Development and characterization of a new rice cultivar, 'Chikushi-kona 85', derived from a starch-branching enzyme IIb-deficient mutant line.

A new super-hard rice cultivar, 'Chikushi-kona 85', which was derived from a cross between 'Fukei 2032' and 'EM129', was developed via bulk method breeding. 'Chikushi-kona 85' showed a higher content of resistant starch than the normal non-glutinous rice cultivar, 'Nishihomare', and a higher grain yield than the first super-hard rice cultivar, 'EM10'. The amylopectin chain length of 'Chikushi-kona 85' and its progenitor line 'EM129' was longer than that of 'Nishihomare', and was similar to that of 'EM10'. This suggests that the starch property of 'Chikushi-kona 85' was inherited from 'EM129', which is a mutant line deficient in a starch branching enzyme similar to 'EM10'. Genetic analysis of 'Chikushi-kona 85' crossed with 'Nishihomare' also showed that the starch property of 'Chikushi-kona 85' was regulated by a single recessive gene. Consumption of processed cookies made from 'Chikushi-kona 85' flour showed a distinctive effect in controlling blood sugar levels in comparison to the normal non-glutinous rice cultivar 'Hinohikari'. These results show that 'Chikushi-kona 85' is a novel genetic source to develop new products made of rice, which could reduce calorie intake and contribute to additional health benefits.

Introduction

In Japan, rice consumption has been steadily declining since 1960 due to Westernization of lifestyle accompanied by an increase in consumption of other foods rich in starch such as bread, noodles, and pasta. Therefore, to boost rice consumption, it is immensely important to develop rice and its products qualitatively. Recently, several new rice cultivars used for rice flour have been developed. ‘Mizuhochikara’ (Sakai 2010) has been used for making bread and distilled liquor and ‘Koshinomenjiman’ (Ishizaki ) for making Japanese udon noodles. Although these cultivars are favored in rice flour markets in Japan, the total production of rice flour has steadily decreased since 2011, when it was at its highest with 36,842 t, to 18,352 t in 2014. These data suggest that the demand for rice flour as an alternative to wheat flour has already been met and novel benefits of rice flour must be sought to increase consumption.

Meanwhile, diabetes mellitus has been growing as a serious condition among the Japanese population; 27.3% of the male and 21.8% of the female population are classified as being either diagnosed with diabetes mellitus or as being prediabetic (Ministry of Health, Labour and Welfare, Japan 2014). As a result, medical costs have increased in order to provide proper care and treatment for these patients. Subsequently, the Ministry of Health, Labour and Welfare proposed guidelines of desirable eating habits for Japanese people to prevent diabetes. Controlling calorie intake is one of the important ways to maintain good eating habits. One of the ways to do this is to consume low-calorie food.

‘EM10’ was developed by Kyushu University, Japan, by mutation breeding using N-methyl-N-nitrosourea (Nishi , Satoh and Oomura 1979, Takahashi ). This cultivar was called super-hard rice because ‘EM10’ contains a substantial amount of resistant starch (Ohtsubo ) and its starch is difficult to be digested by the human body. Grains of ‘EM10’ are opaque, and its starch property is totally different from that of conventional non-glutinous rice cultivars; the amylopectin chain length of ‘EM10’ is longer than that of the wild-type cultivar ‘Kinmaze’ (Nishi ). This specific characteristic of ‘EM10’ is due to the lack of activity of the branching enzyme IIb, which corresponds to the amylose-extender gene (Mizuno , Nishi ). Using rice flour of ‘EM10’, it was attempted to develop wheat/rice bread and noodles (Nakamura ) and tempura flour (Nakamura and Ohtsubo 2010). Although ‘EM10’ was quite promising, its yield is low (60–70% of the wild-type cultivar ‘Kinmaze’), which labeled ‘EM10’ as not being suitable for commercial production for the rice market.

The aim of this study was to develop a super-hard rice cultivar, which is suitable for rice flour production and also shows a high yield. We successfully developed a promising cultivar, named ‘Chikushi-kona 85’.

Materials and Methods

Breeding process

The genealogy of ‘Chikushi-kona 85’ is shown in Fig. 1. The maternal cultivar, ‘Fukei 2032’, is a non-glutinous japonica cultivar with a high yield, developed in Fukuoka Agricultural Research Center. The paternal cultivar, ‘EM129’, is a super-hard rice cultivar with a lower activity of the branching enzyme IIb than its wild-type cultivar ‘Kinmaze’ (Mizuno ). ‘EM129’ also shows similar starch properties to ‘EM10’, developed by the mutation breeding method using a chemical mutagen, N-methyl-N-nitrosourea (Yano ). The endosperm of ‘EM129’ is opaque, but its grain filling is higher than ‘EM10’ (Satoh 1985). The breeding procedure is shown in Table 1. The cross between both cultivars was carried out at Fukuoka Agricultural Research Center in 2005. Individual selection was conducted in 2010 using F4 plants focusing on the filling ratio and endosperm type (opaque or translucent) of the grains, and subsequent selections thereafter were conducted according to the pedigree method. One of the superior lines, which was harvested in 2011, was named ‘Chikushi-kona 85’, and the evaluation of its yield performance and starch properties, as well as its role in controlling blood sugar content were continuously carried out in the following years. Starch properties, except for amylopectin chain length distribution, were evaluated by Niigata Agricultural Research Institute and Torigoe Company Limited. The amylopectin chain length distribution was measured by capillary electrophoresis methods by Kyushu University. The measurement of blood sugar levels was carried out by Torigoe Company based on the evaluation method for glycemic index (Jenkins ).

Fig. 1

Genealogy of ‘Chikushi-kona 85’.

Table 1

Breeding procedure of ‘Chikushi-kona 85’

Year	Generation	Breeding experiments	Name of selected line
2005	(crossing)	Fukei 2032/EM129
2006	F1	Generation advancement
2009	F2–3	Generation advancement
2010	F4	Individual selection (from 256 plants)
2011	F5	Selection of individual lines and yield trial test (from 53 lines)
2012	F6	Yield trial test	Chikushi-kona 85
2013	F7	Yield trial test
2014	F8	Yield trial test
2015	F9	Yield trial test
2016	F10	Yield trial test

Results and Discussion

Agronomic traits

Table 2 shows the agronomic traits of ‘Chikushi-kona 85’. The heading date of ‘Chikushi-kona 85’ was slightly later than ‘Nishihomare’, which is a standard late-ripening cultivar in the warm regions of Japan. The culm length (83 cm) and panicle length (22.9 cm) of ‘Chikushi-kona 85’ were longer than those of ‘Nishihomare’ (75 cm, 19.8 cm), but the number of panicles in ‘Chikushi-kona 85’ (303/m2) was similar to that of ‘Nishihomare’ (308/m2). The lodging degree of ‘Chikushi-kona 85’ was higher than that of ‘Nishihomare’. The yield of ‘Chikushi-kona 85’ was 4.2 kg/a lower than that of ‘Nishihomare’, but 23.1 kg/a higher than that of ‘EM10’, the first rice cultivar with resistant starch. The weight per 1000 grains of ‘Chikushi-kona 85’ was 1.9 g lighter than that of ‘Nishihomare’. The abortive grains ratio of ‘Chikushi-kona 85’ was 1.9%, which was similar to that of ‘Nishihomare’ (1.2%), and lower than that of ‘EM10’ (9.2%). The grains of ‘Chikushi-kona 85’ were opaque, suggesting insufficient filling of starch in the endosperm (Fig. 2). This grain characteristic was similar to that of ‘EM10’. The field blast resistance was unknown because ‘Chikushi-kona 85’ carried an unknown true resistance gene.

Table 2

Agronomic traits of ‘Chikushi-kona 85’a

Cultivar	Year	Heading date	Culm length (cm)	Panicle length (cm)	No. of panicles per unit area (No./m2)	Lodging degreeb	Yield (kg/a)c	1000 grain weightc (g)	Abortive grain (%)	Grain quality
Chikushi kona 85		Sep. 5	83	22.9	303	1.2	47.0	23.3	1.9	opaque
Nishihomare	2011–16	Sep. 1	75	19.8	308	0	51.2	25.2	1.2	translucent
EM10		Aug. 31	72	19.5	373	0.1	23.9	18.2	9.2	opaque

The evaluation of agronomic traits was conducted in Fukuoka Agriculture and Forestry Research Center. Dates of sowing seeds and transplanting seedlings were May 15 to May 21 and June 20 to June 25, respectively. The amount of total nitrogen application was 1.0 kg/a.

Zero means plants stand erect and 5 means plants lodge completely.

Yield and 1000-grains weight were calculated using grains screened with 1.85 mm wire cloth test sieves.

Fig. 2

Plants (A) and grains (B) of ‘Chikushi-kona 85’.

Polishing properties

Table 3 shows the polishing properties of ‘Chikushi-kona 85’. With polishing time, the percentage of polished rice steadily decreased and the whiteness increased in both ‘Chikushi-kona 85’ and ‘Nishihomare’. ‘Nishihomare’ took 60 seconds to pass 90%; however, ‘Chikushi-kona 85’ took only 40 seconds. Nevertheless, the percentage of polished rice with the embryo of ‘Chikushi-kona 85’ remained higher than that of ‘Nishihomare’. This suggests that some of the brown rice grains cracked while polishing. However, from a practical viewpoint, the loss of grains due to cracked rice did not affect the yield of the polished rice (data not shown).

Table 3

Polishing properties of ‘Chikushi-kona 85’a

Cultivar	Brown rice		Measured properties	Polishing time (seconds)
	Moisture content (%)	Whitenessb		20	30	40	50	60	70
Chikushi-kona 85	15.8	30.3	Percentage of polished rice (%)c	92.5	90.5	89.6	88.0	86.6	83.7
			Whiteness of polished riceb	43.7	46.5	47.9	49.8	51.5	53.9
			Percentage of polished rice with embryo (%)	79.0	60.3	15.0	8.3	5.0	4.8
Nishihomare	14.6	20.9	Percentage of polished rice (%)c	92.9	92.0	91.0	90.2	89.4	89.1
			Whiteness of polished riceb	33.6	35.5	36.1	37.3	38.4	39.5
			Percentage of polished rice with embryo (%)	6.3	0.3	0.0	0.3	0.3	0.0

Polishing machine TPII (Kett Company Limited) was used for measurement of polishing properties.

C-300 (Kett Company Limited) was used for measurement of whiteness.

Percentage of polished rice was calculated as follows; polished rice (g) × 100/brown rice (g).

Chemical properties

Table 4 shows the chemical properties of the endosperm of ‘Chikushi-kona 85’. The apparent amylose content of ‘Chikushi-kona 85’ was higher than that of ‘Nishihomare’ and was similar to that of ‘EM10’. The polished grain of ‘Chikushi-kona 85’ indicated lower gelatinization than that of ‘Nishihomare’ in 5 M urea solution. The resistant starch content of ‘Chikushi-kona 85’, which was measured by two different methods (Resistant Starch Assay Kit (Megazyme, Wicklow, Ireland) and artificial digestion method (Homma )), was higher than that of ‘Nishihomare’ and slightly lower than that of ‘EM10’. The damaged starch content, which was measured by two different methods (Starch Damage Assay Kit (Megazyme, Wicklow, Ireland) and acid solution process (Arisaka and Yoshii 1991)), was lower than that of ‘Nishihomare’. The lower content of damaged starch is preferable, otherwise it could affect the quality of post-harvest processed foods made of rice flour (Araki ). The mean diameter of the flour of ‘Chikushi-kona 85’ was smaller than that of ‘Nishihomare’. A smaller flour size is preferable for post-harvest processing rice (Shoji ). All these properties together establish that the grain of ‘Chikushi-kona 85’ produced improved-quality rice flour in comparison to that of normal non-glutinous cultivars. The water absorbance of ‘Chikushi-kona 85’ was higher than that of ‘Nishihomare’ because of the grain conformation of the former, as well as the endosperm of ‘Chikushi-kona 85’ being opaque and containing more air space than that of ‘Nishihomare’.

Table 4

Chemical properties of endosperm and flour of ‘Chikushi-kona 85’a

Cultivar	Apparent amylose content (%)b	Starch disintegration in 5 M urea solutionc	Resistant starch (%)		Damaged starch (%)		Mean diameter of flour (μm)g	Water absorbance (%)
			Kitd	ADMe	Kitd	ASPf		15°C16 hr	40°C60 min
Chikushi kona 85	29.2	L	17.4	22.5	9.5	5.7	63.8	40.9	40.4
Nishihomare	18.9	H	0.3	13.9	11.4	6.8	77.9	30.8	30.2
EM10	31.4	L	25.4	33.4	8.8	4.5	66.5	44.4	43.4

Rice flour was generated with pinmill SRG10A (SATAKE, Corp. Japan) and sieved with 100 mesh.

This value was measured by Auto Analyzer (BL-TEC K. K., Osaka, Japan) based on the method of Juliano (1971).

This evaluation was performed based on the method Sato . L and H indicate that gelatinization is low and high, respectively.

Resistant Starch Assay Kit and Starch Damage Assay Kit (Megazyme, Wicklow, Ireland).

Artificial digestion method (Homma).

Acid solution process (Arisaka and Yoshii 1991).

This value was measured with Helium-Neon Laser Optical System (Sympatec, Clausthal-Zellerfeld, Germany).

Fig. 3 indicates varietal differences in amylopectin chain length distribution of the endosperm of ‘Chikushi-kona 85’. The proportion of short chains (6 ≤ DP ≤ 11) is lower and that of intermediate chains (12 ≤ DP ≤ 28) is higher than that of the normal non-glutinous cultivar ‘Kinmaze’. These changes in amylopectin chain length might contribute to the higher value of apparent amylose content (Table 4). ‘EM129’, which is a paternal parent line of ‘Chikushi-kona 85’, and ‘EM10’ showed a similar pattern of amylopectin chain length distribution to ‘Chikushi-kona 85’, although ‘EM10’ exhibited the lowest proportion of short chains and the highest proportion of intermediate chains among these three cultivars.

Fig. 3

(A) Amylopectin chain length distribution of ‘Chikushi-kona 85’, ‘EM129’, ‘EM10’, and the wild-type cutivar, ‘Kinmaze’. (B) Differences in chain length distribution patterns of the amylopectin of ‘Chikushi-kona 85’, ‘EM129’, ‘EM10’, and ‘Kinmaze’. This analysis was carried out using the capillary electrophoresis methods of O’Shea and Morell (1996), and Fujita in a P/ACE MDQ Carbohydrate System (Beckman Coulters, CA, USA).

Butardo demonstrated that inactivation of the starch branching enzyme IIb by RNA silencing leads to an apparent high amylose content and a high ratio of amylopectin long chain. Asai , Tsuiki , and Itoh also showed that the branching enzyme IIb-deficient mutant lines exhibited a similar higher apparent amylose content than normal glutinous cultivars. Furthermore, Tsuiki and Itoh revealed that their mutant lines also harbored a higher resistant starch content than the wild-type cultivar. These data suggest that all of the starch properties of ‘Chikushi-kona 85’ were due to a lack of activity of the branching enzyme IIb.

Genetic analysis of starch properties

Table 5 shows the segregation ratio of translucent and opaque endosperms of F2 seeds derived from crosses between super-hard rice cultivars (‘Chikushi-kona 85’ and ‘EM129’) and a normal cultivar (‘Nishihomare’). As mentioned above, the grain quality of ‘Chikushi-kona 85’ and ‘EM129’ were opaque. Furthermore, all crosses showed 3:1 segregation ratios of translucent and opaque grains, implying that a single recessive gene, beIIb, which corresponds to the amylose-extender gene, might be involved in the specific starch character of ‘Chikushi-kona 85’ and ‘EM129’.

Table 5

Allelism test of responsible gene for specific starch properties of ‘Chikushi kona 85’ and ‘EM129’.

No.	Cross	Generation	translucent	Opaque	χ2 value (3:1)
1	Chikushi kona 85/Nishihomare	F2	235	69	0.44ns
2	Nishihomare/Chikushi kona 85	F2	229	85	0.41ns
3	Nishihomare/EM129	F2	158	41	1.15ns
4	EM129/Nishihomare	F2	182	55	0.18ns

indicates that χ2 value is not significant for expected segregation ratio.

Effect of consumption of processed cookies on blood sugar content rise

Fig. 4 shows the influence of ingestion of processed stick-type cookies (Fig. 5, Supplementary Table 1) made of rice flour to the change in blood sugar levels in the human body. When thirty minutes passed after consuming cookies made of general non-glutinous cultivar ‘Hinohikari’ and super-hard cultivar ‘Chikushi-kona 85’ flour, the blood sugar levels increased to 27 mg/dL and 14 mg/dL, respectively. This shows that the rise in blood sugar levels after consuming cookies made of ‘Chikushi-kona 85’ rice flour was significantly lower compared to that of the normal non-glutinous rice cultivar ‘Hinohikari’. Since rice generally has a higher glycemic index compared to other carbohydrate foods (Ludwig 2002), its over-consumption tends to lead diabetes mellitus. As shown in Fig. 4, cookies made of ‘Chikushi-kona 85’ could restrict the increase in blood sugar content. This result indicates that rice products made of ‘Chikushi-kona 85’ have a potential as low glycemic index foods.

Fig. 4

Influence of consuming different types of cookies on the increase in blood sugar levels. One female and five male subjects were involved in this experiment. Composition of cookies are shown in Supplemental Table 1. The dietary intake of ‘Chikushi-kona 85’ and ‘Hinohikari’ were set as 84 g and 86 g, respectively, which were equivalent to 50 g carbohydrate intake. Zero in the horizontal axis means fasting, and the following value denotes time post consumption.

Fig. 5

Prototype product cookies made of ‘Chikushi-kona 85’ rice flour which were used for evaluating the impact of consumption on the rise in blood sugar levels.

Future prospects for dissemination of ‘Chikushi-kona 85’

As mentioned above, ‘Chikushi-kona 85’ consists of unique starch properties and has the potential to contribute toward reducing calorie intake and subsequently to prevent obesity and diabetes. On the contrary, brown rice of ‘Chikushi-kona 85’ tends to crack while polishing, and the eating quality of the cooked rice was poor and its resistant starch decomposed while being cooked in a rice cooker (data not shown). Therefore, suitable post-harvest processing methods, including one that can restrict crack of grains and the decomposition of resistant starch and maintain a positive impact on blood sugar levels, are necessary to disseminate this cultivar. This trial is now in progress and a finished product will be launched in the near future.

Itoh and Tsuiki pointed out that double mutant lines (beIIb + GBSSI or SSIIIa deficient) carried a higher resistant starch content than a single mutant line (only beIIb) and the resistant starch content of double mutant lines was higher than a single mutant line after cooking. This demonstrates the possibility that newly improved super-hard rice cultivars harboring stable resistant starch could be developed.