Endosperm Texture in Wheat

Journal of Cereal Science 36 (2002) 327±337 doi:10.1006/jcrs.2002.0468, available online at http://www.idealibrary.com on

1

Endosperm Texture in Wheat K.-M. Turnbull²³ and S Rahman*² ²CSIRO ± Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia; ³Plant Breeding Institute, University of Sydney, PMB, Cobbity, NSW 2570, Australia

ABSTRACT One of the fundamental means of classifying wheat is through its endosperm texture. It impacts signi®cantly on the milling process affecting among other things ¯our particle size and milling yield. Hardness in wheat is largely controlled by genetic factors but it can be affected by the environment and factors such as moisture, lipid, and pentosan content. The principal genetic locus controlling endosperm texture in wheat, Ha, is located on the chromosome 5D. At this locus several genes, notably the puroindolines, have been identi®ed. Puroindolines are the major components of the 15 kDa protein band associated with starch granules that is more abundant in soft wheats than in hard. Recently the puroindolines have been shown to enhance grain hardness in rice. In this review we discuss the structure of hard and soft wheat endosperm with particular emphasis on when differences in endosperm texture can be detected in the developing seed. The role of the environment and other factors that may affect the endosperm texture is also examined together with the role of the puroindoline genes at the Ha locus. Finally, we compare endosperm hardness in wheat and in barley. # 2002 Published by Elsevier Science Ltd

Keywords: grain hardness, wheat, endosperm, Ha locus, puroindoline, starch, barley.

INTRODUCTION Endosperm texture is one of the most important quality characteristics of wheat and it largely determines how a particular wheat cultivar is processed1. In this review we examine the structure, genetics, biochemistry, and molecular biology of endosperm texture. Factors that may have an impact on grain texture and the role of the puroindolines and related proteins in determining the endosperm texture are discussed.

A DEFINITION OF ENDOSPERM TEXTURE The endosperm texture or the relative hardness or softness of a grain can be de®ned as a measure of the resistance to deformation. This de®nition is at the basis of the measurement of hardness by the Single Kernel Characterisation System (SKCS), which measures the force required to crush individual grains of a sample between two surfaces taking into 

Corresponding author.

0733±5210/02/060327 ‡ 11 $35.00/0

account the weight, diameter, and moisture of the grain2. Using the SKCS, hard wheats have hardness scores of around 75 while soft wheats have scores of 30 or less3. More indirect de®nitions of grain texture refer to the manner in which grain breaks down to a meal or ¯our and how that meal or ¯our behaves during processing. For example, the particle size index (psi) is determined by grinding the grain and determining the percentage of the total weight that passes through a sieve3. Hard wheats have smaller psi scores than soft wheats. The hardness of wheats can also be assessed by other means such as near infra-red spectroscopy (NIR)4. The classi®cation of wheat as hard or soft is one of the most fundamental and useful means of classifying wheat and is used wherever wheat is grown on a large scale. To the miller, endosperm texture affects the tempering requirements, ¯our particle size, ¯our density, starch damage, water absorption, and milling yield5. To the processor, the endosperm texture is an indicator of the suitability of a particular ¯our for a particular product and to the grower, endosperm texture is important as generally higher premiums are paid for the harder wheats. # 2002 Published by Elsevier Science Ltd

328

K.-M. Turnbull and S. Rahman

The wheat grain is composed of the husk (fused pericarp and testa), aleurone, endosperm, and embryo. In barley the husk and aleurone have been shown to be harder than the starchy endosperm6 and this is likely to also be the case for wheat. However, because of the greater bulk of the endosperm, the hardness of the grain is largely determined by the properties of the endosperm. The embryo is soft and accounts for only a small proportion of the total grain. The starchy endosperm itself is a composite. The dried endosperm consists of cells ®lled by the protein/starch granule matrix and separated from the neighbouring cells by cell-walls7. The resistance of the grain to deformation would be determined by the weakest phase of this composite. It is possible that many genes may alter the hardness of the grain; however, only those genes that show variation in the genetic analysis performed will be detected. So far the most well characterised source of variation is the Ha locus located on the chromosome 5D, and this will largely form the focus of this review. THE STRUCTURE OF HARD AND SOFT WHEAT Macroscopically, the morphology of hard and soft wheat grains is similar. No differences in the kernel size and weight are observed between near isogenic hard and soft grains grown in similar environments8. The cut surface of a hard cultivar can often be distinguished from a soft cultivar by the amount of vitreousness on the cut surface but this is not always a rigorous predictor of grain texture (see more detailed discussion below). At the light microscope level mature hard and soft grains both contain large lenticular shaped starch granules (A-type granules) and the smaller more spherical B-type starch granules. Using micro-penetrometer tests, Barlow et al.9 showed that there is little difference between the resistance to deformation of starch granules from hard and soft wheat cultivars. Starch granules have been shown to differ between hard and soft wheat cultivars in their mean surface area but the results are equivocal10,11. Using quantitative image analysis some red American winter cultivars have been classi®ed as either hard or soft based on the diameter of the B-type granules12. Differences in the amount of material adhering to the starch granules from hard and soft wheat have been reported9. Hard wheat starches prepared by solvent sedimentation had more material adhering

to their surface than soft wheat starches prepared by the same method when examined by scanning electron microscopy (SEM)9. This observation led to one of the ®rst suggestions that an `extra' protein present in either soft or hard wheat is responsible for the adhesion, or the lack of adhesion, between the starch granules and the protein matrix (see later). The differences in structure between the hard and soft wheat grains are more apparent using SEM or transmission electron microscopy (TEM) (see, for example8,13,14). The cut surface of mature hard wheat examined under SEM reveals a compact uniform endosperm structure with starch granules ®rmly embedded in the surrounding protein matrix8,13. In contrast mature soft wheat has a much more disordered structure with the protein matrix in many cases being pulled away from the starch granules8,13,15. Protein accounts for approximately 10±13% of the grain16 and consists mainly of gliadins and glutenins. Protein content can vary signi®cantly for a given cultivar depending on the environmental conditions under which the wheat is grown but it is also under genetic control17. The composition of the storage protein or its amount (within a broad range of values) is unlikely to have any major effect on grain hardness. In our recent work with nearisogenic Heron lines, the soft cultivar was found to have a higher protein content (13
7%) than the hard cultivar (11
8%) yet it could be clearly distinguished as a soft8. Conversely, wheat lines with different storage protein patterns and dough properties can have very similar hardness scores. Developmental changes in endosperm structure in relation to grain hardness Endosperm texture in wheat and other cereals is usually discussed as a characteristic of the mature grain. This is not unexpected as it is mature grain that is classi®ed as hard or soft for market. However, by examining material throughout the development for grain hardness both Bechtel et al.18 and Turnbull et al.8 found that soft and hard cultivars could be distinguished provided the immature grain was allowed to air-dry. If, however, the material was freeze-dried then the grains could not be distinguished. This demonstrates that factors that cause the difference in grain hardness at maturity are already present in the immature grain. Visual differences between the cut surface of airdried soft and hard wheats can be detected by SEM

Endosperm texture in wheat

from as early as 5 days after ¯owering (DAF) and these differences were evident throughout the development until maturity8. A sub-sample of these wheat lines were tested for hardness using the SKCS and hard and soft lines could be distinguished from as early as 15 DAF. The smaller material at 5 and 10 DAF could not be measured accurately using the SKCS. The air-drying process is particularly important in the manifestation of grain softness or hardness. Using the same samples referred to above, Turnbull et al.8 found that hard and soft grains could not be distinguished on the basis of their structure when fresh developing endosperm sections from 5 to 32 DAF were examined under a light microscope. Similarly, Bechtel and Wilson14 were unable to ®nd signi®cant differences between the ultrastructure of developing hard and soft wheat using TEM of material ranging in age from 14 DAF to maturity; again, in this study freeze-dried material was used. The ®nding that the softness or hardness of the wheat grain is manifested throughout the development poses some interesting questions relating to the possible cause of differing endosperm texture in wheat. Some theories which explain the hardness and softness in wheat suggest that the trait is caused by the differing amounts of adhesion between the starch granules and surrounding protein matrix9,19. Others20 have suggested that the differences in hardness are related to the continuity of the protein matrix and the strength with which it physically entraps starch granules. However, the constitution of the starch/protein matrix formed on air-drying grain at 5 or even 15 DAF is very different from the starch/protein matrix formed in the mature grain. For example, at 15 DAF the number of starch granules in the endosperm is much less than at maturity and the protein complement contains far less storage protein8,14 yet the hardness scores at 15 DAF and at maturity are very similar and the hard and soft wheats can be easily distinguished.

Endosperm texture and vitreousness Hardness and vitreousness are often referred to synonymously21,22 yet they describe different characteristics of the wheat grain. Hardness, as mentioned above, refers to the strength of the grain in regard to its resistance to deformation, whereas vitreousness is a visual description of the appearance of the grain. Vitreous (translucent, horny) kernels appear glassy

329

and under a microscope vitreous endosperms are tightly packed with essentially no air-spaces. Nonvitreous (opaque, ¯oury) kernels appear white and mealy on the surface and have a discontinuous endosperm with numerous air-spaces23. Hosney24 suggests that as the cytoplasm in the endosperm dries it shrinks and either still remains intact (vitreous grains) or ruptures leaving air spaces (opaque kernels). This interpretation may partly explain the results of Parish and Halse25 who found that both the temperature and light intensity during grain ®lling and the rate of drying at maturity can determine if the grain will appear vitreous or opaque. Nitrogen availability during the kernel development also affects kernel appearance26,27. Furthermore, Dexter et al.23 reported that a single durum kernel can contain both opaque and vitreous sections. Glenn and Johnston28 physically measured the vitreous and opaque kernels from a single cultivar and concluded that vitreous kernels were harder. Others have used proportion of vitreousness to classify wheat as hard or soft22. The expression of vitreousness is thus highly in¯uenced by the environmental conditions but in any one environment it is possible to distinguish between the genotypes differing in vitreousness. The trait has been mapped to chromosome 5D and is tightly linked to the Ha locus22. Thus it seems that the environmental expression of the extent of vitreousness is in¯uenced by genetic factors.

OTHER FACTORS AFFECTING THE GRAIN HARDNESS Seed moisture content There is a general agreement that moisture content affects the absolute and relative values of a hardness classi®cation but there is disagreement as to whether it makes a wheat softer or harder21,29. In a comprehensive study comparing the effect of moisture on ®ve different methods of measuring hardness, Obuchowski and Bushuk30 showed that different techniques and different cultivars varied in their sensitivity to increasing moisture content. Many of the dif®culties associated with measuring hardness can be avoided if samples are equilibrated to similar moisture content before the hardness measurements are taken and only the results from apparatus that use similar mechanisms to measure hardness are compared.

330

K.-M. Turnbull and S. Rahman

Pentosans and other seed water soluble material Pentosans are essentially polymers of xylose and arabinose with a b-1,4 linked xylan providing the backbone and arabinose often occurring as 1, 2 or 1, 3 linked substituents; however, there is variation in the nature and number of substituents and this may be the basis of the division into soluble and insoluble pentosan fractions31. Pentosans are thought to play an important role in the water balance of dough in relation to its rheological properties as they can absorb 6±10 times their weight of water on a dry basis32. Approximately 2±3% of wheat ¯our is pentosan with the water-soluble fraction representing around one-third of the total pentosans32. In general, hardness in wheat is positively correlated with higher pentosan content, both soluble and insoluble, and grain hardness tends to increase as water-soluble and total pentosan contents increase33. However, a study by Bettge and Morris34 found that the amount of pentosan appeared to modify the hardness within the soft category much more than within the hard and suggested that this could be one source of variation in grain hardness that was not controlled by the Ha locus. Hong et al.33 suggest that pentosan quality, rather than quantity, may be important in determining different levels of hardness. Water-soluble pentosan levels are more strongly affected by the environment33 than hardness, which is largely cultivar-dependent (see below). Other differences in the water-soluble material from the grain have also been reported. Simmonds et al.19 noted that there were some differences in the total amount of soluble material extracted in a neutral pyrophosphate buffer. The material was identi®ed as a 2:1 mixture of carbohydrate (glucose and maltose) and protein (gliadins and to a lesser extent albumins) but more detailed examination of the data19 revealed that the amount of soluble material did not differ between some of the soft and hard cultivars. Seed lipid content Lipids occur in the various endosperm membranes, the aleurone layer and in starch granules of wheat and in similar structures in other members of the Triticeae such as barley, rye, and triticale35. They are located inside the starch granule (true starch lipids) or on the surface of the starch granules (starch surface lipids). Lipids other than those associated with starch are known as non-starch lipids35. Lipids

play a varied and often important role in many of the processes involved in milling, dough mixing, bread making, and staling. The relationship between endosperm texture and the amount and type of lipid is not clear but there have been several reports showing a correlation between these traits. Panozzo et al.36 found that hard grained Australian wheat cultivars have higher levels of hexane-extractable free lipids (FL) than soft cultivars, while Morrison et al.37, using British wheat cultivars, demonstrated a strong correlation between the increasing hardness and decreasing amounts of free polar lipids (Fpol). At least two genes control Fpol levels in wheat and have been mapped to the long (Fpl-1 ) and short (Fpl-2) arms of chromosome 5D. It has been suggested that Fpl-2 may be identical to the Ha locus and therefore may provide some explanation as to the control of Fpol level in wheat37. It is possible that Fpol levels may be in¯uenced by the puroindolines (the genes for puroindolines are tightly linked to the Ha locus; see later). The linkage between Fpl-2 and Ha, however, has not been examined further.

THE GENETICS OF WHEAT HARDNESS (Ha) LOCUS Genotype vs. environment It is generally accepted that hardness is a highly heritable trait38 that can be affected by the environmental conditions. Environmental conditions can have intermediate to extreme effects on hardness depending on the conditions to which a cultivar is subjected to during growth13,39,40 and the stage of growth under which the adverse conditions are experienced25. In one of the largest studies of this type, the same 15 wheat cultivars were grown in 11 locations in the USA, Europe and Asia39 and the hardness was measured using four different methods: time to grind, resistance to grinding, particle size index, and NIR of ground wheat. Cultivar had much larger effect than growing location on wheat hardness as measured by all four methods. Given the high heritability and, therefore, the strong cultivar effect on hardness it is possible to predict the relative cultivar performance for kernel hardness from a single location or a composite sample from multiple locations41. While the environment can strongly affect the hardness of a particular cultivar it does not seem to affect the relative ranking of cultivars41, with the environment affecting all

Endosperm texture in wheat

cultivars in a similar manner. This effect can be positive or negative depending on the environmental conditions. Until the 1960s the genetics of grain hardness were poorly understood42±45. It is now generally accepted that the grain hardness is controlled by one major gene and that softness is the dominant character37. Symes38 also noted the existence of at least two additional or modifying genes. These genes were thought to account for the different levels of hardness or softness observed within either the hard or soft class with the major gene being responsible for a large effect. The major gene controlling endosperm texture was shown to be located on the chromosome 5D using chromosome substitution lines. This locus is called the Ha (hardness) locus46,47. Using a population from a cross between Synthetic and Opata wheat varieties, Sourdille et al.48 showed that the Ha locus is located at the distal end of 5DS. Other less important loci modifying endosperm texture have been located on the homologous chromosomes 2, 5, 6 and 748. A more recent analysis of the four different mapping populations has revealed that there may also be other regions of the wheat genome that are important in controlling endosperm texture in wheat2. In this study, only two of the four populations showed linkage between the hardness trait (as measured by the SKCS) and the chromosome 5DS locus as de®ned by the DNA markers for the Ha locus region. Other genetic regions showing a signi®cant association with hardness were located on chromosome 4D and 4B2. The markers for hardness locus used in this study were for the puroindoline-a gene and the microsatellite marker wmc233. In crosses between the hard red spring wheats Giroux et al.49, found similarly that factors other than puroindoline-a or puroindoline-b were responsible for the variation in grain hardness in this cross. From these studies it is evident that some effects on grain hardness are not associated with the classical 5DS locus.

THE BIOCHEMISTRY OF WHEAT ENDOSPERM TEXTURE While the genetics of hardness are reasonably well understood much speculation remains about the exact biochemical and molecular basis of hardness. Most research has focused on the isolation and

331

characterisation of a 15 kDa starch granuleassociated mixture of proteins called friabilin. Friabilin, puroindolines and GSP-1 Friabilin is the name given to a 15 kDa polypeptide band observed by SDS-PAGE of protein fractions extracted from the water-washed starch granules with SDS. It was identi®ed as a putative gene product of the Ha locus because it is present in such fractions from all soft wheat cultivars but is absent, or weakly expressed, in fractions from hard and durum wheat cultivars50 and it was the ®rst biochemical marker for grain texture. The 15 kDa band was given the name `friabilin' because of its abundance on the soft `friable' wheats and it has also been called the Grain Softness Protein or GSP50,51. The initial hypothesis was that friabilin acts as a `non-stick' protein that affects the interface between the starch granule and the protein matrix. Thus, the two components are more easily separated in soft wheat than hard wheat52 and this leads to greater softness. The Ha locus and the genes controlling friabilin accumulation are tightly linked as con®rmed in an examination of 100 lines from a cross between Chinese Spring (CS) and a CS substitution line containing chromosome 5D from the hard cultivar `Hope'53. Friabilin has been further separated into different components on the basis of chromatographic mobility54. Jolly et al.51 raised antibodies to the 15 kDa polypeptide puri®ed from the starch granules of a soft wheat and showed that most of the friabilin was associated with the gluten fraction when ¯our was separated into gluten and starch by water washing. Comparisons of the total friabilin content showed that there was no clear distinction between the soft and hard wheats as was observed when only the starch granule-associated friabilin was considered51. Recently Darlington et al.55 have also shown that friabilin is mostly associated with matrix proteins in hard wheats. N-terminal sequencing revealed that the two main components that comprise friabilin are identical to polypeptides, which had been isolated from wheat endosperm using Triton X100 and named puroindoline-a and -b50,55. A third minor component has been identi®ed as GSP-156. Other components have also been identi®ed57. Puroindolines are basic cysteine-rich polypeptides with a mean molecular weight of 12
8 kDa and contain a unique amphiphilic tryptophan-rich domain58. The name puroindoline derives from

332

K.-M. Turnbull and S. Rahman

`puros' the Greek word for wheat and `indoline' describing the indole ring of tryptophan59. It was thought initially that puroindolines had a role in the plant defence mechanisms due to the similarity in structure and extraction properties to wheat thionins, which are known to be toxic to various microorganisms and animal cells58. Puroindolines and purothionins have been shown to bind to starch granules speci®cally in vitro; but no difference in binding to starch granules from soft and hard cultivars was observed60. The genes encoding puroindoline-a and -b share 55% nucleotide similarity. The molecular mass of mature puroindoline-b (13
6 or 12
9 kDa depending on C-terminal processing) is slightly higher than that of puroindoline-a (13
4 or 12
8 kDa ) and both are less than the 16 kDa estimated from SDS-PAGE59. The tryptophan-rich domains differ between the two polypeptides. In puroindoline-a this domain consists of ®ve tryptophan residues and three basic residues (WRWWKWWK) whereas in puroindoline-b there are three tryptophan residues and two basic residues (WPTKWWK). Both puroindoline-a and -b are believed to be synthesised as preproteins59. They contain a cysteine skeleton that is also found in lipid transfer proteins (LTP) and a-amalyse inhibitors and the similarities between the primary and secondary structure of puroindolines and LTPs have been noted59,61. Both puroindoline-a and -b show a high level of homology to a protein initially described as an oat seed avenin62, particularly in the presence of an identical cleavable N-terminal peptide and the presence of a tryptophan-rich domain59,63. Puroindoline-a and -b are expressed speci®cally in developing seeds of T. aestivum from early in development but not in tetraploid T. durum59. This is consistent with the genes for puroindolines being located on the D-genome of the modern bread wheat. Although the actual donors of the A and B genomes in tetraploid and hexaploid wheat cannot be identi®ed with certainty, all of the likely candidates contain the puroindoline genes63,64. Puroindoline homologues are also found in barley55,64, oat and rye65 but not in maize, rice, or sorghum66. The reason for the apparent lack of puroindoline genes on the A and B genomes of wheat is not clear. The third and minor component of friabilin is GSP-1 and it accounts for one of the peptides obtained from a friabilin digest by Jolly et al.51. The deduced amino acid sequence of GSP-1 has a high amino acid sequence identity to the oat seed avenin protein (50%) and also to the puroindolines

(45% and 49% to puroindoline-a and -b, respectively)56,58; however, there are only two tryptophan residues in the region of the polypeptide (WIFPRTW) corresponding to the tryptophan-rich domain in the puroindolines. GSP-1 also shares sequence homology with a 30 kDa arabinoxylanase isolated from wheat67. The signi®cance of this homology has yet to be determined.

Location of puroindolines within the endosperm Immunolocation studies using monoclonal antibodies raised against puroindoline-a and -b have shown that puroindoline-a is located primarily in the starchy endosperm. Puroindoline-b in comparison, is located in the aleurone and possibly also in the starchy endosperm68. In the aleurone, puroindoline-b is located in the small inclusion bodies and in the endosperm puroindoline-a is located at the interface between the starch granule and the protein matrix. Interestingly, no immunolabelling of puroindoline-a occurred when starch granules were not surrounded by proteins68. These results are consistent with puroindoline-a, at least, being associated with starch granules. In transgenic rice plants the puroindoline-b promoter was found to drive GUS expression in the aleurone, starchy endosperm, scutellum and the pericarp69. No expression was found in the stems, roots, leaves, or pollen69. These results corroborate those of Dubriel et al.68.

Puroindoline and GSP-1 genes are tightly linked to the Ha locus. Puroindoline-a has been mapped to the end of the short arm of 5D and is tightly linked to the Ha locus in two populations48,70. Puroindoline-b has been shown to co-segregate with puroindoline-a and is also tightly linked to the Ha locus48,70. To date, puroindoline-a remains the closest marker to the Ha locus and can account for up to 70% of the variability in hardness between the two parent lines48. As mentioned above, other chromosome regions are also important in determining the hardness particularly in crosses between the hard wheat varieties2,49. The expression of the puroindoline-a gene is affected more by growing conditions than the puroindoline-b gene but the expression of both of these genes is highly heritable71. The genes for GSP-1 ( gsp-1) have

Endosperm texture in wheat

also been shown to be tightly linked to the Ha locus in two populations72,73.

Puroindolines bind lipids While the biological function of puroindolines is still a matter for further research, the mode of action of these proteins may be through their lipid binding properties. This may explain the association of a locus controlling the level of free polar lipids in the grain (Fpl-2) with the Ha locus36. The evidence suggesting a lipid-binding role for puroindolines relies on the homology between the primary structure of puroindolines and wheat LTPs. With the exception of the tryptophan rich domain four of the ®ve disulphide bridges of puroindolines are in identical positions to that found in LTPs74. It has been proposed that puroindolines interact with lipids via the tryptophan-rich domain where the domain forms a membrane-anchoring loop between a-helices74. The interaction of puroindolines with lipids in solution suggests that lipid-binding occurs at the air±water interface74±76. Puroindolines are unique in that they are capable of forming very stable foams, which show a high level of resistance to destabilisation by polar and neutral lipids77. The lipid binding capacity of puroindolines is important in many aspects of the cereal processing. In bread, puroindolines are thought to prevent destabilisation of foams by oil globules. Adding small amounts of puroindoline to a dough results therefore in a loaf with a ®ne crumb structure77. Puroindolines also affect dough extensibility and tenacity68 and, thereby, affect the texture of baked products78. In beer making, puroindolines can restore foam destabilised by the neutral and polar lipids (reviewed in 78).

MOLECULAR BIOLOGY OF WHEAT ENDOSPERM TEXTURE Both puroindoline-a and -b genes have been sequenced from wheat cultivars that are grown throughout the world79±81. A mutation in the tryptophan rich domain of the puroindoline-b gene has been found to be highly correlated with the grain texture. This mutation was ®rst reported by Giroux and Morris70 who sequenced the puroindoline-b gene from two wheat lines differing in grain hardness and found a single base change that resulted in a mutation from

333

glycine to serine in the encoded polypeptide. No recombination was found between the puroindolineb glycine to serine mutation and hard grain texture in 83 homozygous 5D recombinant lines derived from the soft cultivar CS and the substitution line `CS' containing the 5D chromosome from the hard cultivar `Cheyenne'70. Further investigation indicated that not all hard cultivars (9 out of 13) contained the mutation although all cultivars with the mutation are hard81. However, the hard wheats that did not contain the mutation also did not contain puroindoline-a transcripts. The absence of puroindoline-a was also linked to grain hardness in a population of near-isogenic lines created from the soft cultivar Heron and the hard cultivar Falcon81. Other mutations in puroindoline-b have also been reported79. These involve a leucine to proline change and a tryptophan to arginine change. The leucine to proline mutation was common in germplasm from the Northern hemisphere while the tryptophan to arginine mutation was only found in the winter wheats from Sweden and the Netherlands79. Each of the three mutations are proposed to cause a `loss-of-function', resulting in a hard wheat79. Giroux and Morris suggested that either the loss of puroindoline-a or the alteration of the puroindoline-b sequence might alter the lipid binding capacity of the puroindoline complement and this could affect the way in which the membranes collapse during the desiccation leading to alterations in the grain hardness70. All Australian cultivars that contained the glycine to serine mutation or were lacking puroindoline-a transcripts were also found to be hard80. However, two of the cultivars surveyed that did not have the glycine to serine mutation were found to have `normal' amounts of puroindoline-a80. These results suggest that alterations in the sequence and amount of puroindolines of the type identi®ed by Giroux and Morris70,81 may not be necessary for the wheat endosperm to be hard, although the results are consistent with the hypothesis that such changes are suf®cient80. A further factor may therefore also be responsible for the hard phenotype. Accordingly, hard wheats can be divided into three groups: those with no puroindoline-a accumulation in the grain, those with mutations in puroindoline-b and those with unknown lesions. In contrast to the several mutations reported in the puroindoline-b gene, the sequence of the puroindoline-a gene appears to be highly conserved across cultivars (K. R. Gale pers. comm. and authors' pers. observ.).

334

K.-M. Turnbull and S. Rahman

Large insert bacterial arti®cial chromosome (BAC) type clones have been isolated that contains all three Ha locus marker genes (puroindoline-a, puroindoline-b, and gsp-1) from T. monococcum82 and T. tauschii83. This demonstrates that these genes are physically within 100 kb of each other in the genome and recent analysis of the promoter regions from puroindoline-a, puroindoline-b, and gsp-1 genes has revealed a high level of sequence identity (72%) between the upstream regions83. Puroindoline-a and -b can also act singly to moderate grain hardness as has been shown recently in transgenic rice seeds84. However, some synergistic enhancement of the rice grain softness is found when both puroindoline-a and -b are expressed84. The result is consistent with the hypothesis that puroindolines can act collectively to enhance grain softness. Interestingly, the addition of chromosome 5D to the durum wheat line Langdon (which lacks chromosome 5D) alters the grain texture signi®cantly more than the introduction of the puroindoline-a and -b genes alone to rice84. It is thus possible that other genes carried on the short arm of chromosome 5D in wheat are also important in regulating the grain hardness.

A COMPARISON WITH BARLEY In barley the term used to describe grain hardness is milling energy. A good correlation between the milling energy and malting quality has been reported for barley cultivars grown in Scotland and Germany86,87. Swanston et al.87 found a correlation between the appearance of the cell wall and milling energy. This was also noted by Brennan et al.88 who found, using British cultivars, that poor malting barleys retained their cell-wall structure longer than good malting barleys. This was not due to any difference in the wall-degradative enzymes. An examination of the mature barley endosperm by SEM showed that more protein seemed to be associated with the starch granules of poor quality barley, reminiscent of the situation with hard wheats88. Under the environmental conditions experienced in Britain, the ease with which the endosperm structure can be disrupted mechanically seems to be a good indicator of the ease with which the structure of the endosperm can be broken down by enzymes during malting89. Thus, in the British isles, soft textured barley cultivars are preferred to hard as they are more evenly modi®ed during malting. However, the correlation between malting quality and milling energy found for Scotland did not hold so well for

the same cultivars grown in Spain87 suggesting that different genes determined the milling energy or malting quality in the two environments. In barley, the genetic control of milling energy is complex with chromosome 5H playing a signi®cant role89,90 and RAPD markers for milling energy have been located to barley chromosome 5H91. Interestingly, in a Steptoe  Morex barley mapping population, a small QTL for malting quality has been located on barley chromosome 5H, tightly linked to the genes for hordoindolines, the barley orthologues of puroindoline91. It is possible that the controlling mechanism for hardness encoded at these loci for barley and wheat are similar.

CONCLUDING REMARKS Endosperm texture is an important yet complex characteristic of wheat and other cereals that is of importance to growers, millers, and bakers. There are reliable and direct measurements of grain texture. Grain hardness is a characteristic expressed early in grain development when very few storage proteins and starch granules are present in the cell. Any theories explaining grain texture should be considered in the light of these observations. Grain texture can be modi®ed by the environment, yet the environment rarely has suf®cient effect to cause a re-classi®cation of a wheat between classes. Many factors affect grain hardness measurements such as seed moisture content, seed water soluble materials, lipid content, and pentosans. The traits of seed vitreousness and grain hardness are highly correlated yet not all hard wheat necessarily contains vitreous seeds. Seed vitreousness and hardness therefore should be considered as separate traits that are most likely linked as seen by overlapping QTLs on chromosome 5DS. A major locus controlling grain hardness is the Ha locus, on the short arm of chromosome 5D of wheat. Genes for puroindoline-a, -b and GSP-1 are tightly linked to the Ha locus. While the ®rst transgenic plants have been produced that show decreased endosperm texture through the introduction of the puroindoline genes there are still likely to be other factors that are important in determining or at least moderating grain texture that are yet to be explored and exploited. Other research is likely to focus on the mode of action of the puroindolines as their mechanism for affecting grain hardness is still poorly understood. Their role may be related to their ability to bind lipids. The very hard durum wheats provide

Endosperm texture in wheat

a model system for future transformation studies as they are the hardest of the wheat classes and do not contain the D-genome that carries the genes for grain softness. Any modi®cation to the durum varieties with candidate genes should enhance grain softness and provide conclusive proof of the role of the puroindolines and other candidate genes from the Ha locus. Other sources of variation in grain hardness may become evident as different mutations in wheat become available.

REFERENCES 1. Moss, H.J., Edwards, C.S. and Goodchild, N.A. Small scale tests of soft wheat quality. Australian Journal of Experimental Agriculture and Animal Husbandry 13 (1973) 299. 2. Osborne, B., Turnbull, K.-M., Anderssen, R.S., Rahman, S., Sharp, P.J. and Appels, R. The hardness locus in Australian wheat lines. Australian Journal of Agricultural Research 52 (2001) 1275±1286. 3. Worzella, W.W. and Cutler, G.H. A critical study of techniques for measuring granulation in wheat meal. Journal of Agricultural Science 58 (1939) 329±341. 4. Williams, P.C. Screening wheat for protein and hardness by near Infrared relfectance spectroscopy. Cereal Chemistry 56 (1979) 169±172. 5. Delwiche, S.R. Measurement of single-kernel wheat hardness using Near-Infrared Transmittance. Transactions of the ASAE 36 (1993) 1431±1437. 6. Ellis, R.P., Camm, J.-P. and Morrison, W.R. A rapid test for malting quality in barley. HGCA Report No. 63 (1992). 7. Mares, D.J. and Stone, B.A. Studies on wheat endosperm I. Chemical composition and ultrastructure of cell walls. Australian Journal of Biological Science 26 (1973) 793±812. 8. Turnbull, K.-M., Marion, D., Gaborit, T., Appels, R. and Rahman, S. Early expression of grain hardness in the developing wheat endosperm. Planta (2002) in press. 9. Barlow, K.K., Butrose, M.S., Simmonds, D.H. and Vesk, M. The nature of the starch±protein interface in wheat endosperm. Cereal Chemistry 50 (1973) 443±454. 10. Glenn, G.M., Pitts, M.J., Liao, K. and Irving, D.W. Block surface staining for differentiation of starch and cell walls in wheat endosperm. Biotechnology and Histochemistry 67 (1992) 88±97. 11. Pitts, M.J., Liao, K. and Glenn, G. Classifying wheat kernel milling performance via starch granule size. American Society of Agricultural Engineers. Paper number 893566 (1989). 12. Bechtel, D.B., Zyasas, I., Dempster, R. and Wilson, J.D. Size-distribution of starch granules isolated from hard red winter and soft red winter wheats. Cereal Chemistry 70 (1993) 238±240. 13. Stenvert, N.L. and Kingswood, K. The in¯uence of the physical structure of the protein matrix on wheat hardness. Journal of the Science of Food and Agriculture 28 (1977) 11±19. 14. Bechtel, D.B. and Wilson, J.D. Ultrastructure of developing hard and soft red winter wheats after air- and freezedrying and its relationship to endosperm texture. Cereal Chemistry 74 (1997) 235±241.

335

15. Glenn, G.M. and Saunders, R.M. Physical and structural properties of wheat endosperm associated with grain texture. Cereal Chemistry 67 (1990) 176±182. 16. Salisbury, F.B. and Ross, C.W. In `Plant Physiology'. Second edn. Wadsworth Publishing Company, Inc. 208, Belmant California (1978). 17. Bietz, J.A. Genetic and biochemical studies of nonenzymatic endosperm proteins. In `Wheat and Wheat Improvement', (E.G. Heyne, ed.), American Society of Agronomy Inc. Crop Science Society of America Inc. Soil Science Society of America Inc., Madison, Wisconsin (1987) pp 215±241. 18. Bechtel, D.B., Wilson, J.D. and Martin, C.R. Determining endosperm texture of developing hard and soft red winter wheats dried by different methods using the single-kernel wheat characterisation system. Cereal Chemistry 73 (1996) 567±570. 19. Simmonds, D.H., Barlow, K.K. and Wrigley, C.W. The biochemical basis of grain hardness in wheat. Cereal Chemistry 50 (1973) 553±562. 20. Stenvert, N.L. and Kingswood, K. The in¯uence of the physical structure of the protein matrix on wheat hardness. Journal of Science, Food and Agriculture 28 (1977) 11±19. 21. Symes, K.J. Classi®cation of Australian wheat varieties based on the granularity of their wholemeal. Australian Journal of Experimental Agriculture and Animal Husbandry 1 (1961) 18±23. 22. Nelson, J.C., Sorrells, M.E., Van Deynze, A.E., Lu, Y.H., Atkinson, M., Bernard, M., Leroy, P., Faris, J.D. and Anderson, J.A. Molecular mapping of wheat: major genes and rearrangements in homeologous groups 4, 5 and 7. Genetics 141 (1995) 721±731. 23. Dexter, J.E., Marchylo, B.A., MacGregor, A.W. and Tkachuk, R. The structure and protein composition of vitreous piebald and starchy durum wheat kernels. Journal of Cereal Science 10 (1989) 19±32. 24. Hosney Principles of cereal science and technology, American Association of Cereal Chemistry, St Paul Minnasota (1986). 25. Parish, J.A. and Halse, N.J. Effects of light, temperature and the rate of desiccation on translucency in wheat grain. Australian Journal of Agricultural Research 19 (1968) 365±372. 26. Hadjichristodoulou, A. Genetic and environmental effects on vitreousness of durum wheat. Euphytica 28 (1979) 711±716. 27. Pomeranz, Y. and Williams, P.C. Wheat hardness: Its genetic, structural and biochemical background, measurement and signi®cance. In `Advances in Cereal Science and Technology,' volume 10, (Y. Pomeranz, ed.) American Association of Cereal Chemistry, St Paul, MN (1990) pp. 471±557. 28. Glenn, G.M. and Johnston, R.K. Water Vapor diffusivity in vitreous and mealy wheat endosperm. Journal of Cereal Science 20 (1994) 275±282. 29. Grosh, G.M. and Milner, M. Water penetration and internal cracking in tempered wheat grains. Cereal Chemistry 36 (1959) 260. 30. Obuchowski, W. and Bushuk, W. Wheat hardness: Comparison of methods of its evaluation. Cereal Chemistry 57 (1980) 421±425. 31. D'Appolonia, B.L., Gilles, K.A., Osman, E.M. and Pomeranz, Y. In `Wheat: Chemistry and Technology', (Y. Pomeranz, ed.), American Association of Cereal Chemists (1978) pp 301±392.

336

K.-M. Turnbull and S. Rahman

32. Jelaca, S.L. and Hylnka, I. Water-binding capacity of wheat ¯our crude pentosans and their relation to mixing characteristics of dough. Cereal Chemistry 48 (1971) 211±222. 33. Hong, B.H., Rubenthaler, G.L. and Allan, R.E. Wheat pentosans I cultivar variation and relationship to kernel hardness. Cereal Chemistry 66 (1989) 369±373. 34. Bettge, A.D. and Morris, C.F. Relationship among grain hardness, pentosan fractions and end-use quality of wheat. Cereal Chemistry 77 (2000) 241±247. 35. Morrison, W.M. Lipids in Cereal Starches. Journal of Cereal Science 8 (1988) 1±15. 36. Panozzo, J.F., Hannah, M.C., O'Brien, L. and Bekes, F. The relationship of free lipids and ¯our protein to breadmaking quality. Journal of Cereal Science 17 (1993) 47±62. 37. Morrison, W.R., Law, C.N., Wylie, L.J., Coventry, A.M. and Seekings, J. The effect of group 5 chromosomes on the free polar lipids and breadmaking quality of wheat. Journal of Cereal Science 9 (1989) 41±51. 38. Symes, K.J. The inheritance of grain hardness in wheat as measured by the particle size index. Australian Journal of Agricultural Research 16 (1965) 113±123. 39. Pomeranz, Y., Peterson, C.J. and Mattern, P.J. Hardness of winter wheats grown under widely different climatic conditions. Cereal Chemistry 62 (1985) 463±467. 40. Bushuk, W. Wheat Breeding for end-product use. Euphytica 100 (1998) 137±145. 41. Hazen, S.P. and Ward, R.W. Variation in soft winter wheat characteristics measured by the single kernel characterisation system. Crop Science 37 (1997) 1079±1086. 42. Beard, B.H. and Poehlman, J.M. A study of quality, as measured by the pearling test, in crosses between hard and soft wheats. Agronomy Journal 46 (1954) 220±223. 43. Millington, A.J. and Remilton, E. The strength of Australian wheat varieties. Journal of Australian Institute of Agricultural Science 20 (1954) 24±35. 44. Worzella, W.W. Inheritance and interrelationship of components of quality, cold resistance and morphological characters in wheat hybrids. Journal of Agricultural Research 65 (1942) 501±522. 45. Thompson, J.B. and Whitehouse, R.N.H. Studies on the breeding of self pollinating cereals.4. Environment and inheritance of quality in spring wheats. Euphytica 11 (1962) 181±196. 46. Mattern, P.J., Morris, R., Schmidt, J.W. and Johnson, V.A. Location of genes for kernel properties in the wheat variety `Cheyenne' using chromosome substitution lines. In `Proceedings of the 4th International Wheat Genetics Symposium, Colombia, MO (1973). 47. Law, C.N., Young, C.F., Brown, J.W.S., Snape, J.W. and Worland, A.J. The study of grain-protein control in wheat using whole chromosome substitution lines. In `Seed Protein Improvement by Nuclear Techniques'. International Atomic Energy agency, Vienna (1978). 48. Sourdille, P., Perretant, M.R., Charmet, G., Leroy, P., Gautier, M.F., Jourdrier, P., Nelson, J.C., Sorrels, M.E. and Bernard, M. Linkage between RFLP markers and genes affecting kernel hardness in wheat. Theoretical and Applied Genetics 93 (1996) 580±586. 49. Giroux, M.J., Talbert, L., Habernicht, D.K., Lanning, S., Hempill, A. and Martin, J.M. Association of puroindoline sequence type and grain hardness in hard red spring wheat. Crop Science 40 (2000) 370±374.

50. Greenwell, P. and Scho®eld, J.D. A starch granule protein associated with endosperm softness in wheat. Cereal Chemistry 63 (1986) 379±380. 51. Jolly, C.J., Rahman, S., Kortt, A.A. and Higgins, T.J.V. Characterisation of the wheat Mr 15 000 `grain softness protein' and analysis of the relationship between its accumulation in the while seed and grain softness. Theoretical and Applied Genetics 86 (1993) 590±597. 52. Greenwell, P. Biochemical studies of endosperm texture in wheat. Chorleywood Digest 118 (1992) 74±76. 53. Greenwell, P. and Scho®eld, J.D. The chemical basis of grain hardness and softness. In `Proceedings of the ICCC Symposia', University of Helsinki and the Department of Food Chemistry and Technology, Helsinki (1989). 54. Day, L., Greenwell, P., Lock, S. and Brown, H. Analysis of wheat ¯our proteins related to grain hardness using capillary electrophoresis. Journal of Chromatography A 836 (1999) 147±152. 55. Darlington, H.F., Tesci, L., Harris, N., Griggs, D., Cantrell, I. and Shewry, P.R. Starch granule associated proteins in barley and wheat. Journal of Cereal Science 32 (2000) 21±29. 56. Rahman, S., Jolly, C.J., Skerritt, J.H. and Wallosheck, A. Cloning of a wheat 15-KDa grain softness protein (GSP). GSP is a mixture of puroindoline-like polypeptides. European Journal of Biochemistry 223 (1994) 917±925. 57. Oda, S. and Scho®eld, J.D. Characterisation of friabilin polypeptides. Journal of Cereal Science 26 (1997) 29±36. 58. Blochet, J.-E., Chevalier, C., Forest, E., Pebay-Peyroula, E., Gautier, M.-F., Joudrier, P., Pezolot, M. and Marion, D. Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophanrich domain, isolated from wheat endosperm by Triton X-114 phase partitioning. FEBS Letters 329 (1993) 336±340. 59. Gautier, M.-F., Aleman, A.-E., Guirao, A., Marion, D. and Joudrier, P. Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence and developmental gene expression. Plant Molecular Biology 25 (1994) 43±57. 60. Bloch, H.A., Darlington, H.F. and Shewry, P.R. In vitro binding of puroindolines to wheat starch granules. Cereal Chemistry 78 (2001) 74±78. 61. Le Bihan, T., Blochet, J.E., Desmoreaus, A., Marion, D. and Pezelot, M. Determination of the secondary structure and conformation of puroindolines by infrared and Raman spectroscopy. Biochemistry 39 (1996) 12712±12722. 62. Fabijanski, S., Chang, S.-C., Dukiandjiev, S., Dukiandjiev, M.B., Bahramian, P., Ferrara, P. and Altosaar, I. The nucleotide sequence of a cDNA for a major prolamin (avenin) in oat (Avena sativa L. cultivar Hinoat) which reveals homology with oat globulin. Biochemistry and Physiology P¯anzen 183 (1998) 143±152. 63. Tanchak, M.A., Schernthaner, J.P., Giband, M. and Altosaar, I. Tryptophanins: Isolation and molecular characterisation of oat cDNA clones encoding proteins structurally related to puroindoline and wheat grain softness proteins. Plant Science 137 (1988) 173±184. 64. Jagtap, S.S., Beardsley, A., Forrest, J.M.S. and Ellis, R.P. Protein composition and grain quality in barley. Aspects of Applied Biology 36 (1993) 51±60. 65. Morrison, W.R., Greenwell, P., Law, C.N. and Sulaiman, B.D. Occurrence of friabilin, a low molecular weight protein associated with grain softness on starch granules

Endosperm texture in wheat

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

isolated from some wheat and related species. Journal of Cereal Science 15 (1992) 143±149. Gautier, M.F., Cosson, P., Guiaro, A., Alary, R. and Joudrier, P. Puroindoline genes are highly conserved in diploid ancestor wheats and related species but absent in tetraploid Triticum species. Plant Science 153 (2000) 81±91. Cleemput, G., Van Laere, K., Hessing, M., Van Leuven, F., Torrekens, S. and Delcour, J.A. Identi®cation and characterisation of a novel Arabinoxylanase from wheat ¯our. Plant Physiology 115 (1997) 1619±1627. Dubriel, L., Gaborit, T., Bouchet, B., Gallant, D., Broekaert, W.F., Quillien, L. and Marion, D. Spatial and temporal distribution of puroindolines and non speci®c lipid transfer protein in Triticum aestivum seeds. Relationships with their in vitro antifungal properties. Plant Science 138 (1998) 121±135. Digeon, J.F., Guiderdoni, E., Alary, R., Michaux-Ferriere, N., Jourdrier, P. and Gautier, M.F. Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-speci®c expression in transgenic rice seeds. Plant Molecular Biology 39 (1999) 1101±1112. Giroux, M.J. and Morris, C.F. A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch-surface friabilin. Theoretical and Applied Genetics 95 (1997) 857±864. Igrejas, G., Gaborit, T., Oury, F.-X., Chiron, H., Marion, D. and Branlard, G. Genetic and environmental effects on the contents of puroindoline-a and puroindoline-b, lipid binding proteins from bread wheats. Relationship with hardness and breadmaking quality. Cereal Chemistry 34 (2000) 37±47. Jolly, C.J., Glenn, G.M. and Rahman, S. GSP-1 genes are linked to the grain hardness locus (Ha) on wheat chromosome 5D. Proceedings of the National Academy of Sciences USA 93 (1996) 2408±2413. Turner, M., Mukai, Y., Leroy, P., Charef, B., Appels, R. and Rahman, S. The Ha locus of wheat: identi®cation of a polymorphic region for tracing grain hardness in crosses. Genome 42 (1999) 1±9. Marion, D., Gautier, M.-F., Joudrier, P., Ptak, M., Pezolet, M., Forest, E., Clark, D.C. and Broekaert, W. Structure and function of wheat lipid binding proteins. In `Wheat Kernel Proteins, Molecular and Functional Aspects', Universita delgi studi della Tuscia, Viterbo, Italy (1994). Wilde, P.J., Clark, D.C. and Marion, D. The in¯uence of competitive absorption of lysopalmitoyl phosphatidylcholine on the functional properties of puroindoline, a lipid binding protein isolated from wheat ¯our. Journal of Agriculture and Food Chemistry 41 (1993) 1570±1576. Kooijman, M., Orsel, R., Hessing, M., Hamer, R.J. and Bekkers, A.C.C.P.A. Spectroscopic characterisation of the lipid-binding properties of wheat puroindolines. Journal of Cereal Science 26 (1997) 145±159. Dubriel, L., Compoint, J.P. and Marion, D. The interaction of puroindolines with wheat polar lipids determines their foaming properties. Journal of Agriculture and Food Chemistry 45 (1997) 108±116.

337

78. Douliez, J.-P., Michon, T., Elmorjani, K. and Marion, D. Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels. Journal of Cereal Science 32 (2000) 1±20. 79. Lillemo, M. and Morris, C.F. A leucine to proline mutation in puroindoline-b is frequently present in hard wheats from Northern Europe. Theoretical and Applied Genetics 100 (2000) 1100±1107. 80. Turnbull, K.-M., Gaborit, T., Marion, D. and Rahman, S. Variation in puroindoline polypeptides in Australian wheat cultivars in relation to grain hardness. Australian Journal of Plant Physiology 2 (2000) 153±158. 81. Giroux, M.J. and Morris, C.F. Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b. Proceedings of the National Academy of Science USA 95 (1998) 6262±6266. 82. Tranquilli, G., Lijavetzky, D., Muzzi, G. and Dubcovsky, J. Genetic and physical characterisation of grain texturerelated loci in diploid wheat. Molecular and General Genetics 262 (1999) 846±850. 83. Turnbull, K.-M. Genomic and Environmental Analyses of Grain Hardness in Wheat. Ph. D. Thesis. University of Sydney (2000). 84. Krishnamurthy, K. and Giroux, M.J. Expression of wheat puroindoline genes in transgenic rice enhances grain softness. Nature Biotechnology 19 (2001) 162±166. 85. Chandrashekhar, A. and Mazhar, M. The biochemical basis and implications of grain strength in sorghum and maize. Journal of Cereal Science 30 (1999) 193±207. 86. Alison, J.M., Borzucki, R., Cowe, I.A. and McHale, R. Variation in a barley collection for endosperm attributes that relate to malting quality. Journal of the Institute of Brewing 85 (1979) 89±88. 87. Swanston, J.S., Ellis, R.P., Rubio, A., Perez-Vendrell, A. and Molina-Cano, J.L. Differences in malting performance between barleys grown in Spain and Scotland. Journal of the Institute of Brewing 101 (1995) 261±265. 88. Brennan, C.S., Amor, M.A., Harris, N., Smith, D., Cantrell, I., Griggs, D. and Shewry, P.R. Cultivar differences in modi®cation patterns of protein and carbohydrate reserves during malting of barley. Journal of Cereal Science 26 (1997) 83±93. 89. Chalmers, K.J., Barua, U.M., Hackett, C.A., Thomas, W.T.B., Waugh, R. and Powell, W. Identi®cation of RAPD markers linked to genetic factors controlling the milling energy requirements of barley. Theoretical and Applied Genetics 87 (1993) 314±320. 90. Rouves, S., Boeuf, C., Zwickert-Menteur, S., Gautier, M.-F., Jourdrier, P., Bernard, M. and Jestin, L. Locating supplementary RFLP markers on barley chromosome 7 and synteny with homeologous wheat group 5. Plant Breeding 115 (1996) 511±513. 91. Beecher, B., Smidansky, E.D., See, D., Balke, T.K. and Giroux, M.J. Mapping and sequence analysis of barley hordoindolines. Theoretical and Applied Genetics 102 (2001) 833±840.