- Email: [email protected]
Barley genetics — not only here for the beer
IOMMENT
Barley genetics - not 0nly here for the beer BENGT O. BENGTSSON DEPARTMENTOFGENErics,LL'NOUNIVERSITY, SOLVEGATAN29, S-223 62 Lt'XD,SWEDEN. How do you like your science? Good, of course, but which flavour? Pure and elegant, with the beauty of abstract models and dean-cut results? Or crude and wide-ranging, with the messy but sensual pleasures of real life? If you are of the second type, then you would have enjoyed attcnding the Sixth International Barley Genetics Symposium, held at Helsingborg, Sweden, in July 1991. Here you would have learnt about cloudy beer, hormone promoter sequences, snow damage to crops in Hokuriku, an electric gun to decapitate aphids, and lots more in a strange but wonderful mixture. To understand why these conferences are so special, one must first know something about barley itself.
A crop for both rich and poor Together with primitive forms of wheat, barley was the first plant to become properly domesticated. A thick barley paste - maza or puis - constituted the staple food in Greek and Italian antiquity. The tradition of eating barley porridge continued among European peasants well into our century, particularly in the Nordic countries. The loss of this habit is gastronomicaily understandable but deplorable from a dietary point of view, since both barley bran and oil are excellent at reducing serum cholesterol values (R.K. Newman, Montana; F. Weber, Miller Brewing Co., Milwaukee). As food, barley is today mainly used by the poor for whom rice, maize and wheat are impossible to grow and too expensive to buy. Its broad ecological adaptability sets it apart from the other cereals. Barley prefers a cool and moderately dry temperate climate, but it can be found in south China as a winter crop, at high altitudes in the Andes, in Iceland's short and windy summer season, and in the heat of central Spain. It can be argued that barley is the world's most
important crop for farmers trying to live off agriculturally marginal areas. Special attention to breeding for such areas is given by the International Center for Agricultural Research in the Dry Areas (ICARDA) at Aleppo, Syria. Barley is an important crop in the rich world too, particularly in Europe. Globally it ranks as the fourth of the cereal crops, after wheat, rice and maize, with a yearly production of around 170 million tons. Most of this goes to feed pigs, chicken and, to some extent, cattle. The trade in barley is not extensive, since most farmers keep the harvest on the farm to give to their animals. The straw is normally not used, though Lars Munck (Carlsberg, Copenhagen) gave a spirited plea for its use in paper production. Of very special importance is the best of the barley: it is used for brewing beer. Beer should be made from malted barley, yeast, hops, water and nothing else. Other cereals can be used for beer making, but none is as suitable as barley for a number of reasons. One is that beer brewed from barley tastes good, while another is that the husk, which covers the barley seed and which wheat and rye lack, is helpful as a filtration agent in the brewing process. Barley malt is also used for making whisky, which is, however, of little direct agricultural or scientific relevance.
A plant for geneticists Barley (Hordeum t,ulgare) has a combination of specific advantages that have made it a model organism for many plant geneticists and physiologists. It is easy to cultivate, both on a small and a large scale, and it has a reasonably short generation time (about 4 months in the greenhouse). Its chromosome number is diploid (2n = 14), and it normally reproduces by self-fertilization. The last property is important since genetically T1G JANUARY 1 9 9 2 VOL. 8 NO. 1
1992 Elsevier Scient'e Publishers Ltd ( ! :K I
n
well-defined barley lines can easily be maintained without any direct inbreeding effects. There are now standard methods for producing haploid plants, which after chromosome doubling become perfectly homozygous. A very wide range of genetic variation is available for every agriculturally important character in barley. The domesticated form is also fully fertile with its wild relative, ssp. spontaneum, which grows in west and central Asia. Nowadays, extensive efforts are directed at tapping this natural resource for useful characters, in particular genes that improve disease resistance (A.H.D. Brown, CSIRO, Canberra; G. Fischbeck, Mfinchen; L. Lehmann, Sval6v, Sweden: and many others). Paradoxically, given the amount of natural variation available, it is in the field of mutation research that barley has been particularly important. From Stadler's early studies on the effects of X rays on plants, barley has been used to measure the physiological and genetic effects of mutagenic treatments. Huge numbers of gene and chromosome mutations have over the years been collected, analysed and maintained. This pool of well-defined mutants may constitute barley's greatest asset as a model organism. Most genetic research in barley has been performed in close contact with breeding problems. Undou3tedly, this has given barley genetics a somewhat slow pace and an unfashionable style. But barley research has, at the same time, been able to capitalize on the broad and often fascinating questions that the breeders turn up. plus the willingness of the barley community to exchange information and stocks.
Cloudy beer A good example of the broad nature of barley research and the u~efulnc'~s of barley mutants was given at the conference by Barbro
[I]OMMENT than 700 mutants lacking anthocyanin and other flavonoid substances were used to work out the relevant part of the proanthocyanidin pathway (Fig. 1). From this information, mutants in a structural (ant 26) and a regulatory gene (ant 27), both with wild-type amounts of anthocyanin but without any proanthocyanidins, were chosen as the most promising candidates for further breeding. It will still take some years before it is known if lines with these mutants will fulfil the demand for a high yield, but the first field trials have given promising results.
Jende-Strid, who described the efforts made at the Carlsberg Research Center to breed better malting barley. The problem they have tackled is the protein haze that sometimes forms in beer, giving it a cloudy look. Their starting point was an observation chat this unwanted effect is avoided if the malt comes from seeds low in certain flavonoid compounds related to the pigment anthocyanin. At first the breedet's tried to base their new lines on mutants identified by their lack of anthocyanin and low amounts of many of the flavonoids. However, after intensive breeding it was found that such lines do not, in general, give the high yield needed in modern varieties. A new attempt was therefore made to produce plants exclusively lacking the proanthocyanidins which are the prime culprits in the haze formation. More
COOX 0 c x~ CO- SCoA 3 MALONYL-CoA
coAs-c "f ~ 0 4- COUMAROYL;CoA
Pathways and regulation Mutant-based genetic analyses have also been applied to the biochemical pathways of, for example, nitrite reduction (J. Wray, St Andrews) and the synthesis of waxy lipids (P. yon Wettstein-
.ooc ~ ~
. o o c 'd ~
4-COUMARIC ACID
.ooc
CINNA.IC ACID PHENYLALANINE
OH 0 NARINGENIN CHALk.ONE
Knowles, Carlsberg, Copenhagen). This approach becomes particularly interesting and powerful when the aim is not just to find the relevant enzymes, but to investigate the way in which they are developmentally regulated. Most attention has so far been given to the regulation of genes expressed in the seed during its maturation and germination (P.R. Shewry, Bristol). The work on hormone regulation of amylases in aleurone cells (P.M. Chandler, CSIRO, Canberra; F. Lok Olsen, Carisberg, Copenhagen) was particularly impressive, while many other studies mainly communicated frustration over the lack of good expression systems in barley. This is now changing (see below), and I am sure that tissue-specific promoters will be one of the key subjects at the next conference. As often in barley research, this question is not only of academic interest. If an increased understanding of the germination process makes a better fine-tuning possible, then there is a lot of practical molecular breeding to be done. One aim will, for example, be to combine an improved ease of germination in the malting house with stronger resistance to preharvest sprouting in the spike.
Rules from monsters NARINGENIN
ERIOOICTYOL
OH 0 DIHYDRO(]UERCETIN
IX)
OH OH
1tO
OH OH
CYANIOIN
140
OH
OH OH
2,3- trans- 3,4- cis* LEUCOCYANIDIN
CATECHIN
n3 PROCYANIDIN C2
FIG[[I The pathway to the clouds. ProcyanidinB3 and procyanidin C2 are two of the proanthocyanidins in barley that promote haze formation in beer. Illustration kindly provided by B. Jende-Strid (Carlsberg, Copenhagen). TIC; JANt:ARY 1 9 9 2 VOI.. 8 NO. 1
n
A beautiful example of how the established collection of barley mutants can be used for interesting scientific questions was given by Francesco Salamini (KOin). What to others look like hopeless monsters (see Fig. 2), have in his eyes become examples of disturbances of the normal developmental rules. On the basis of genetic and histological studies by Udda Lundqvist (SvalOf AB, Sweden) and Gerd Bossinger (K61n), Salamini gave an outline of how a grass of barley's type is built exclusively from a repetition of 'phytomers', consisting of an internode associated with a leaf at its upper end and a bud opposite the leaf at its lower end. This takes a bit of imagination to believe when it comes to such parts as the spike and its flowers, but Salamini used a set of different malformed mutants to illustrate the range of transformations possible for the basic phytomeric unit.
L~OMMENT Towards a better future
beginning of a real breakBarley has not only adthrough, since there are vantages as a genetic ormany interesting characters ganism but also some among the wild barleys, severe disadvantages. The such as salt tolerance, that first is that, together with all one would like to see other cereal species, it incorporated into cultivated lacks an endogenous transbarley. formation system. Hartmut Schreiber (Quedlingburg, Getting the genes in order FRG) and colleagues deThere is an older, single scribed an interesting genexception to the statement etic instability that they have that there have been no studied using the pollenfertile interspecies crosses expressed waxy gene. Their involving barley. An expresentation showed the tremely useful set of wheat advantage of performing plants with single additional genetic analysis at the hapbadey chromosomes was loid stage, but whether any produced some years ago transposable element is by Rafiqul Islam and Ken associated with the effect is Shepherd (Adelaide), and still unknown. Another case they reported on the conof instability was shown by struction of recombinants Alberto Prina (Castelar, that, in a wheat backArgentina) to depend on a ground, contain further recessive nuclear mutation ~. reduced parts of the barley that increases the mutation genome. These lines will. rate in the chloroplast. of course, be of particular blGm The lack of an endogenimportance in the gene mapous transformation system Morphological abnormalities,as in the spike to the right, are ping work that has always is no longer so critical, used to understand the rules governing the development of been strong in barley. normal barley plants (left). Photograph kindly provided by In this field, the most since transformation sys- U. Lundqvist(Sval6fAB, Sweden). important news concerned tems developed in other the recent completion of species can - in principle be used. They also work in prac- independent of the genetic consti- three restriction fragment length tice, at least sometimes, and at the tution of the material. For studies polymorphism (RFLP) maps based conference there were many dis- on gene regulation, it will also be on more than 100 clones produced cussitms of the relative advantages important to develop techniques by European and US groups in a of electroporation and particle gun for the cultivation of as many mixture of competition and coordination. From both practical and methods for getting DNA into dif- tissues as possible. Some people claim that the theoretical points of view these ferent types of cells. The most severe disadvantage strongest argument for studying maps are most welcome, and so with barley has been the impossi- badey is that this is the best are the efforts to coordinate the bility of regenerating plants from approach to wheat, rye, oats and results from the different data sets. protoplast cultures. But here the other important grasses that are Because of its large size and comsituation is now much brighter, polyploid or in other ways difficult plex nature, the barley genome is since a number of groups reported to study directly. This argument not a candidate for early sequencthat they have been able to de- would be further strengthened if it ing. Instead one can hope that a velop normal fertile plants from was possible to move genetic ma- concentrated effort will go into protoplasts (P.A. Lazzeri and H. terial between the species, but here coordinating the genetic, cytoL6rz, Hamburg: N. Satoh and col- there have always been problems. logical and physical maps of the leagues, Sapporo Breweries, Barley can be crossed with many species. It seems to me that barley Japan). No-one at the conference, other grasses, but the result is is an ideal organism for sorting out, however, stood up and showed a invariably a sterile hybrid. This has for example, the exact relationship barley plant derived from a trans- been the truth up to the time of the between gene distribution, crossff~rmed protoplast culture (though conference, when Richard Picker- over points and chiasmata in higher according to whispers in the corri- ing (Christchurch, New Zealand) plants. I am sure there will be somedors such plants exist). Anyway, it and Ken Kasha (Guelph, Canada) must now only be a question of reported convincing evidence of thing on this, plus a lot more. in months before such plants are pre- recombination between domesti- the rich brew that will make up the sented. Then the real work will cated barley and its closest species Seventh International Barley Genbegin to make the transformation relative, Hordeum bulbosum, in etics Symposium, due to be held in and the regeneration methods some specially constructed hybrids. Canada in 1996. Will you be strong more efficient and, in particular, It is to be hoped that this is the enough for it? TIGJANL'ARY1992 rOE 8 xo. 1
I1
Barley genetics - not 0nly here for the beer BENGT O. BENGTSSON DEPARTMENTOFGENErics,LL'NOUNIVERSITY, SOLVEGATAN29, S-223 62 Lt'XD,SWEDEN. How do you like your science? Good, of course, but which flavour? Pure and elegant, with the beauty of abstract models and dean-cut results? Or crude and wide-ranging, with the messy but sensual pleasures of real life? If you are of the second type, then you would have enjoyed attcnding the Sixth International Barley Genetics Symposium, held at Helsingborg, Sweden, in July 1991. Here you would have learnt about cloudy beer, hormone promoter sequences, snow damage to crops in Hokuriku, an electric gun to decapitate aphids, and lots more in a strange but wonderful mixture. To understand why these conferences are so special, one must first know something about barley itself.
A crop for both rich and poor Together with primitive forms of wheat, barley was the first plant to become properly domesticated. A thick barley paste - maza or puis - constituted the staple food in Greek and Italian antiquity. The tradition of eating barley porridge continued among European peasants well into our century, particularly in the Nordic countries. The loss of this habit is gastronomicaily understandable but deplorable from a dietary point of view, since both barley bran and oil are excellent at reducing serum cholesterol values (R.K. Newman, Montana; F. Weber, Miller Brewing Co., Milwaukee). As food, barley is today mainly used by the poor for whom rice, maize and wheat are impossible to grow and too expensive to buy. Its broad ecological adaptability sets it apart from the other cereals. Barley prefers a cool and moderately dry temperate climate, but it can be found in south China as a winter crop, at high altitudes in the Andes, in Iceland's short and windy summer season, and in the heat of central Spain. It can be argued that barley is the world's most
important crop for farmers trying to live off agriculturally marginal areas. Special attention to breeding for such areas is given by the International Center for Agricultural Research in the Dry Areas (ICARDA) at Aleppo, Syria. Barley is an important crop in the rich world too, particularly in Europe. Globally it ranks as the fourth of the cereal crops, after wheat, rice and maize, with a yearly production of around 170 million tons. Most of this goes to feed pigs, chicken and, to some extent, cattle. The trade in barley is not extensive, since most farmers keep the harvest on the farm to give to their animals. The straw is normally not used, though Lars Munck (Carlsberg, Copenhagen) gave a spirited plea for its use in paper production. Of very special importance is the best of the barley: it is used for brewing beer. Beer should be made from malted barley, yeast, hops, water and nothing else. Other cereals can be used for beer making, but none is as suitable as barley for a number of reasons. One is that beer brewed from barley tastes good, while another is that the husk, which covers the barley seed and which wheat and rye lack, is helpful as a filtration agent in the brewing process. Barley malt is also used for making whisky, which is, however, of little direct agricultural or scientific relevance.
A plant for geneticists Barley (Hordeum t,ulgare) has a combination of specific advantages that have made it a model organism for many plant geneticists and physiologists. It is easy to cultivate, both on a small and a large scale, and it has a reasonably short generation time (about 4 months in the greenhouse). Its chromosome number is diploid (2n = 14), and it normally reproduces by self-fertilization. The last property is important since genetically T1G JANUARY 1 9 9 2 VOL. 8 NO. 1
1992 Elsevier Scient'e Publishers Ltd ( ! :K I
n
well-defined barley lines can easily be maintained without any direct inbreeding effects. There are now standard methods for producing haploid plants, which after chromosome doubling become perfectly homozygous. A very wide range of genetic variation is available for every agriculturally important character in barley. The domesticated form is also fully fertile with its wild relative, ssp. spontaneum, which grows in west and central Asia. Nowadays, extensive efforts are directed at tapping this natural resource for useful characters, in particular genes that improve disease resistance (A.H.D. Brown, CSIRO, Canberra; G. Fischbeck, Mfinchen; L. Lehmann, Sval6v, Sweden: and many others). Paradoxically, given the amount of natural variation available, it is in the field of mutation research that barley has been particularly important. From Stadler's early studies on the effects of X rays on plants, barley has been used to measure the physiological and genetic effects of mutagenic treatments. Huge numbers of gene and chromosome mutations have over the years been collected, analysed and maintained. This pool of well-defined mutants may constitute barley's greatest asset as a model organism. Most genetic research in barley has been performed in close contact with breeding problems. Undou3tedly, this has given barley genetics a somewhat slow pace and an unfashionable style. But barley research has, at the same time, been able to capitalize on the broad and often fascinating questions that the breeders turn up. plus the willingness of the barley community to exchange information and stocks.
Cloudy beer A good example of the broad nature of barley research and the u~efulnc'~s of barley mutants was given at the conference by Barbro
[I]OMMENT than 700 mutants lacking anthocyanin and other flavonoid substances were used to work out the relevant part of the proanthocyanidin pathway (Fig. 1). From this information, mutants in a structural (ant 26) and a regulatory gene (ant 27), both with wild-type amounts of anthocyanin but without any proanthocyanidins, were chosen as the most promising candidates for further breeding. It will still take some years before it is known if lines with these mutants will fulfil the demand for a high yield, but the first field trials have given promising results.
Jende-Strid, who described the efforts made at the Carlsberg Research Center to breed better malting barley. The problem they have tackled is the protein haze that sometimes forms in beer, giving it a cloudy look. Their starting point was an observation chat this unwanted effect is avoided if the malt comes from seeds low in certain flavonoid compounds related to the pigment anthocyanin. At first the breedet's tried to base their new lines on mutants identified by their lack of anthocyanin and low amounts of many of the flavonoids. However, after intensive breeding it was found that such lines do not, in general, give the high yield needed in modern varieties. A new attempt was therefore made to produce plants exclusively lacking the proanthocyanidins which are the prime culprits in the haze formation. More
COOX 0 c x~ CO- SCoA 3 MALONYL-CoA
coAs-c "f ~ 0 4- COUMAROYL;CoA
Pathways and regulation Mutant-based genetic analyses have also been applied to the biochemical pathways of, for example, nitrite reduction (J. Wray, St Andrews) and the synthesis of waxy lipids (P. yon Wettstein-
.ooc ~ ~
. o o c 'd ~
4-COUMARIC ACID
.ooc
CINNA.IC ACID PHENYLALANINE
OH 0 NARINGENIN CHALk.ONE
Knowles, Carlsberg, Copenhagen). This approach becomes particularly interesting and powerful when the aim is not just to find the relevant enzymes, but to investigate the way in which they are developmentally regulated. Most attention has so far been given to the regulation of genes expressed in the seed during its maturation and germination (P.R. Shewry, Bristol). The work on hormone regulation of amylases in aleurone cells (P.M. Chandler, CSIRO, Canberra; F. Lok Olsen, Carisberg, Copenhagen) was particularly impressive, while many other studies mainly communicated frustration over the lack of good expression systems in barley. This is now changing (see below), and I am sure that tissue-specific promoters will be one of the key subjects at the next conference. As often in barley research, this question is not only of academic interest. If an increased understanding of the germination process makes a better fine-tuning possible, then there is a lot of practical molecular breeding to be done. One aim will, for example, be to combine an improved ease of germination in the malting house with stronger resistance to preharvest sprouting in the spike.
Rules from monsters NARINGENIN
ERIOOICTYOL
OH 0 DIHYDRO(]UERCETIN
IX)
OH OH
1tO
OH OH
CYANIOIN
140
OH
OH OH
2,3- trans- 3,4- cis* LEUCOCYANIDIN
CATECHIN
n3 PROCYANIDIN C2
FIG[[I The pathway to the clouds. ProcyanidinB3 and procyanidin C2 are two of the proanthocyanidins in barley that promote haze formation in beer. Illustration kindly provided by B. Jende-Strid (Carlsberg, Copenhagen). TIC; JANt:ARY 1 9 9 2 VOI.. 8 NO. 1
n
A beautiful example of how the established collection of barley mutants can be used for interesting scientific questions was given by Francesco Salamini (KOin). What to others look like hopeless monsters (see Fig. 2), have in his eyes become examples of disturbances of the normal developmental rules. On the basis of genetic and histological studies by Udda Lundqvist (SvalOf AB, Sweden) and Gerd Bossinger (K61n), Salamini gave an outline of how a grass of barley's type is built exclusively from a repetition of 'phytomers', consisting of an internode associated with a leaf at its upper end and a bud opposite the leaf at its lower end. This takes a bit of imagination to believe when it comes to such parts as the spike and its flowers, but Salamini used a set of different malformed mutants to illustrate the range of transformations possible for the basic phytomeric unit.
L~OMMENT Towards a better future
beginning of a real breakBarley has not only adthrough, since there are vantages as a genetic ormany interesting characters ganism but also some among the wild barleys, severe disadvantages. The such as salt tolerance, that first is that, together with all one would like to see other cereal species, it incorporated into cultivated lacks an endogenous transbarley. formation system. Hartmut Schreiber (Quedlingburg, Getting the genes in order FRG) and colleagues deThere is an older, single scribed an interesting genexception to the statement etic instability that they have that there have been no studied using the pollenfertile interspecies crosses expressed waxy gene. Their involving barley. An expresentation showed the tremely useful set of wheat advantage of performing plants with single additional genetic analysis at the hapbadey chromosomes was loid stage, but whether any produced some years ago transposable element is by Rafiqul Islam and Ken associated with the effect is Shepherd (Adelaide), and still unknown. Another case they reported on the conof instability was shown by struction of recombinants Alberto Prina (Castelar, that, in a wheat backArgentina) to depend on a ground, contain further recessive nuclear mutation ~. reduced parts of the barley that increases the mutation genome. These lines will. rate in the chloroplast. of course, be of particular blGm The lack of an endogenimportance in the gene mapous transformation system Morphological abnormalities,as in the spike to the right, are ping work that has always is no longer so critical, used to understand the rules governing the development of been strong in barley. normal barley plants (left). Photograph kindly provided by In this field, the most since transformation sys- U. Lundqvist(Sval6fAB, Sweden). important news concerned tems developed in other the recent completion of species can - in principle be used. They also work in prac- independent of the genetic consti- three restriction fragment length tice, at least sometimes, and at the tution of the material. For studies polymorphism (RFLP) maps based conference there were many dis- on gene regulation, it will also be on more than 100 clones produced cussitms of the relative advantages important to develop techniques by European and US groups in a of electroporation and particle gun for the cultivation of as many mixture of competition and coordination. From both practical and methods for getting DNA into dif- tissues as possible. Some people claim that the theoretical points of view these ferent types of cells. The most severe disadvantage strongest argument for studying maps are most welcome, and so with barley has been the impossi- badey is that this is the best are the efforts to coordinate the bility of regenerating plants from approach to wheat, rye, oats and results from the different data sets. protoplast cultures. But here the other important grasses that are Because of its large size and comsituation is now much brighter, polyploid or in other ways difficult plex nature, the barley genome is since a number of groups reported to study directly. This argument not a candidate for early sequencthat they have been able to de- would be further strengthened if it ing. Instead one can hope that a velop normal fertile plants from was possible to move genetic ma- concentrated effort will go into protoplasts (P.A. Lazzeri and H. terial between the species, but here coordinating the genetic, cytoL6rz, Hamburg: N. Satoh and col- there have always been problems. logical and physical maps of the leagues, Sapporo Breweries, Barley can be crossed with many species. It seems to me that barley Japan). No-one at the conference, other grasses, but the result is is an ideal organism for sorting out, however, stood up and showed a invariably a sterile hybrid. This has for example, the exact relationship barley plant derived from a trans- been the truth up to the time of the between gene distribution, crossff~rmed protoplast culture (though conference, when Richard Picker- over points and chiasmata in higher according to whispers in the corri- ing (Christchurch, New Zealand) plants. I am sure there will be somedors such plants exist). Anyway, it and Ken Kasha (Guelph, Canada) must now only be a question of reported convincing evidence of thing on this, plus a lot more. in months before such plants are pre- recombination between domesti- the rich brew that will make up the sented. Then the real work will cated barley and its closest species Seventh International Barley Genbegin to make the transformation relative, Hordeum bulbosum, in etics Symposium, due to be held in and the regeneration methods some specially constructed hybrids. Canada in 1996. Will you be strong more efficient and, in particular, It is to be hoped that this is the enough for it? TIGJANL'ARY1992 rOE 8 xo. 1
I1