+12 1B/1R N - 2+12 + Grana N 6+8 2+12 Jubilatka 2* 6+8 2+12 Begra N 7+9 5+10
‘Pavon’), was un der taken in pres ent study. Anal y sis of electrophoregrams re vealed that Glu A3 lo cus lo cal ized on 1AS chro mo some is re spon si ble for syn the sis of N-1-2 sub units, Glu B3 gene on 1BS chro mo some en codes 6-13 sub units, and Glu D3 oc cur ring on short arm of 1D chro mo some en codes sub unit 15 (Ta ble 2). How -ever, electrophoregrams were un clear and dif fi cult for in ter pre ta tion. It was only pos si ble to as sess the com po si tion of sub units for wheat, triticale and two ad di tion lines. In ‘Grana’ wheat, lowmo lec u lar weight glutenin frac tion con sisted of sub -units 1-6+9-15, and in ad di tion lines 2R and 6RL sub -units N-6+9-15 were in cluded (Ta ble 2). Octoploid triticale ‘Grana’ × ‘Dañkowskie Z³ote’ was not uni form re gard ing to the com po si tion of lowmo lec u lar weight glutenin frac tion. Com po si -tion of 1-6+9-15 was ob served in one case, 1-11+13-N in an other. On a ba sis of re sults achieved and poor data in avail able lit er a ture, it is hard to univocally ac -count for the rea son of such poly mor phism. Ac cord ing to Rozynek et al. (1998) who an a lyzed elec tro pho retic sep a ra tion of gliadins and secalins in pri mary forms of octoploid triticale, dif fer en ti a tion of gene ex pres sion en cod ing those pro teins can re sult from the interaction between parental rye and wheat genomes, effect of foreign cytoplasm as well as high level of aneuploidy.
CONCLUSIONS
Ad di tion of rye chro mo somes to wheat had no ef fect on high-mo lec u lar weight glutenin com po si tion. In ‘Grana’ wheat and all ad di tion lines, the same com po si -tion of glutenin frac -tion (N-6+8-2+12) was ob served. In an a lyzed ad di -tion lines, no poly mor phism re fer ring to high-mo lec u lar weight glutenin com po si tion was found. Rye stor age pro teins (secalins) were iden ti fied in 1R and 2R ad di tion lines and sub sti tu tion line 1B/1R. Re sults achieved al low con clud ing that in rye cv. Dañkowskie Z³ote Sec genes re spon si ble for secalin syn the sis are localized on 1R and 2R chromosomes.
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