Published in Crop Sci. 44:1608-1614 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Differentiation of ß-Amylase Phenotypes in Cultivated Barley
Wensheng Zhanga,
Takafumi Kanekob,
Makoto Ishiic and
Kazuyoshi Takedac,*
a Shijiazhuang Institute of Agricultural Modernization, Chinese Academy of Sciences, 286 Huaizhong Road, Shijia-zhuang, Hebei 050021, China
b Plant Bioeng. Res. Lab. Sapporo Brew. Ltd., 37-1, Kizaki, Nitta, Gunma 370-0393, Japan
c Research Institute for Bioresources, Okayama Univ., Chuo 2-20-1, Kurashiki, Okayama 710-0046, Japan
* Corresponding author (takeda{at}rib.okayama-u.ac.jp).
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ABSTRACT
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ß-Amylase types coded by the Bmy1 locus were investigated by analyzing the thermostability and isoelectric focusing (IEF) patterns in 8270 accessions of cultivated barley (Hordeum vulgare L.) from different regions of the world. The ß-amylase types were classified into three main thermostability types (A, high; B, medium; C, low) and three major IEF patterns (I, Ia, and II) together with several rare mutant types. By combined analysis of the thermostability types and IEF patterns, we found 14 ß-amylase phenotypes. Among them, A-II, B-I, B-Ia, B-II, and C-II were confirmed to be the major types comprising more than 99% of cultivated barley, and accessions belonging to types A-Ia, A-IIa, C-I, C-Ia, and C-IV were reported here for the first time. We also confirmed a clear geographical differentiation of ß-amylase phenotypes: In East Asia, nearly 90% of the accessions were of the A-II type, while 97% of the Ethiopian accessions were of the C-II type. The B-I type accessions were mostly restricted to the former USSR and Europe. In Nepal, Bhutan, India, and Pakistan, 90% of the B type accessions were of the B-Ia type. The B-II type barley seems to have originated in Turkey and then mainly spread to North Africa, Europe, and the former USSR.
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INTRODUCTION
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BARLEY is the fourth largest of the cereal crops after wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.); it is also the major material for beer production. In the grain of malting barley, ß-amylase (
-1,4-glucan maltohydrolase; EC 3.2.1.2) is one of the most important hydrolytic enzymes. During malting, not only ß-amylase activity but also its thermostability plays an important role. The genetic variation in ß-amylase thermostability has been studied by different research groups, and a close correlation has been found between thermostability and the apparent attenuation limit (AAL), which is a major malting quality parameter (Eglinton et al., 1998; Kihara et al., 1998, 1999). Since it would be an effective method to enhance the fermentability of malting barley by increasing the thermostability of ß-amylase, more attention has been devoted to the analysis of this parameter.
ß-Amylase in barley seed is controlled by Bmy1 locus, which is located on the long arm of chromosome 4H (Nielsen et al., 1983; Kreis et al., 1988; Netsvetaev, 1992). By thermostability analysis, the ß-amylase could be classified into A, B, and C types which possess high, medium and low thermostability, respectively (Kihara et al., 1998, 1999). On the basis of the analyses of gene expression (Kaneko et al., 2000) and QTL mapping (Kaneko et al., 2001b), it was concluded that Bmy1 locus controls the protein structure and thermostability of ß-amylase.
Kaneko et al. (2002) investigated 1800 accessions of A, B, and C type barley by IEF analysis and reported that ß-amylase could be classified into two (I and II) main patterns with two mutants. Combined findings of ß-amylase thermostability type and IEF pattern suggested that four kinds of allelic ß-amylase forms A-II, B-I, B-II, and C-II were observed. Thermostability Types A and C were found only in IEF Pattern II accessions. The gDNA sequences of ß-amylase in ‘Haruna Nijo’ (A-II type), ‘Harrington’ (B-I type), and ‘Schooner’ (C-II type) have been analyzed to find amino acid substitutions (Yoshigi et al., 1995; Kaneko et al., 2000). The effects of the amino acid substitutions among three different allelic ß-amylase forms have been evaluated by site-directed mutagenesis (Ma et al., 2001). However, the relationship between thermostability type and IEF pattern is still unclear; analysis of limited accessions could not exclude the possibility of A-I and C-I type ß-amylase.
Because thermostability and IEF analysis are rapid and relatively inexpensive, they are suitable for evaluation of large germplasm collections. These analyses would provide valuable information regarding phylogenetic relationships for breeding. Previously, we reported the varietal variation and geographical distribution of ß-amylase thermostability (Kaneko et al., 2001a). In this paper, we present the summary data of 8270 barley accessions for the thermostability in combination with IEF pattern of ß-amylase.
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MATERIALS AND METHODS
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Plant Materials
We used 8270 accessions of cultivated barley collected from different countries and regions of the world (Table 1). These accessions are preserved in the Barley Germplasm Center, Okayama University (http://www.rib.okayama-u.ac.jp/barley/; verified 6 April 2004). These accessions were grown in the field of Research Institute for Bioresources, Okayama University, Kurashiki, Japan. Harvested seeds were used for ß-amylase analysis. Several morphological and physiological characteristics including kernel row type, caryopsis type, rachilla hair type, brittleness of rachis, and growth habit have been investigated, and a database of barley germplasm has been established.
ß-Amylase Extraction
Crude ß-amylase was extracted according to the method reported by Kihara et al. (1998). The powder of a crushed barley seed was incubated in 300 µL acetate buffer (50 mM, pH 5.5) containing 10 mM dithiothreitol (DTT) at 4°C for 10 to 15 h with agitation. The extract was centrifuged at 20 000 x g at 4°C for 10 min., and the supernatant was used as the crude enzyme solution both for the analysis of thermostability and IEF.
Thermostability Analysis
One microliter of the extract of each sample was diluted 100-fold with 50 mM MOPS buffer (pH 7.1) containing 1% (v/v) bovine albumin (Fraction V, 99%, Sigma Chemical Co., St. Louis, MO). Fifty microliters of diluted sample was incubated in a water bath at 57.5°C for exactly 30 min., and immediately cooled in water and ice mixture for about 3 min. Twenty microliters of Betamyl (Megazyme Ltd., Wicklow, Ireland) substrate solution was dispensed into the bottom of 0.2-mL thin-walled 8 multitubes and preincubated in water bath at 40°C for approximately 5 min. ß-Amylase preparation was also preincubated at 40°C for approximately 5 min. Then 20 µL of ß-amylase preparation of each sample was added to the bottom of the multitubes and incubated at 40°C for exactly 10 min. At the end of the 10-min incubation period, 160 µL stopping reagent [1% (w/v) Trizma base, Sigma T 1503] was added. After stirred the reaction solutions, 100 µL was transferred into a 96 well plate. The absorbance of the reaction solutions and a reaction blank were read at 410 nm. The relative remaining activity (%) was calculated as the absorbance with heat treatment/absorbance without treatment (Kihara et al., 1998). Haruna Nijo, Harrington, and Schooner were set as control varieties of types A, B, and C, respectively.
Isoelectric Focusing Analysis
The ß-amylase extract was analyzed by electrophoresis by means of the Phastsystem and ready-made gel (PhastGEL IEF 4.0-6.5; Amersham Bioscience, Uppsala, Sweden). The electrophoresed gels were incubated in 3% (w/v) starch solution at 37°C for 10 min., followed by staining in a KI-I2 solution for about 15 min. Harrington and Haruna Nijo were set as control varieties of IEF Patterns I and II, respectively. In our previous study (Kaneko et al., 2002), the incubation time in starch solution was 30 min. We shortened the incubation time from 30 min to 10 min and obtained clearer images in this investigation. By the improved method, we could classify the IEF pattern in a more exact way.
Statistical Analysis
Gene diversity (H) at Bmy1 locus was calculated by the gene diversity index of Nei (1973): H = 1 – 
pi2, in which pi is the frequency of the ith allele of the locus.
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RESULTS AND DISCUSSION
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ß-Amylase Thermostability and Isoelectric Focusing (IEF) Analysis
As shown in Table 1, a wide variation in ß-amylase was found in the 8270 accessions of cultivated barley. On the basis of thermostability, most of the accessions could be grouped into the three distinct thermostability types. After heat-treatment at 57.5°C for 30 min., the relative remaining activity of types A, B, and C was 60 ± 2%, 30 ± 5%, and 3 ± 2%. Barley accessions OUI429 and OUI462, from India and Afghanistan, respectively, showed thermostability type intermediate to type A and B (Fig. 1)
. More than half of the accessions (56%) expressed Thermostability Type A, 21% had type B, and 23% exhibited type C. Intermediate (A–B) and superior (A+) thermostability types (Kaneko et al., 2001a) accessions were extremely rare.
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Fig. 1. Frequency distribution of ß-amylase thermostability types in cultivated barley (n = 8263). Relative remaining activity after heat-treatment at 57.5°C for 30 min: A+: 68%; A: 58–62%; A–B:38–47% (OUI462 and OUI429, respectively); B: 25–35%; C: 1–5%.
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The IEF pattern of ß-amylase segregated into seven patterns: I, Ia, II, IIa, IIb, IIc, and IV (Fig. 2)
. IEF Pattern III was only found in H. vulgare subsp. spontaneum (C. Koch.) Thell. (Zhang et al., 2004). Among these seven patterns, IIa (OUC1152) and IV (OUU421) were observed for the first time. IEF Pattern Ia lack the top band when compared with Pattern I but showed an additional band on the alkaline side. Although the IEF Patterns Ia and II are easily confused, they are distinguishable by comparing their band position.
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Fig. 2. Zymograms of ß-amylase in cultivated barley. Isoelectric focusing was done with a Phastsystem (Pharmcia, Sweden) and ready-made acrylamide gel (PhastGEL IEF 4.0-6.5; Amersham Bioscience, Uppsala, Sweden). Lane 1: Harrington; 2: OUNI096; 3: OUC1152; 4: Haruna Nijo; 5: OUI429; 6: OUI462; 7: OUU421.
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Accessions with Pattern II was predominant at 86%; Pattern Ia was 10% and Pattern I was 4%. Patterns IIa, IIb, IIc, and IV were rare mutants.
Combined analysis of thermostability types and IEF patterns (Table 1) revealed that Thermostability Type A+ accessions associated with IEF Pattern II; Thermostability Type A accessions consisted of IEF Patterns Ia, IIa, and II; Type A–B varieties included IIb and IIc; Type B accessions included IEF Patterns I, Ia, II, while type C included IEF Patterns I, Ia, II, and IV. In general, A-II (55.5%), B-I (3.6%), B-Ia (9.5%), B-II (8.3%), and C-II (22.5%) were the five major ß-amylase phenotypes in cultivated barley. A+-II, A-Ia, A-IIa, A-B-IIb, A-B-IIc, C-I, C-Ia, C-IV, and ß-amylaseless types were very rare (Table 1). Among these 14 ß-amylase types, we report here for the first time accessions with the A-Ia, A-IIa, C-I, C-Ia, and C-IV types. In addition, the B-Ia type was confirmed to be one of the major types of cultivated barley for the first time. Most (98%) of the Patterns I and Ia accessions were restricted to the Thermostability Type B group, while IEF Pattern II accessions were separated into A-II (64.2%), B-II (9.7%), and C-II (26.1%) three groups.
The detailed information of accessions possessing rare ß-amylase phenotypes are shown in Table 2. C-Ia type accessions formed a relatively large group (14 accessions) and were found in Azerbaijan, Georgia (former USSR), Yugoslavia, USA, Tunisia, and Egypt. A-Ia, A-B-IIb, A-B-IIc, and C-IV types were only detected in one accession each and in Mongolia, India, Afghanistan, and France, respectively. Cultivated barley in China had the highest diversity of rare types: A+-II, A-IIa, and ß-amylaseless. Table 2 also showed that the majority of rare-type accessions were six-rowed, covered, long rachilla hair, and spring growth habit. Short rachilla hair and winter growth habit barleys were only found in C-Ia type accessions. No notable tendency was found in brittleness type of rachis.
To date, we have confirmed that there are three main thermostability types (A, B, and C) and three major IEF patterns (I, Ia, and II) in barley ß-amylase phenotypes through evaluation of an extensive collection of germplasms. By combination of these three thermostability types and three IEF patterns, theoretically there could be nine possible kinds ß-amylase phenotypes (A-I, A-Ia, A-II, B-I, B-Ia, B-II, C-I, C-Ia, and C-II). We have found all these nine types in barley except that the A-I type, which was only found in wild barley H. vulgare subsp. spontaneum (Zhang et al., 2004). This indicated that the thermostability and IEF pattern are independent factors of ß-amylase. Besides, in H. vulgare subsp. spontaneum (the ancestor of cultivated barley), the three main thermostability types (A, B, and C) and the three major IEF patterns (I, Ia, and II) of ß-amylase already existed (Zhang et al., 2004). Hordeum vulgare subsp. spontaneum had A-I, A-II, B-I, B-Ia, B-II, and C-II six major types, which is one more than the five major types in cultivated barley. This result confirmed our suggestion that the main differentiation of ß-amylase preceded the domestication of barley. However, since barley has been cultivated for more than 8000 yr, the evolution of ß-amylase might have continued during this period. This may be why we observed several unique rare phenotypes (such as A-B-IIb, A-B-IIc, and C-IV) in cultivated barley. Moreover, the A-I type accession was frequently observed in H. vulgare subsp. spontaneum (12%) but was not found in H. vulgare subsp. vulgare (cultivated barley), even though these two subspecies are easy crossable.
According to the results of Nielsen et al. (1986) and Netsvetaev (1992), Bmy1 Br, Bmy1 Ar, and Bmy1 Al represent three kinds of ß-amylase allelic forms (from varieties Birka, Aramir, and Algerian, respectively) that could be classified by zymograms of electrophoresis. In our study, Birka, Aramir, and Algerian possess C-II, B-I, and B-Ia type ß-amylase, respectively; Eglinton et al. (1998) reported that three ß-amylase allelic forms (Bmy1 Sd2L, Bmy1 Sd1, and Bmy1 Sd2H) were identified in cultivated barley by thermostability assays in conjunction with IEF and molecular mapping. In this study, allelic forms Sd2L (from Schooner, Hiplroy etc.), Sd1 (from Harrington, Franklin et.), and Sd2H (from Haruna Nijo, Namoi et.) correspond to C-II, B-I, and A-II type ß-amylase, respectively. Thus, our classification of barley ß-amylase is more detailed and comprehensive and corroborates previous independent studies.
Diversity Index of ß-Amylase Phenotype
Table 1 shows the genetic diversity of ß-amylase phenotype in different regions. The total diversity index of investigated accessions was 0.624. Barley accessions in the Americas showed the highest diversity (0.796), followed by the former USSR accessions (0.765) and European accessions (0.759). North African accessions also showed high diversity (0.747). East Asian (Japan, Korea, and China) barley exhibited quite similar diversity. The lowest diversity (0.06) was observed in Ethiopian accessions, suggesting a bottleneck effect of Ethiopian barley origin. The barley accessions in the Americas had the highest diversity probably because they were mainly introduced from Europe and together with a part of barley accessions from other regions.
Relationship between ß-Amylase Types and Some Other Characteristics
We also analyzed the relationships between ß-amylase types and several morphological and physiological characteristics of barley, including kernel row type, caryopsis type, rachilla hair type, brittleness of rachis, and growth habit (Table 3). In general, barley accessions possessing the A-II type ß-amylase were six-rowed, long rachilla hair, East type brittleness, and a significant part of A-II accessions were intermediate or winter type. Naked barley had a large ratio (61%) only in A-II type accessions. By contrast, most of the B-I and C-II type accessions were covered spring habit barley with West type brittleness. Nearly 90% of B-II type accessions were six-rowed, covered and spring habit barley. The majority of B-Ia type accessions are six-rowed, covered and long rachilla hair barley. However, this general tendency was not common in all areas. For example, in Iran, Iraq, and Turkey, 72% of the B-Ia accessions were two-rowed; in India, Pakistan, and Afghanistan, 73% of the B-Ia type accessions were naked (Table 4). In the present study, a large ratio of A-II type accessions was of naked barley because a large number of barley accessions from West China (Tibet, Sichuan, and Qing-hai province) were included, and about 99% of them were naked and A-II type. In fact, covered barleys were the majority in A-II type accessions in the other regions except for China and Nepal. In North Africa, the ratios of long rachilla hair and East type brittleness in C-II type accessions were about 84 and 92%, respectively. In contrast, in Ethiopia about 72 and 99% of C-II type accessions had short rachilla hair and West type brittleness (detailed data not shown). On the basis of this result, we supposed that the ß-amylase type might be independent of other examined characteristics in barley. Such genetic diversity might be caused by natural crossing.
Geographical Distribution of Five Main ß-Amylase Types
Previously, we reported the geographical distribution of ß-amylase thermostability types in the world (Kaneko et al., 2001a). A more detailed description of ß-amylase type is presented here on the basis of the combined analysis of thermostability types and IEF patterns. The geographical distribution of the five major ß-amylase types (A-II, B-I, B-Ia, B-II, and CII) in the world is shown in Fig. 3
. Nearly 90% of accessions were of the A-II type in East Asia including Japan, Korean peninsular, and China. The B-II type accessions accounted for more than 55% of the rest, while the B-I, B-Ia, and C-II type accessions were rare in these countries. In Japan, a total of 20 C-II type accessions were detected (Table 1), 17 of them were two-rowed and covered, and most of them were malting barley. Only two accessions were six-rowed and naked and the last one was six-rowed and covered. Among the 20 C-II type accessions, 12 accessions were examined for brittleness, and only one accession was of the East type (Brt1Brt1brt2brt2, Takahashi, 1955) and 11 were of the West type (brt1brt1Brt2Brt2). Similar results were found in Korea and China (detailed data not shown). These results indicate that the C-II type accessions in East Asia were not original but introduced.
From the east to west, the ratio of the A-II type accessions gradually decreased, the lowest ratio being found in Turkey (0.8%) and Ethiopia (0.7%), while the ratios of the B-Ia and C-II type accessions increased. In Ethiopia, 97% of the accessions were of the C-II type. Only seven accessions being of the A-II type and all showed Oriental barley characters: six-rowed, long rachilla hair and East type brittleness. Although the ratio of A-II type accessions in Nepal and Bhutan was quite similar to that in East Asia, the B-Ia type accessions accounted for 88% of the remainder in Nepal and Bhutan. This is an obvious difference from East Asian barley. Takahashi (1955) and Takahashi et al. (1968) reported that Nepalese barley could be divided into two types, i.e., the highland naked barley and lowland covered barley. Table 4 shows that 93% of the B-Ia type accessions were covered barley in Nepal and Bhutan. Tibet is a highland area and Tibetan people prefer naked barley for food; thus, we propose that for these two reasons, the B-Ia-type (covered) barley was only recently introduced into Tibet. The exchange of barley between Tibet side and Nepal, Bhutan, and India side was mainly restricted in the A-II-type barley (as described previously, about 99% of the Tibet barleys were naked and of the A-II type). The B-Ia type accessions hold a major part in India, Pakistan, Iran, Iraq, and Turkey and North Africa cultivars. It is notable that B-Ia type accessions in Americas were mainly observed in Guatemala, where 79% of the accessions were of the B-Ia type (detailed data not shown).
B-I type accessions were mainly concentrated in Europe, former USSR, and North America. Kaneko et al. (2002) reported that B-I type accessions were predominant in North Europe, especially in Denmark (93% in landrace and 100% in commercial variety). Netsvetaev (1996) reported that in 179 spring barley varieties in Euro-Asia regions (the former USSR), Bmy1Ar allele (B-I type) in the northern region exceeded 50%, while in the southern region it was only 2%, and this distribution tendency did not change from West to East. This indicated that barley accessions carrying the B-I allele originated in and adapted to northern regions. Our results support the proposition that the B-I type accessions in cultivated barley originated in North Europe (Kaneko et al., 2002).
In addition, as shown in Fig. 3, the frequencies of different ß-amylase types were very similar among the East Asian countries (Japan, Korean peninsular, and China) and among the countries of Europe and the Americas, indicating a close correlation of their barley resources. In Mongolia, the ratio of ß-amylase types seems to reflect the influence of barley germplasm from its neighbors: the C-II allele was from the former USSR and the A-II allele was mainly from China.
In conclusion, on the basis of the classification of ß-amylase types, cultivated barley could be divided into five major types from five respective centers of origin in the world: A-II type barley center was in East Asia, B-I type center was in North Europe, B-Ia type center was in the Middle East and Southwest Asia, B-II type barley was spread from Turkey, and the C-II type center was in Ethiopia. This result is consistent with the geographical differentiations of various traits which have been investigated in barley, including morphological and physiological characteristics (Takahashi, 1955, 1987), phenol reaction (Takeda and Chang, 1996), and diazinon sensitivity (Takeda, 1996). The newly observed rare ß-amylase phenotypes provide us with new resources for improving the fermentability of malting barley. Future analysis of the DNA sequence and amino acid sequence of these 14 ß-amylase types should provide more information about the mechanism of ß-amylase thermostability.
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ACKNOWLEDGMENTS
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We are grateful to the Brewers Association of Japan and the Ohara Foundation for Agricultural Science.
Received for publication May 8, 2003.
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ABSTRACT
INTRODUCTION
