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J. lnst. Bre11·.. Jul y-A ugust. 1983. Vol. 89, pp. 299-302

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299

VARIETAL ID ENTIFICATI ON Of 11..\RLE\' AND l\ IALT* Bv A.

MoN TBtBA U L T, J .

C. ..\t.:TRAN . P. J ouoRJER

(Laboratoire de Technologie des Céréales. /.VR.-1. 9 Place 1ïala. F-3./060 l1fo11tpe//ier Cedex. France) AND

M . Mou.

(Centre de Recherches TEPR..t L, 1 Rue Gabriel Bour. F- 5::/150 Champig11eu//es. France) Rcceired 9 Dcœmhcr 1982

A vertical polyacrylamide - SOS electrophoretic technique, including whole protein extraction and staining steps, was improved with a view to d e veloping it for routine laboratory use with single barley kernels. The pattern consisted of 4 zones: A (albumins-globulins), Band C (hordeins) and D (possibly glutelins) displaying unequal varietal polymorphisms (1, 13, 13 and 4 types re'spectively). 28% of the barley samples (77 varieties), including most cultivars grown in France, could be unambiguously identified from qualita t ive differences only, in the 8, C and D zones. Adding three other characteristics (hairs and furrow h a iriness, peroxidase, zymogram, esterase zymogram), as many as 78% of the varieties could b e identified, the other 22% consisting of very closely related barleys. After slight modifica tion of protein extraction conditions, the same methods could be used with malt, based on the same e lectrophoretic types. A graphie tablet connected to a microcomputer was used for automatic acquisitions of records and comparisons of electrophoretic data. · JiOicultics due to problems in the extraction ofstorage protein and to the biological process ofma lting have delayed the use of clcctrophoresis as a routine laboratory proced ure for \':trie tal identification. Morco\·er. the method has to be val id for both barlcy and malted barlcy. ln se\'crnl studies barley hordeins have been investigated in regard to thcir bchav iour in vario us electrophoretic s) stcms. Sta rch geP·v poorly resol\'ed P-hordcin type componcnts. The use of convcntiona l polyacrylamidc gel citha in sla bh or in disc 7 apriaratus gcnerally showed insufticient seriar:ition of protcins in the case of ma lts. Isoelectric focus ing 11 • 1 ~ allowed good fractionation s of malt proteins but did not lc:id to identical patterns with barley and malt. Aecordingl y. wc suggestcd th:it clectrophoretic mcthods b:ised on diffcrences in mol ccular sizc on ly, instead ofelectrical cha rge. might be more likcly to minimise the elfect of mailing on kerncl protein pattcrns.M Following S h ewry's 1 i. 1 ~ st udics on vertica l electrophoretic systems in pol yacryl:i mide-SDS medium , we developed a single kcrnel method of varietal identification based on

Key words: barh•.1'. 1·aric•t_1'. malt. elcctrop/10resi.\'. compwer a11al.rsis. JN TRODL'CTI0:-1

13ecause the effect of variety on mailing properties ofbarley is especial ly important in the m~dting and brcwing industries. there is a need for methods or va rictal identification of barky and malt. Morrihological charaetcrisation is a most uscful mcthod. but it rcquircs skilkJ oricrators and is not val id \\ith m:i lt duc to thc disappcar:incc or somc morphologic:i l characteri stics. From sevcr:i l studics on whc:it. 1 2 clcctrophoresis of proteins seemed to be thc most suitable method fo r positi\·c identification since protcins arc direct products of genc tr:inscri ption :iml thc reforc rctkct the genotype of the orga nis m. 1 ~ ln thc case of b:irlc y, howe\·er. •rn:scntcc.l at the 7th Worlc.J Cereal and Bread Congrcss, Pragu.:. :?8 Junc- 2 Ju ly, 1982.

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Fig. 1. Polyacrylamide-SDS clcctrophcrograms or wholc protcins extracted from bark } single sceds. Culti vars: · 1- r-.lcnuct: 2-Polka; 3-Adorra: 4- Dragon: 5-l'irolinc; 6-,\lpha: 7- Thi baud: 8- Sonja: 9- Voguc; 1 0-C~ tr i s.

300

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Fig. 2. Comparison of the diffcrcnt types of barley electrophorctic patterns.

.wholc kemcl protein (hordein. glutelin, soluble protein) patterns. Simplified extraction and staining procedures were worked out wi th n view to routine labor:llory use. Elcctrophoretic componcnts of ail va ricties wcre passed through a graphie tablct connected to a mieroeomputcr and a program allowing the reeovery of the name of the variety from an electrophoretie pattern was dcvc lopcd . ln the case of wry closely related varicties, the use of enzyme polymorphisms and morphologieal eharacteristics has also becn eonsidcred for further difTercntiation.

M ATERIALS AND METllODS

Representative samples of 77 cultivars covering the barleys most extensively grown in France, were obtained from several brecders. Thirty micromalts and 10 industrial malts processcd from some of these above mentioned culti vars were obtaincd from TEPRAL"s laboratorics.

Proteins were extraeted from single seeds ofbarley or malt by 8 ~ilnng of seed of the the following sol ut ion: Tris-SOS buffer ~-f\ 1crcaptocthanol Dimcthylformamide Distilled water

llARLEY

MALT

3·40 ml 0·95 ml 1·80 ml to 12·00 ml

3·40 ml 1·20ml 1·00 ml to 12·00 ml

Tris-SOS bufîer was obtained by mixing 6·25 ml IM Tris pH 6·8 so lution. 10 ml glycerol, 2 g SDS and 10 mg pyronin (migration marker) and was brought to 28·3 ml by distilled water. Extraction , including a prote in reduction step, was earried out for 150 s (barley) or 90 s (malt) in a boiling water bath. Electrophoresis was performed in vertical polyacrylamidc-SDS slab gels accord ing to Laemi li's conditi ons. A 31lo polyacrylamide pH 6·8 concentration gel and a l 31Vo

8· l · N

Fig. 3. lnheritance of electrophoretie types in the genealogy ofbarl ey varieties, e.g. 6-2-N = type 6 in B zone, type 2 in C zone and type N in D zone.

JI. 89. 1983]

301

MONTEMllAUL TET AL: BA RLEY AND MALT \IAR IET AL IDENTIFI CATION

TABLE 1. Pcrccntaec of Discrimination of the 77 French Cultivars Rcsulting from Cumulating 1, 2, 3 or 4 Methods of Varietal Identification Mcthod: 1

Protein elcctrophoresis

Methods: 1 + 2

Mcthods: 1 + 2 + 3

Mcthods: 1 + 2 + 3 + 4

Protein electrophorcsis + morphologica l characteristics

Protein electrophoresis + morphological characteristics + peroxidase

Protein electrophoresis + morphological characteristics + peroxidase +este rase

Percentage Numbcr of of varietal groups discrimination

Percentage Number of of varietal groups discrimination

:

Number of varicties in the group 1 2 3 4 5

6 7 8

Perccntage Pcrccntage of varictal Numbcr of of va rictal Nu mbcr of groups groups discrimination discrimination 22 8 3 2 1 1 1 1

28·5 21·0 12·0 5·0 6·5 8·0 9·0 10·0

35 7 4 1

45·4 18·0 16·0 5·0

2

15·5

polyacrylamide pH 8·8 separation gel were used in a LKB "lectrophoresis appa rat us. Migration was carried out for 16 h l 8:C under constant intensit y (3·5 mA per gel slab). Pro1eins were fi xed for 30 min in a 15% (w/v) trichloroacetic acid solution and then sta ined for 30 min at 90°C in a O· I75% (w/v) Coomassie Blue BBR 250 solution (acetic acid /cthanol/water; 10135155: v/ v/ v). Complemcntary enzy me fractionations wcre carried out according to the M ac ko~ procedure for cathodic peroxidases and the met hod ofScandalios 10 fo r anodic u- and p-estcrases.

49 6 4 1

64·0 15·5 15·5 5·0

60 7 1

78·0 18·0 4·0

distinguished and , by add in g the fou r possible types ofbarley estcrases, a differentiation of 78% of the cultiva rs could be obtained (Table 1).

.\/ails.- The dccrease of about 40% of horde in du ring germination and the insolubilisation of protein during kilning made the investigation of malt more difficult. However, the use of a higher concentration of reducing agent in the extracting solution allowed a good resolution of the patterns to be obtained and the same qualitati ve elcctropherograms to be ohtained for better ba rley and mal t. Sorne quantitati ve modifications occurrcd. cspccia ll y in very modificd kernels from industrial malts. but the barlcy classi fication based on R ESCL TS AND DISCUSSION ·1r. ·c and ·o· clectrophorctic rcgions and the number of Bar/e.rs.-Firstl y it was confi rmed that SOS electrophero- types wc rc prcserved (Fig. 4) al lowing the sa me key of grams of whole barl ey proteins we re not intluenced by va rictal identification for both barleys and malts lo be environmcntal factors (of growth and cultural conditions). proposcd. The clcctrophcrograms of Fig. 1 show tha t componcnts se parated on the basis of molecula r weight in polyacrylCvmp111erised acq111.wron of e/ecltrop horetic data. amidc-SDS gels arc distributed i nto fo ur groups: A·. s·, C': C lassica ll y, the electrop herogram interpretation is bascd on (C 1 +C~l and ·o·. According to prcvious nomenclatures. only two dilfcrcn t criteria, mobility and relative concentration of ·s· and ·c,' secm to correspond to the !nain storage protcins. each component. Making a dic hotomie identification key From .preliminary ph ysico-chcmica l studesK however. the from these two criteria requires a complete knowledge of the nroup ' ..\' and ·c; co mponcn ts cou Id be assigned to diagrams. Moreove r, with the an nuai addition of new cu ltiùumins-globulins -wnereas ' D', which was not system- vars to the catalogue it is necessary to modify the key more aticall y rc portcd in other s tudies , 13 • 1 ~ could probably or less compl etcly. Accordingly, wc fcel that an open identicorrespond to a glutclin fraction . fication kc y and a n au tomatic intcrpretation procedure are Ali groups. excepti ng 'A', displayed substant ial qualitati\'e highl y dcsirablc. intervarietal polymorphism so that the 27 barley cult ivars For this purpose, a microcomputer has been coupled with cou Id be classi fi ed into 13 types from the 'B' group onl y. into a gra phie tablet and magnetic stylo system. so that it is pos13 other types from the 'C' group on ly and into 4 other types sible to ca lculate automaticall y the distance between two from the ·o· group only (Fig. 2). If the three regions ·s·. ·c points. From any electropherogram (gel or photograph) a and ·o· were taken into acco unt simultaneously, 22 cu ltivars starting point (electrophoretic slot) was defin ed; then, using (28·5% of the sampling of French ba rleys) could be identi- the magnetic stylo, all components of the pattern were plotfied unambiguously. T he other cu ltivars fell into groups of tcd successively. The relati ve (or absolute) mobilities (M) 2, 3, 4. 5, 6, 7 or 8 un its that were always made up by we re automaticall y recorded and appeared on a screen. close ly related gcnotypes, as is evidenced by the Fig. 3 famil y Optiona l coeffi cients such as relative concentrations (k) of tree of barley va rieties in Fig. 3. components (for instance scored 1, 3. 5.) through visual Furthcr identi fica tions wit hi n these groups could be examination of the patterns cou Id also be introduced. From attempted from quantita tive charac teristics (intensity of thcse data, several types of calculat ions could be carried out bands) of the eleetropherograms. lt was however. preferred by the rnicrocomputer: to exa mine two stable and discrcte morphological characters such as 'rachilla ha irs' (long and straigh t or short and wooll y) -numbcr of componcnts: n and 'furrow hai rincss' (glabrous or short dense hairs). An -sum of mohi liti es of the n components: L Mi extra discriminat io n of thi s kinu allowcd 45·5'Vo of the culti - -sum of mobilities of th e n components bearing concenvn·~ to be distinguished unambiguously. If neccssary, a trat ion coe fli cient: L ki, Mi. 1 plementary step co uId be bascd on enzyme fractionation and dctection. Tak in g into account the live possible types of T hcsc th rcc last paramcters werc recorded for ail cultivars. ba rley peroxidase patterns, 64% of the cul tivars could be.: Through a comparison programme, therefore it was

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ln conclusion. polyacryla mid e-SDS electrophoresis of barlcy protcins is a powcrful tool for displaying interva rielal pol ~ morphism and brings new pot en Lia i into Lhe variclal id.:ntif1cat ion o f c ult ivars. The biochemical distribution bascd on 'Ir. ·c and ·o· electrophoretic rcgions p roved Lo tic ra rti cu larly di sc rimin ant and appears also to be consisLcnl with the genct ical classificat ion (Hord 2 genc: B rnmponcnts. Hord 1 gc ne: C components). The approach using comput erised acqu isition o f electrophoretic data is of grca t promise a nd should be cmphasised in both genetic and q uality st udics.

o{

B

A

+ 2

3

4

5

6

Fig. 4. J>olyacrylam idc-SDS dcctrophcrograms of wholc protcins cx1rac1cd from sinclc mail crains. Culli\'ars: 1-0..:tina: 2-D.:1>1cr; 3-Alpha: -1--Càrina: 5-Dcisi.:r: 6--Carina. possible to know whe ther the va lues tha t were com pulcd fro m a ny unk nown pattern agrecd wi th those from previously recorded cu lti var. If a ll va lues (n, ~ Mi. ~ ki .f\li) agreed. th e name oflhe cult ivar was aulomatica ll y displa yed. ln a n opliona l programme. the theorelica l sc heme o f the pa tte rn (regions ·ir. ·c. 'D') was simultaneously di srla~·cd. which a llowcd a fina l ,·cry simrle c hccking of the diagno~ i s.

j

lll.H.RISCES 1. :\ ut ra n. J. C.. 13crricr. R.. Jcanjcan. M. F.. Joudricr, P. & Kohrchcl. K.. !nd11.1·1ries dt•s Ci!r i·alcs. 198 1. 8, 3. .., Autran. J. C. & Bourdet. A.. A1111alcs de /'Améliora1io11 des Plantes. 1975. 25. 277. 3...\ut ran . J. C. & Scriban , R.. E11rvt1ca11 Bre1ra.1· Co11re111io11, l 'mcc,·d111g.1· ()(lhl' l