Trop. Agr. Develop. 60（3）：172 － 178，2016

Variation and Geographical Distribution of Perisperm Starch in Grain (Amaranthus spp.), and the Origin of Waxy Perisperm Type

Kazuhiro NEMOTO1, *, Mineo MINAMI1, and Tsukasa NAGAMINE2

1 Graduate School of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan 2 National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Abstract Excluding , grain amaranths are known as the only crop with non-waxy and waxy starch types in seeds. We examined the amylose content stored in the perisperm and starch types; non-waxy, waxy and low-amylose types in 266 accessions of three grain species i.e. Amaranthus hypochondriacus L., A. cruentus L. and A. caudatus L. from around the world. In the accessions from the New World where these species were domesticated, a wide range of amylose contents and all the starch types were detected. Conversely, very limited variation of this trait was observed in the accessions from Asia and Africa. We concluded that the waxy type originated in the New World before being disseminated into Asia and Africa. However, the selection pressure for the waxy type in the perisperm of grain amaranths in the New World was considered to differ from that acting on crops because of differences in the use and people’s preference for sticky food. Key words: Amaranthus spp., Amylose content, Geographical distribution, Grain amaranth, Waxy type

In previous reports, the waxy starch type of a grain Introduction amaranth, which stained reddish brown with iodine The starch-rich seeds of grain amaranths (Amaran- solution, had been observed in A. leucosperma S. Wats. thus spp.), which belong to the family in (= A. hypochondriacus) (Wolf et al., 1950) and A. cruentus the , are used as cereals, as well as and (MacMasters et al., 1955). Since then, intraspecific dif- grain. The seed storage starch of grain ama- ferentiation of the types, non-waxy and waxy in A. hy- ranths is located in the diploid perisperm (2n), which is pochondriacus (Sakamoto, 1982), low-amylose type in A. a modified nucellus of the embryo sac, while in the grass caudatus (Tomita et al., 1980) and non-waxy, waxy and family (), starch stored in cereal grains is depos- low-amylose types in A. cruentus (Inouchi et al., 1999) ited in the triploid endosperm (3n). They consist of three has been reported. Inheritance of the waxy perisperm cultivated species i.e. A. hypochondriacus L., A. cruentus trait is controlled by a single major structural gene with L. and A. caudatus L., all of which were domesticated the waxy allele, which is recessive to the Waxy allele in from early time in the New World (Sauer, 1976). A. hypochondriacus (Okuno and Sakaguchi, 1982). Starch is a complex polysaccharide composed of After the discovery of the New World by Columbus, amylose and amylopectin molecules. Non-waxy (non- the cultivation of grain amaranths was disseminated glutinous) starch contains both amylose and amylopec- throughout Asia and Africa far away from their area of tin, while waxy starch (glutinous) is composed of only origin. Especially, the southern foot of the Himalayas in amylopectin, or amylopectin with a very little amylose. the Indian subcontinent is considered to be the second- In grain amaranths, the perisperm starch has the waxy ary center of genetic diversity (Sauer, 1976), which con- type, which was differentiated from the non-waxy type trasts markedly with the decline of amaranth cultivation by spontaneous mutation (Sakamoto, 1997). This prop- in the New World. erty of the grain amaranths is unique in the Eudicots, The waxy/non-waxy trait which is correlated to the in other words, except for cereals such as , foxtail amylose content of the starch granules in grains, is an , common millet, Job’s tears, , and important characteristic for the breeding of cereal crops, . particularly in rice, because differences in the amylose content affect the texture of the processed grains.

Communicated by K. Irie However, research on this aspect in grain amaranths is Received Oct. 13, 2015 limited. In addition, variations of the amylose content of Accepted Jan. 8, 2016 these grain amaranths and the geographical distribution * Corresponding author [email protected] have not been revealed yet. In the present study, we Nemoto et al.: Variation of perisperm starch in grain amaranths 173 described the variations of the amylose content and the A. hypochondriacus, 99 of A. caudatus, and 70 of A. cruen- geographical distribution in three grain species using tus, collected from Central and South America, Asia, and amaranth genetic resources collected from around the Africa, were used in the present study (Table 1). Among world. We also analyze of the origin of the waxy peri- them, 219 strains were provided from the USDA-ARS sperm type in grain amaranths. National Germplasm Amaranth Collection and 47 accessions of A. hypochondriacus and A. caudatus from Materials and Methods around Asia were collected by Shinshu University. Some Plant materials accessions consisted of mixtures of seeds with different A total of 266 accessions of grain amaranths, 97 of seed coat color and/or appearance of the perisperm. In

Table 1. Geographical distribution of perisperm starch types identified by I2KI staining in three grain amaranth species. No. of Perisperm starch type Origin strains Non-waxy Low-amylose Waxy A. cruentus Central and South America Mexico 33 1 2 30 Guatemala 15 14 - 1 United States 6 1 - 5 Peru 3 3 - - Asia India 4 4 - - Africa Nigeria 2 2 - - Ghana 3 3 - - Zambia 3 3 - - Zaire 1 1 - - Total 70 32 2 36 A. caudatus South America Peru 37 9 3 25 Bolivia 29 1 6 22 Argentina 7 6 1 - Ecuador 1 1 - - Asia Nepal 18 - 18 - India 5 - 5 - Pakistan 1 - 1 - Bhutan 1 - 1 - Total 99 17 35 47 A. hypochondriacus Central and South America Mexico 46 35 - 11 United States 2 - - 2 Guatemala 1 1 - - Puerto Rico 1 1 - - Brasil 1 - - 1 Chile 1 - - 1 Asia Nepal 22 2 - 20 Pakistan 6 - - 6 India 5 - - 5 China 5 - - 5 Bhutan 2 - - 2 Afganistan 1 - - 1 Africa Nigeria 2 1 - 1 Uganda 1 - - 1 Zambia 1 - - 1 Total 97 40 - 57 Grand total 266 89 37 140 174 Trop. Agr. Develop. 60（3）2016 that case, seeds were separated and analyzed as a differ- distribution of the amylose content in A. cruentus and A. ent accession. hypochondriacus showed two clear peaks. In A. caudatus, the number of accessions tended to decrease when the Measurement of amylose content amylose content increased.

The amylose content of perisperm starch was mea- I2KI staining and seed appearance of A. cruentus sured colorimetrically using a TECHNICON SOLID- and A. caudatus revealed the existence of three types PREPTM II autoanalyzer (Instruments Co., NY). Given of perisperm starch (waxy, low-amylose and non-waxy) the large number of samples that had to be measured, in these species (Table 1). On the other hand, the ac- whole grain powder instead of purified starch granules cessions of A. hypochondriacus only contained two types from the grains was used for the analysis. Twenty milli- (waxy and non-waxy). The three types, waxy, low-amy- grams of grain powder passed through a 50-mesh screen lose and non-waxy ranged from 0 to 2.9%, 1.8 to 2.7% and was gelatinized in a mixture of 0.5N NaOH for more than 4.4 to 12.0% in A. cruentus, 0 to 1.9%, 0.9 to 5.9% and 8.0 to two hours at room temperature, and then applied to the 17.7% in A. caudatus, respectively. In A. hypochondriacus, autoanalyzer. The amylose content was calculated using the two types, waxy and non-waxy ranged from 0 to 2.7% a calibration curve made from using values obtained and 6.8 to 15.9%, respectively. Because the value of the from starch granule from Oryza sativa L. and A. cruentus amylose content is relative, waxy and low-amylose types (Table 2). The measurements of all the accessions were could not be differentiated clearly from each other. The conducted in triplicate. variation range of both types in the two species over- lapped, although both types could easily be identified by Identification of perisperm starch type the appearance of the perisperm (See Fig. 2). Among the Accessions were classified into three groups based three types, the waxy type predominated in all the three on the perisperm starch type, i.e. non-waxy, low-amylose species (Table 1). Whereas only 2 accessions consisted and waxy, which could be clearly distinguished from of the low-amylose type in 70 A. cruentus, 33 low-amylose one another by I2KI staining; grain powder of non-waxy, accessions were found in 99 A. caudatus. low-amylose and waxy types stained blue, purple and reddish brown, respectively (Fig. 1). In addition, these Geographical distribution of amylose content and peri- types could also be distinguished based on the perisperm sperm starch type appearance, when the seed was not covered with the The geographical distribution of the amylose con- seed coat colored dark brown or black. Non-waxy and tent and perisperm types is shown in Fig. 4 and Table low-amylose types of perisperms were translucent, while 1. Wide variation of both traits was observed in Central the waxy type of it was opaque, respectively (Fig. 2). and South America where these species had been do- mesticated, while limited variation was observed in Asia Results and Africa. Variation of amylose content and perisperm starch type In A. cruentus, all the accessions from Asia and Af- The amylose content of the accessions used in the rica belonged to the non-waxy type. In Central America, present study ranged from 0 to 17.7%. More specifically, 30 of 33 accessions from Mexico belonged to the waxy A. cruentus showed a range from 0 to 12.0%, A. hypo- type, while only one of 15 accessions from Guatemala chondriacus from 0 to 15.9%, and A. caudatus from 0 to belonged to the waxy type. In A. caudatus, all the peri- 17.7%, respectively (Fig. 3). The range of the amylose sperm types were found in accessions from Peru and Bo- content in A. cruentus was thus slightly narrower than livia in South America, while, with the exception of two that observed in the other two species. The frequency waxy accessions from India, all the accessions from Asia

Table 2. Amylose content of crops and varieties/strains used to construct the calibration curve. Crop Variety /strain Amylose content (%) Oryza sativa L. Mochiminori 0 Tankei 1915 9.6 Koshihikari 16.5 Kochihibiki 21.5 Amaranthus cruentus L. A.C.R104 0 A.C.K112 15.8 Nemoto et al.: Variation of perisperm starch in grain amaranths 175

Fig. 1. I2KI staining of different starch types. A: non-waxy starch type, B: low-amylose starch type, C: waxy starch type.

0 5 10 15

Fig. 3. Frequency distribution of amylose content and waxy starch type in three grain amaranth species.

the world. The amylose content in grain amaranths had Fig. 2. Seed appearance of grain amaranths. A: Pale yellow seed with non-waxy perisperm type. The been reported previously in a few samples (Sugimoto et perisperm is translucent (Central part). Since the low- al., 1981; Tomita et al., 1981; Konishi et al., 1985; Inouchi amylose type shows the same appearance (D), both et al., 1999). These studies can indicated that the content starch types can not be distinguished from each other by the appearance. B: Whitish yellow seed with waxy of amylose in A. cruentus ranged from 0 to 28%, in A. perisperm type. The perisperm is opaque and is whiter caudatus from 5.0 to 6.9% and in A. hypochondriacus than that of the non-waxy type (A). C: The seed is covered from 0 to 21.8%, respectively. In the case of A. caudatus, with a dark brown seed coat. Perisperm starch types can not be differentiated into the non-waxy or waxy type a narrow variation of the amylose content and only the by the appearance. D: A. caudatus with low-amylose low-amylose type were reported (Tomita et al., 1982). perisperm type. Most of the accessions in Asia show this Consequently, our study is the first to show the existence appearance. of a wider variation of the amylose content and of both waxy and non-waxy types. belonged to the low amylose type. In A.hypochondriacus, Compared to previous studies, the relatively low while the non-waxy type was dominant among the ac- amylose content we observed can be ascribed to the use cessions from Mexico, all of the accessions from Asia of whole grain powder for the analysis and the calculation and Africa belonged to the waxy type, except for three of the amylose contents were using a calibration curve of non-waxy accessions from Nepal and Nigeria. starch granules (Table 2). In grain amaranths, generally, the starch content of whole grains ranges approximately Discussion from 50 to 60% (Saunders and Becker, 1984). Then, as- Variation of amylose content and perisperm type suming a value of 50%, the amylose content in the starch Wide variation of the amylose content in the peri- granule becomes just double. Based on this assumption, sperm of the three species was revealed in the present the amylose content ranged from 0 to 35.4%, corroborat- study, using materials collected from different parts of ing previous reports, in which almost the same range in 176 Trop. Agr. Develop. 60（3）2016

Fig. 4. Geographical variation of amylose content and perisperm starch type in three grain amaranth species. rice (0 to 30%, Katsuta et al., 1989) and foxtail millet (0 ‘Love-Lies-Bleeding’ etc. in Europe, and is grown as an to 25.1%, Takei et al., 1989; Nakayama et al., 1998) was ornamental plant. According to Sauer (1950), this variety observed. may have been introduced into Asia from South America via Europe. In addition, it is assumed that this variety Geographical distribution was selected in Europe based on the characteristics of The obvious gap in the geographical distribution of the inflorescence and not for grain use with a low amy- the amylose content and perisperm types was revealed lose content. However, after the introduction into Asia, between the New World and the Old World in three spe- especially to the Indian subcontinent, A. caudatus has cies. The accessions from Central and South America been considered to be one of the grain crops and not an exhibited a wide variation of amylose content and all the ornamental one. perisperm types. Conversely, accessions from Asia and In A. cruentus, the accessions from Asia and Af- Africa showed a limited variation of amylose content and rica showed only dark brown or black seed coats and a particular type of perisperm; A. cruentus had only the morphologically similar inflorescences. In addition, the non-waxy type, A. caudatus only the low-amylose type seed size was significantly smaller than that of the seeds except for two accessions from India, and A. hypochon- with a pale-yellow seed coat (data not shown). These driacus only the waxy type except for three accessions characteristics indicate that these accessions had been from Nepal and Nigeria, respectively. Interestingly, used as vegetables in the regions. In this case, the starch different perisperm starch types were introduced from type of the perisperm cannot directly be differentiated the area of origin to the Old World, i.e. non-waxy type of into non-waxy and waxy types by the appearance of A. cruentus, low amylose type of A. caudatus and waxy the perisperm because of the colored seed coat (Fig. type of A. hypochondriacus, respectively. These results 2-A). In other words, the perisperm starch type is not suggest that the geographical gap was developed due important for vegetable use. In Central America, the to a bottle neck, namely a limited population was dis- proportions of non-waxy and waxy accessions from seminated from Central and South America to Asia and Guatemala and Mexico were completely different, even Africa, and also the frequency of dissemination might though the countries are adjacent. In Guatemala, 14 out have been limited. of 15 accessions belonged to the non-waxy type and 10 Indeed, all the A. caudatus accessions in Asia with of the accessions showed the same dark brown or black the low-amylose type shoes the same morphological seed coat as the accessions from Asia and Africa. In traits; a drooping inflorescence and red color, and the Guatemala, amaranth is typically called ‘bledo’ in Spanish seeds had a translucent perisperm with a red rim (em- and is generally used as a relict vegetable (Sauer, 1967). bryo part) (Fig. 2-D), while most accessions from South Conversely, in Mexico, 30 out of 33 accessions belonged America display an erect or semi-erect inflorescence to the waxy type and amaranth is a staple grain crop. It and a variation of the seed color (data not shown). The is considered that the difference in main use between drooping inflorescence type is commonly referred to as Guatemala and Mexico caused such results. Nemoto et al.: Variation of perisperm starch in grain amaranths 177

A. hypochondriacus showed different characteristics waxy form of seven cereal crops, such as rice, foxtail from these of A. cruentus. Almost all the accessions from millet, common millet, Job’s tears, sorghum, barley and Asia and Africa belonged to the waxy type and showed maize was developed (Sasaki, 1982; Sakamoto, 1989). In a whitish yellow seed coat. In the foothills of the Hima- this area, the sticker trait of waxy endosperm starch has layan range in Asia (e.g. Nepal, India, Pakistan and Bhu- played an important role in the particular food culture. tan), A. hypochondriacus is widely cultivated as a staple Generally, waxy grains are associated with own recipes grain crop (Sauer, 1967; Sakamoto, 1997). Similarly, an using the sticky trait and are clearly different from those investigation of more than 200 Nepalese accessions of of the non-waxy grains. On the other hand, in the case A. hypochondriacus by the author in 1994 revealed the of A. hypochondriacus in the Mesoamerica, Sakamoto presence of only three non-waxy accessions (K. Nemoto (1982, 1997) considered that the waxy periperm type unpublished). However, in a survey conducted in Kath- might have developed as a result of the preference for mandu and Langtang valley in Nepal in 1975, Sakamoto the lighter seed coat (See Fig. 2-B, C) rather than for (1982) reported that 16 samples included three acces- the sticky trait. Because popped seeds are used mostly sions with the non-waxy type and one with a mixture of for food preparation, it does not matter whether the both types. These findings indicate that, compared to non-waxy type or the waxy type is used when cooking the present, the proportion of non-waxy type accessions amaranth grains. Indeed, except for the Chepang, may have been higher than several decades ago. a minor ethnic group in Nepal, the utilization of the stickier waxy starch compared to that of the non-waxy Origin of the waxy and low-waxy types starch has never been reported (manuscript in prepara- In the present study, the amylose content and peri- tion). Consequently, as proposed by Sakamoto (1989), sperm starch type were investigated using a large number the selection pressure to develop the waxy perisperm of accessions from different regions of the three species. starch type of grain amaranths is different from that for The results revealed that the diversity of the perisperm the waxy endosperm types of cereal crops in East Asia starch type in the New World was evident. Sakamoto where there is a distinct preference for sticky food. (1982) assumed that the waxy type of A. hipochondriacus The low-amylose types of both A. cruentus and A. had already differentiated from the non-waxy type in the caudatus were also considered to have originated from New World in pre-Columbian times. This assumption the New World. However, the perisperms of the low- was based on the fact both the waxy and non-waxy types amylose and non-waxy types are both translucent and had been found in Central America where the species they cannot be clearly distinguished from each other by had been domesticated. The results of our study not the appearance. Consequently, the selection for the low- only support the hypothesis of Sakamoto, but also indi- amylose type instead of the non-waxy type cannot to be cate that the waxy and low-amylose perisperm types of ascribed to a particular preference for sticky food or for A. cruentus and A. caudatus were developed in the New a lighter seed coat. This suggests that the low-amylose World. Maize, which had been domesticated in the New type was not selected consciously from the non-waxy World as well as grain amaranths, also shows a waxy type at all. Indeed, the accessions of the low-amylose endosperm type. A waxy mutant was first discovered in type in A. caudatus were mostly localized in Asia. It was China (Collins, 1909), and waxy landraces were selected thus deduced that the small samples introduced into and particularly widely distributed in East Asia after Europe as an ornamental plant, which show a long and dissemination to the Old World. However, unlike grain drooping inflorescence, as mentioned before, belonged amaranth, the waxy mutant has been selected in the Old to the low-amylose type by chance and were then dis- World (Sakamoto, 1982), although some mutants were seminated to Asia. found in North America and Argentina (Bregger, 1928; Acknowledgement Mangeksdorf, 1924). Grain amaranths are, therefore, the only crop, in which the waxy type was developed by We would like to thank Dr. D. Brenner, USDA-ARS- human selection in the New World. MWA-PIRU, Iowa State University, USA, for providing However, for what was the waxy type selected in the accessions used in the present study. grain amaranth? The waxy endosperm type in cereal References crops was selected for the preference for the sticker trait (Sakamoto, 1989). A specific food preference for waxy Bregger, F. G. 1928. Waxy endosperm in Argentin maize. J. Hered- endosperm starch can be found in East Asia where the ity 19: 111. 178 Trop. Agr. Develop. 60（3）2016

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