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Introduction

An Indica Group rice (Oryza sativa L.) cultivar, IR64, was developed at the International Rice Research Institute (IRRI) by introducing a semi-dwarf gene, sd-1, from the Chinese dwarf cultivar Dee Geo Woo Gen, and it has been cultivated widely in tropical and subtropical countries (Khush 1987). To improve yield potential in rice, ideotype traits such as shoot type (including plant height and panicle number) have been the main focus of study or modification in breeding programs (Khush 2013, Peng and Khush 2003, Peng et al. 1999, 2008). Recently, breeders have considered genetic improvement of root morphological traits to be an important challenge in crop yield production (de Dorlodot et al. 2007). The genetic and physiological mechanisms behind root morphological traits have been identified, but the effectiveness of these traits in rice breeding programs is unclear (Wu and Cheng 2014).

Root angle distribution, which is regulated by the growth angle of the crown roots, is one of the root morphological traits used to determine the area of soil over which root cap-ture water and nutrients (Uga et al. 2015a). For example, deep rooting is useful for extracting more water from the soil and minimizing drought stress under upland and rainfed lowland conditions (Fukai and Cooper 1995, Uga et al. 2013a, Yoshida and Hasegawa 1982). In contrast, under waterlogged conditions, shallow rooting can help plants to absorb oxygen on the soil surface and helps avoid hypoxic conditions (Hanzawa et al. 2013, Ueno and Sato 1989). It is also useful for absorption of nutrients such as phosphorus that accumulate at the soil surface in untilled culture sys-tems (Hanzawa et al. 2013, Uga et al. 2012). However, few studies have demonstrated genetic variations in root angle distribution and the adaptation of root distribution in rice plants in response to soil problems and different rice culti-vation systems. The relationship between genetic variation in rice root angle distribution and diversity of ecosystems for rice cultivations will needs to be clarified if we establish breeding strategies for suitable cultivars adapted to various field conditions.

Several studies have investigated genetic variations in

Breeding Science 67: 181–190 (2017) doi:10.1270/jsbbs.16185

Research Paper

Genetic variation of root angle distribution in rice (Oryza sativa L.) seedlings

Asami Tomita1), Tadashi Sato2), Yusaku Uga3), Mitsuhiro Obara4) and Yoshimichi Fukuta*5)

1) Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan2) Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 981-8555, Japan3) Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8602, Japan4) Biological Resources Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan5) Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences, Ishigaki, Okinawa 907-0002,

Japan

We developed a new method of using seedling trays to evaluate root angle distribution in rice (Oryza sativa. L), and found a wide genetic variation among cultivars. The seedling tray method can be used to evaluate in detail the growth angles of rice crown roots at the seedling stage by allocating nine scores (10° to 90°). Unlike basket methods, it can handle large plant populations over a short growth period (only 14 days). By using the method, we characterized the root angle distributions of 97 accessions into two cluster groups: A and B. The numbers of accessions in group A were limited, and these were categorized as shallow rooting types including soil-surface root. Group B included from shallow to deep rooting types; both included Indica and Japonica Group cultivars, lowland and upland cultivars, and landraces and improved types. No relationship between variation in root vertical angle and total root number was found. The variation in root angle distribution was not related to differentiation between the Japonica and Indica Groups, among ecosystems used for rice cultiva-tion, or among degrees of genetic improvement. The new evaluation method and associated information on genetic variation of rice accessions will be useful in root architecture breeding of rice.

Key Words: evaluation method, genetic variation, rice (Oryza sativa L.), root angle distribution, seedling tray.

Communicated by Toshio YamamotoReceived November 28, 2016. Accepted December 29, 2016.First Published Online in J-STAGE on May 10, 2017.*Corresponding author (e-mail: [email protected])

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the root angle distribution of rice cultivars. Ueno and Sato (1989) evaluated the number of soil-surface roots in 56 rice cultivars grown under three conditions (submerged, sub-merged and aerated, and control) in paper pots. They found that the Indonesian Japonica Group landrace ecotype Bulu (ecotype classified by Ueno et al. 1990) formed soil-surface roots under all three conditions. Ueno and Sato (1992) ex-amined the growth angles of four crown roots at 5 days after sowing on solidified agar in 130 rice cultivars by using a five-score scale [–90° (opposite direction to gravity), –45°, 0°, 45° and 90° (direction of gravity)]. They found a wide variation in the growth angles of crown roots in Indica Group cultivars, including Indian (Aus, Aman, and Boro) and Indonesian (Bulu and Tjereh) ecotypes and Japanese lowland and upland cultivars. Oyanagi et al. (1993) devel-oped a method of measuring the growth angles of wheat cultivar roots emerging from a meshed hemispherical basket buried in the soil. Kato et al. (2006) introduced the basket method to investigate for the root distribution of rice culti-vars and found genetic variation in the frequencies of higher root growth angle (>50°), defined as deeper roots, in 12 rice cultivars (9 Japonica cultivars, 2 Indica, and 1 Aus). On the basis of this information, Uga et al. (2009) investigated the ratios of deeper roots in 59 rice cultivars by using the basket method. They found no significant difference in deep root-ing among one Japonica and two Indica Groups, as classi-fied by using DNA polymorphism data. These studies fo-cused on the frequencies of soil-surface or deeper roots in rice plants, and did not consider the whole distribution of roots in detail. Moreover, the relationship between genetic variation in root angle distribution and differentiation among Japonica and Indica Groups, and among ecosystems for rice cultivation (such as irrigated and rainfed lowland, upland, swampy, and deep water), have not been fully clarified.

Hanzawa et al. (2013) used the basket method and classi-fied the growth angles of crown roots on a four-score scale [<0° (Soil-surface area), 0°–30°, 30°–60° and 60°–90°)] to evaluate varietal differences in the whole distribution of roots in two rice cultivars. However, this method is not suit-able for handling large populations of rice plants because it needs a long growth period and a large area of space for cultivation. To evaluate the root angle distributions of large number of rice plants, a simpler method of evaluating whole root distributions is needed.

Our objective here was to develop a new method for evaluating root angle distribution at the seedling stage of rice, and to clarify the genetic variation among rice culti-vars. We also discuss the wide variations root angle distribution in terms of the differentiation of Japonica and Indica Groups and adaptation to rice cultivation systems.

Materials and Methods

Plant materialsWe used an Indica Group rice (Oryza sativa L.) cultivar,

IR64, and eight accessions with the common genetic back-

ground of IR64 and consisting of four introgression lines (INLs) and four near isogenic lines (NILs), to develop a new method for evaluating root angle distribution. YTH16 and YTH34 were developed at IRRI from BC3 progeny by introducing chromosome segments of New Plant Type (NPT) cultivars IR65600-87-2-2-3, and YTH183 was IR69093-41-2-3-2 (Fujita et al. 2009, 2010). NERICA-L-19 was developed from BC2 progeny by using an interspecific hybrid cross between the African rice cultivar TOG5681 (O. glaberrima Steud.) and IR64 as a New Rice for Africa (NERICA) cultivar for irrigated or rainfed lowland (WARDA 2008). The four NILs—NIL-SPIKE, IR64-Pup1-H, IR64-Pup1-M, and Dro1-NIL—were developed by backcross breeding to introduce the QTLs or gene, namely SPIKE to increase total spikelet number (Fujita et al. 2013), Pup1 to increase phosphorus uptake (Wissuwa et al. 2002), or DRO1 to increase the ratio of deeper roots (Uga et al. 2011, 2013a).

Evaluation of root angle distributionWe used bottomless seedling trays (Naedoko, 602 × 274

× 23 mm, 37 × 2 planting stripes per tray Minoru Industrial Co., Ltd., Okayama, Japan) to investigate rice root angle distribution at the seedling stage (Fig. 1). Seeds were soaked in 1/200 Benrate T wettable powder 20 (Hokko Chemistry Industrial Co., Ltd., Tokyo, Japan) at 25°C for 24 h to sterilize the seeds, and then in tap water for another 24 h to promote uniform germination. A germinated seed was sown at the center of each planting stripe in the seed-ling tray filled with sterilized soil. The tray was then placed in a plastic box (860 × 530 × 150 mm) with the meshed bottom covered by paper. The tray was then placed in a plastic container filled with tap water to a depth of 3 cm and set in a greenhouse at 25°C under natural light. Fourteen-day-old seedlings were collected from the seedling tray, and the growth angles (°) of the crown roots in water were mea-sured on a nine-score scale [10° (0°–10°), 20° (20°–30°), 30° (20°–30°), 40° (30°–40°), 50° (40°–50°), 60° (50°–60°), 70° (60°–70°), 80° (70°–80°) and 90° (80°–90°)] from the horizontal line of water-surface with a protractor. The aver-age values of 22 plants in YTH16 and Dro1-NIL, and 12 plants in the other seven, were used as representative data.

The basket method of Hanzawa et al. (2013) was also used to investigate root angle distribution, with minor modi-fications. Twelve stainless-steel wire baskets (6 cm diame-ter × 3 cm depth) were buried just under the soil surface (open side up) to a depth of 11 cm in a plastic mesh box (40 × 30 × 15 cm) filled with sterilized soil. A germinated seed was sown at the center of each stainless basket at a depth of 1 cm from the soil surface. The basket containing each 28-day-old seedling was dug out carefully, and the growth angles of the crown roots emerging from each basket were counted on a four-score scale [0° (<0°), 15° (0°–30°), 45° (30°–60°) and 75° (60°–90°)]. The average value of 6 plants in each accession was used as the representative data.

The sum of the number of crown roots at each score on

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the scale on each plant was defined as the total root number (TRN). The root vertical angle (RVA) of each plant was cal-culated by using the following equation: RVA (°) = (Sum of scale values in all crown roots/TRN). The average value of Mode for root angle distribution in each accession was also considered.

Genetic variation of rice germplasm on the basis of DNA marker polymorphism data

For analysis of genetic variation we used a total of 97 accessions, including Indica and Japonica Groups, different ecosystems for rice cultivation [such as lowland (68 acces-sions) and upland (29)], and landrace (20) and improved (77) types (Table 1). The Japanese cultivar Nipponbare and the Aus cultivar Kasalath were included as controls for the Japonica and Indica Groups, respectively. A NIL, Dro1-NIL (Uga et al. 2011, 2013a); and the three INLs NERICA-L-19 (WARDA 2008), YTH16 and YTH183 (Fujita et al. 2009, 2010), with the common genetic background of IR64, were included as the improved Indica Group. As the Japonica Group, Akihikari, Koshihikari, and Sasanishiki were includ-ed as irrigated lowland cultivars, and Azucena, Davao, Moroberekan, and Owarihatamochi were included as up-land cultivars. Eighteen upland NERICAs and these par-ents’ cultivars; WAB56-50, WAB56-104 and WAB181-18, as Japonica Group upland lines, and an African rice CG14 (O. glaberrima S.), were also included. Namai et al. (2009) categorized rice germplasm into landrace and improved types (defined as those developed by crossbreeding after 1922), and here we classified the rice accessions by using the same method.

DNA polymorphism patterns were investigated by using 78 SSR markers (McCouch et al. 2002), which were distrib-uted over 12 rice genome chromosomes. Whole genomic DNA was extracted from a young leaf (around 1 cm2) from each accession. Leaf tissue was ground in 100 μl of 0.25 N

NaOH with zirconium beads in 2.0-ml tubes. A volume of 400 μl of 100 mM Tris-HCl (pH 7.5) was added to each tube. The sample was then mixed and centrifuged for 10 min at 10,000 rpm. The supernatant was poured into a fresh 1.5-ml tube. PCR was performed on a 10-μl PCR mix-ture containing 1 μl sterile H2O, a total of 1.5 μl forward primer (2 μM) and reverse primer (2 μM), 7.5 μl of 2× Quick Taq HS DyeMix (Toyobo Co., Ltd., Osaka, Japan), and 5 μl DNA concentrated to about 5 to 10 ng/μl. PCR am-plification was performed with the following profile: 94°C for 2 min, 40 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 68°C. To detect polymorphism, the amplified products were separated by electrophoresis on 3% agarose gels in 1 × TAE buffer at 150 V for 60 min, and the DNA fragment was detected with ethidium bromide. Extracted data were exported as allele sizes and formatted for further statistical analysis.

Classification of rice accessionsThe 91 accessions were classified on the basis of these

data for polymorphisms of 78 SSR markers and the 97 accessions were on the data for the number of roots on each score of the scale by using Ward’s hierarchical clustering method (Ward 1963) with the computer program JMP 11.2.0 (JMP Statistics and Graphics Guide, SAS Institute, Inc., Cary, NC, USA).

Results

Development of a new method of evaluating root angle distribution

We used the seedling tray method to investigate the dis-tributions of crown roots angle in IR64 and eight accessions with the common genetic background of IR64, and found a wide variation (Fig. 2). The roots of IR64 were distributed over eight scores from 20° to 90°, with a peak of 50°; the

Fig. 1. Seeding tray method for evaluation of crown root angle distribution in rice seeding stage. (A) Set up the seeding tray without bottom in the container. Seedling tray is divided 17 raws and 2 steps, and 34 seeding rice plants are cultivated at the same time. (B) Investigation of growth angle of crown roots from horizontal line, at 14 days after sowing.


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RVA was 52.4°. The roots of two accessions, YTH16 and YTH34, were distributed over all scores; the frequencies of roots with a score of 10° were higher than in the other accessions. The RVAs of YTH16 and YTH34 were 47.5° and 53.4°, respectively, and the root angle distributions dif-fered between the two accessions. The mode of YTH16 was 40°, and the distribution of YTH34 had three peaks, at 40°, 60°, and 80°. The RVAs of the three accessions Dro1-NIL, IR64-Pup1-M, and NERICA-L-19 ranged from 60.9° to 71.4°; these values were higher than those of the others. The

frequencies of roots at the deepest score of 90° in these three accessions were higher than in the other accessions, and NERICA-L-19 had the highest values. Two peak distri-butions were observed, at scores of 60° to 70° and at 90°, in IR64-Pup1-M; Dro1-NIL had three peaks, at 40°, from 60° to 70°, and at 90°. The remaining three accessions—NIL-SPIKE, YTH183, and IR64-Pup1-H—had similar RVA val-ues, in the range from 56.4° to 57.4°. NIL-SPIKE had distri-bution peaks at the two scores of 40° and 60°, and IR64-Pup1-H had three at 30°, 60°, and 80°. The frequencies

Table 1. Rice accessions used in this study

Cluster groups

Classified by DNA polymorphism data

Name and No. of rice accessions (%)TotalCluster groups classified by root angle distribution

A BI a Asanohikari Ikuhikari Sasanishiki

Aichiasahi IshikariShiroge Shin2Akihikari K60 Tainou67CSC-194 Kibi Toride1Fujisaka5 Koshihikari ToyonishikiDontokoi Kotobukimochi TsukinohikariChugoku40 Kusabue TsuyuakeFukunishiki Nipponbare YashiromochiHitomebore NorinPL6 ReihoHokkai188 OM576 HokkaiPL9

Sum 0 (0.0) 30 (30.9) 30 (30.9)b Gemdjah Benton Ingngoppor-Tinawon NERICA1 NERICA13

Trembese Lijiangxintuanheigu NERICA2 NERICA14NERICA4 Azucena NERICA3 NERICA15

Chubu32 NERICA5 NERICA16Davao NERICA6 NERICA17Moroberekan NERICA7 NERICA18Kamenoo NERICA8 WAB56-104Jamaica NERICA9 WAB56-50Kahei NERICA10 WAB181-18Oiran NERICA11Owarihatamochi NERICA12

Sum 3 (3.1) 31 (32.0) 34 (35.1)Total 3 (3.1) 61 (62.9) 64 (66.0)

II IR36 Basmati217 Kaluheenati SurjamkuhiCG14 Kanto51 Suweon258Dular Kasalath TadukanGuizhao2 Mahsuri Taichung Native1Hokuriku193 Milyang23 TakanariIR8 Milyang42 TetepIR24 Mudgo US-2IR64 Nanjing11 YTH183IR74 SR-1

Total 1 (1.0) 26(26.8) 27 (27.8)Others Tohoku U-3-7 IR65600-87-2-2-3 Tohoku U. No. 34

YTH16 Dro1-NIL NERICA-L-19Total 2 (2.1) 4 (4.1) 6 (6.2)

Grand total 6 (6.2) 91 (93.8) 97 (100.0)

A total of 97 accessions were classified into three cluster groups, Ia, Ib, and II, on the basis of DNA polymorphism data, and into two cluster groups, A and B, on the basis of data on the numbers of roots with each of nine angle scores. Others: Non-analysis by SSR markers. Tohoku U-3-7 (Hanzawa et al. 2013) and Tohoku U. No. 34 (unpublished material) have the genetic background of a Japonica Group cultivar, Nipponbare. YTH16 (Fujita et al. 2009), Dro1-NIL (Uga et al. 2011, 2013a) and NERICA-L-19 (WARDA 2008) have the genetic background of an Indica Group cultivar, IR64. IR65600-2-2-3 is a NPT cultivar developed from a cross between the Chinese Japonica-Group cultivar Shen Nung 89-366 and the Indonesian Japonica-Group cultivar Ketan Lumbu (Fujita et al. 2009, 2010).


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of YTH183 varied from 11.9 % to 15.6 % at scores from 30° to 90°, without clear peaks.

The TRN of IR64 was 6.6. These of YTH16, YTH34, SPIKE-NIL, Dro1-NIL, and NERICA-L-19 ranged from 6.5 to 6.9—similar to that of IR64. The TRNs of IR64-Pup1-M, IR64-Pup1-H and YTH183 were 7.6, 7.9 and 9.1, respec-tively—higher than the others. Thus the TRNs of IR64 and most of the INLs and NILs ranged basically from 6.5 to 6.9, whereas those of three of the accessions were different.

Even when the RVA values in each accession were simi-lar, the distributions of root angles differed among the nine accessions. There was no significant correlation between RVA and TRN (r = 0.12; ns).

We also performed an investigation using the basket

method of Hanzawa et al. (2013) for comparison with the results of the seedling tray method. We found different dis-tributions over the four scores—0°, 15°, 45° and 75°—among the nine accessions (Supplemental Fig. 1). The peaks of root distribution were 45° in seven accessions: IR64, NIL-SPIKE, YTH183, IR64-Pup1-H, Dro1-NIL, IR64-Pup1-M and NERICA-L-19. The modes of the re-maining two accessions—YTH16 and YTH34—were 15°; in these accessions the roots were found at the lowest score (0°) at higher frequencies than in the other accessions. The RVAs of YTH16 and YTH34 were 17.6° and 21.1°, respec-tively—lower than those of the others. The RVAs of Dro1-NIL and NERICA-L-19 were the highest at 47.6° and 51.7°, respectively. The remaining five accessions—IR64, IR64-Pup1-H, NIL-SPIKE, IR64-Pup1-M and YTH183—had similar RVA values and no clear differences in their distri-butions. TRNs varied from 13.5 to 22.8, and there was a significant correlation between RVA and TRN among the nine accessions (r = 0.64*).

The RVA, TRN and mode values in the nine accessions were compared between the two methods. The RVA values of the two methods were significantly positively correlated (r = 0.86**) (Fig. 3). In both methods, YTH16 had the lowest RVA and NERICA-L-19 the highest, but the orders of the values among the other seven accessions differed between the two methods. The TRN values of the two meth-ods showed a positive association, but not significantly (r = 0.56, ns). The Mode values of each accession were also significantly correlated (r = 0.71*).

Fig. 2. Root angle distributions in IR 64 and eight accessions with the IR 64 genetic background were investigated by using the seedling tray method. The averages of YTH16 and Dro1-NIL were calculated by using 22 plants, and the others were calculated by using 12 plants. [ ]: Average ± SD of RVA. ( ): Average ± SD of TRN. TRN, total root number. RVA, root vertical angle. Values denoted by different letters are significantly different at P = 0.05 by Turkey-Kramer test.

Fig. 3. Root vertical angle (RVA) relationship between the basket and seedling tray methods in IR64 and eight accessions with the IR64 genetic background. Average values of RVA for each accession were used as representative data in the seedling tray method and in the bas-ket method of Hanzawa et al. (2013). Error bars indicate SD for each accession. **; significant at P = 0.01.


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Classification of rice accessions by using DNA marker polymorphism data

A total of 358 DNA polymorphism patterns were ob-served in 78 SSR markers distributed over the 12 rice ge-nome chromosomes (McCouch et al. 2002), and the 91 rice accessions were classified into two major groups, namely I (64 accessions) and II (27), including two subgroups, Ia and Ib, by cluster analysis (Supplemental Fig. 2). Group I in-cluded the Japonica Group cultivar Nipponbare and group II included the Indica Group cultivar Kasalath; these corre-sponded to the Japonica and the Indica Groups, respectively.

A total of 32 accessions were included in group Ia; these consisted mainly of the improved Japonica Group cultivars for irrigated lowland that originated from Japan, such as Akihikari, Nipponbare, Koshihikari and Hitomebore. The 35 accessions in group Ib consisted mainly of upland culti-vars and landraces of the Japonica Group. An Indonesian cultivar, Gemdjah Benton, categorizing into Bulu ecotype (Ueno and Sato 1989, 1992, Uga et al. 2012), 18 upland NERICAs (Jones et al. 1997), and Azucena, Moroberekan and Owarihatamochi were included in this cluster group. The 30 accessions of group II consisted of 19 Indica Group improved cultivars and 10 landraces for lowland. In addi-tion, CG14 (O. glaberrima S.) was included in this group. The improved types consisted of semi-dwarf cultivars bred at IRRI (such as IR 8 and IR64) or cultivars developed by using IRRI cultivars, and also Mahsuri from Malaysia. The Indian aromatic landrace Basmati217 was also included in this group.

Genetic variation of root angle distribution in rice cultivarsWe found wide the variation for root angle distributions

of 97 rice accessions by using the seedling tray method (Figs. 4, 5). A total 91 accessions were common materials which were used for the classification by polymorphisms of SSR markers, and 6 accessions, Tohoku No. 34, Tohoku U-3-7, NTP cultivar (IR656000-87-2-2-3), YTH183, Dro1-NIL and NERICA-19. Tohoku U-3-7, is a mutant line of Nipponbare harboring a gene for soil-surface rooting, sor1 (Hanzawa et al. 2013), and Tohoku No. 34, a recombinant inbred line developed by introducing a QTL for soil-surface rooting, qSOR1 (Uga et al. 2012) into Nipponbare genetic background (unpublished material).

The RVA values of these accessions varied from 52.3° to 81.2° (average 71.8°). TRN varied from 3.3 to 8.8 (average 6.1). There was no correlation between RVA and TRN (r = –0.13, ns). This result confirmed that variation in RVA was not related to that of TRN.

We performed a cluster analysis by using distribution data for the number of roots among the 97 accessions with each of the nine scores. They were classified into two clus-ter groups, A and B (Table 1, Supplemental Fig. 3). Six accessions; Gemdjah Benton, NERICA4 and Trembese in group Ib, IR 36 in group II, and Tohoku U-3-7 and YTH16 in others, in group A had the lower RVA and the higher TRN values (average 58.2° and 7.0) than those of group B

(Fig. 5). The roots were distributed over all the scores, and the mode of root frequency was 60°. Gemdjah Benton har-bors qSOR1, a QTL for soil-surface rooting (Uga et al. 2012) in its genetic background. Tohoku U-3-7 harbors sor1, a mutant gene for soil-surface rooting, in the Nippon-bare genetic background (Hanzawa et al. 2013). Thus, the accessions in group A were categorized as shallow rooting type including soil-surface roots. The other 91 accessions were classified into group B. The RVA and TRN of group B were 72.7° and 6.0, respectively, and the frequencies of deep root scores from 60° to 90° in group B were higher than those in group A (Fig. 5). Tohoku No. 34 harboring a QTL, qSOR1, for soil-surface rooting (Uga et al. 2012) with

Fig. 4. Relationship between TRN and RVA in 97 rice accessions. TRN, total root number. RVA, root vertical angle. Average values for six plants in each accession were used as representative data. These were classified into two cluster groups: A (closed triangles) and B (open squares), on the basis of data on the numbers of roots with each of nine angle scores. ns: not significant at P = 0.05.

Fig. 5. Root angle distributions in the two cluster groups. Average values for each cluster group were used as representative data. n: No. of accessions. [ ]: Average ± SD of RVA. ( ): Average ± SD of TRN. TRN, total root number. RVA, root vertical angle.


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a Nipponbare genetic background; four accessions (IR64, YTH183, Dro1-NIL and NERICA-L-19) with the common genetic background of IR64; and the 17 upland NERICAs and three Asian upland cultivars as recurrent parents were included in this group. NERICA-L-19 had the highest RVA (81.2°) among the 97 accessions.

Discussion

A new method of evaluating root angle distributionWe developed a new method of evaluating root angle

distribution in rice at the seedling stage by using seedling trays to examine root growth angles and numbers of crown roots. To evaluate root angle distribution in detail, we classified growth angle in seedling trays into nine scores (10°–90°) from the horizontal. We observed wide distribu-tions of crown roots in each accession, from shallow to deep roots (Fig. 2). Even if the RVA values were similar, the dis-tribution of roots differed among accessions. We investigat-ed the relationship between the results of our seedling tray method and the basket method of Hanzawa et al. (2013), and positive correlations for RVAs (r = 0.86**) (Fig. 3), and modes (r = 0.71*) were found among IR 64 and the eight accessions. Thus, the results by the seedling tray method corresponded with those of the basket method by Hanzawa et al. (2013).

And the seedling tray method can thus be used to evalu-ate the growth angles of crown roots and gives more detail than the basket method in terms of the genetic variation of root angle distribution. The seedling tray method can be used to investigate 137 plants m–2 in 14 days, whereas the basket method can be used to investigate only 44 plants m–2 and takes 28 days. Therefore, the seedling tray method can handle larger populations of plants in less time. We thus es-tablished a simple and improved method of evaluation for root angle distribution at the seedling stage in rice.

Genetic mechanisms in rice accessions with common ge-netic background of IR64

The eight accessions with the IR 64 genetic backgrounds consisted of four INLs and four NILs. They were developed by backcross breeding between their donor parents and IR64 as a recurrent parent, and the numbers of chromosom-al segments introgressed from the donor parents were limit-ed in these genetic backgrounds. These varietal differences in root angle distribution from IR 64 were controlled by the genetic factors on these chromosomal segments. Dro1-NIL, harboring DRO1, a QTL for deep rooting (Uga et al. 2011, 2013a), had a higher RVA than that of IR64, and was confirmed to be deep rooting. NERICA-L-19 had the highest RVA value—significantly higher than that of Dro1-NIL. These results suggested that NERICA-L-19 harbored a different genetic mechanism for deep rooting from that of Dro1-NIL. In contrast, YTH16 and YTH34 had low RVA values, and root distributions with the lowest score (10°) were found at higher frequencies in these accessions than in

the others. YTH16 had a high frequency of roots with a score of 30°, and YTH34 had three peaks (at scores of 40°, 60° and 80°); the root angle distributions thus differed between these two accessions. They were developed as INLs from the BC3 progeny of IR64 and an NPT cultivar, IR65600-87-2-2-3 (Fujita et al. 2009, 2010). YTH16 and YTH34 differ in terms of the presence and length of intro-gressed segments on chromosomes 2, 4, 5, 7, and 8 (Fujita et al. 2009, 2010). Genetic factors on these chromosomes might play a role in differences in the root angle distribu-tions between YTH16 and YTH34. YTH16 had a lower RVA than those of the parents, IR65600-87-2-2-3 and IR64 (Fig. 4). Our results suggest that the root angle distribution in YTH16 was the result of transgressive segregation through the interaction of genetic factors from both parents. We therefore estimated that the wide variation in root angle distribution among IR 64 and the eight accessions was the result of the action of only a small number of genetic factors on limited chromosome segments originated from the donor cultivars and IR 64. To clarify these genetic mechanisms and genetic factors, genetic analyses of hybrid populations derived from crosses between IR 64 and these accessions will be needed.

YTH16 and YTH34 had high frequencies of the roots with a score of 10° by the seedling tray method (Fig. 2). They had also soil-surface roots in the field (unpublished data). These findings suggested that the high frequencies of roots at score 10° were corresponded with appearances of soil-surface rooting. These accessions can be used to elucidate the relationship between root angle distribution and soil-surface rooting.

Genetic variation in rice germplasmUse of the seedling tray method also revealed a wide var-

iation in root angle distribution among the 97 rice acces-sions; they were classified into two cluster groups, A and B. (Figs. 4, 5, Table 1, Supplemental Fig. 3). These groups showed differences in RVAs, TRNs, and root distributions. Group A consisted of six accessions: Gemdjah Benton, Trembese, YTH16, IR 36, NERICA4 and Tohoku U-3-7. Ueno and Sato (1989) reported that Gemdjah Benton was an Indonesian Japonica Group cultivar and included in the ecotype Bulu; it has soil-surface roots from the seedling stage to the ripening stage. Uga et al. (2012) found a major QTL for soil-surface rooting, qSOR1, in Gemdjah Benton. The Bulu cultivars have been grown in Indonesia as low-land landraces, and they have phenotypes similar to those of upland rice, i.e. a long panicle, low tiller, and wide leaf (Tsunoda 1987). Ueno et al. (1990) found that Bulu culti-vars showed resistance to potassium chlorate and were sim-ilar to the upland Japonica Group cultivars in Indonesia. The donor parent of YTH16—the NPT cultivar IR 65600-87-2-2-3—was bred from a cross between the Chinese Japonica-Group cultivar Shen Nung 89-366 and the Indo-nesian Japonica-Group cultivar Ketan Lumbu (Fujita et al. 2009, 2010). Trembese is also an Indonesian Japonica


188

Group cultivar (Thomson et al. 2007). IR 36 is an Indica Group cultivar bred at IRRI; its pedigree involves 16 land-races, including an Indonesian landrace Benong (Khush and Virk 2005). These accessions in cluster group A might harbor genetic factors originating from Indonesian Japonica Group cultivars or other upland cultivars, and these might have contributed the shallow rooting type of this group. We found here that the number of accessions classified into group A was limited. Thus the numbers of shallow rooting type accessions might be limited in natural variations of rice. We will need to confirm the frequencies of shallow rooting type accessions by using much more rice germplasm from different ecosystems used in regions ranging from tropical to temperate.

Tohoku U-3-7 harbors a mutant gene for soil-surface roots, sor1, in a Nipponbare genetic background; it lacks the gravitropic response of seminal roots (Hanzawa et al. 2013). Ueno and Sato (1992) found that the crown roots of Bulu cultivars, including Gemdjah Benton, did not respond to gravity under dark condition. They also found that the root gravitropic response was related to the growth angle of the crown roots under dark conditions, i.e. a physiological factor. Therefore, the shallow rooting type of group A may be controlled by genetic factors for root gravitropic re-sponse. We will need to identify the genetic factors, develop isogenic lines for them with a common genetic background, and then confirm the gravitropic response of these lines.

A total of 91 accessions were classified in group B. Tohoku No. 34, which harbors a soil-surface rooting QTL, qSOR1 (Uga et al. 2012), with a Nipponbare genetic background (unpublished material), and its recurrent parent Nipponbare, Dro1-NIL, with DRO1, a QTL for deep rooting in the IR64 genetic background (Uga et al. 2011, 2013a), NERICA-L-19 and IR64 were included in this group. These results indicated that the accessions of group B had a wide variation in root angle distribution from shallow to deep rooting types. The substitution line CSC-194 was much higher RVA than those of its parents, Akihikari and Milyang23. These results indicated that CSC-194 was a transgressive segregation progeny for deep rooting, and that this phenotype was controlled by genetic factors from both parents. The 18 upland NERICAs developed from BC2 progeny between an African cultivar CG14 (O. glaberrima S.) and three Asian upland cultivars as recurrent parents (Jones et al. 1997) had the different RVA values. The num-bers of introgressed chromosomal segments from donors are limited in the genetic backgrounds of the upland NERICAs (Fukuta et al. 2012). The three accessions YTH16, YTH183 and NERICA-L-19, with the common genetic background of IR 64, also had different RVA values. These results suggest that root angle distributions in rice cultivars are controlled easily by a limited No. of genetic factors, but that the effects are also influenced by the genetic background. Six major QTLs for deep rooting have been identified: DRO1 (Uga et al. 2011), DRO2 (Uga et al. 2013b), DRO3 (Uga et al. 2015b), DRO4 and DRO5

(Kitomi et al. 2015), and qRDR-2 (Lou et al. 2015). The QTLs or their alleles might be associated with the wide var-iations in the distribution of the root angles.

The classification of the accessions into two root distri-bution groups was not related to the classification into the three cluster groups Ia, Ib, and II according to the DNA polymorphism data (Table 1, Supplemental Fig. 2). Group Ia consisted mainly of improved Japonica Group cultivars for irrigated lowlands, originating from Japan. The acces-sions in group Ib included upland cultivars and landraces of the Japonica Group. Group II included both improved types and the landraces of the Indica Group for lowlands. Uga et al. (2009) found no significant difference in deep rooting between Japonica and Indica Groups in 59 Asian cultivars classified by DNA polymorphism data. Therefore, the wide variation in root angle distribution was not related to the differentiation between Japonica and Indica Groups or to adaptation to lowlands or uplands, or to whether the acces-sion was a landrace or had been improved.

Relationship of variations in growth angle and number of crown roots

A wide variation in TRN was also observed among IR 64, the eight accessions with IR64 genetic backgrounds, and the 97 accessions, and there was no correlation between RVA and TRN (Figs. 2, 4). Kato et al. (2006) also found no significant correlation between number of roots per stem and frequency of deeper roots in 12 rice cultivars. Uga et al. (2012) reported that soil-surface rooting was not related to TRN in recombinant inbred lines derived from a cross be-tween Gemdjah Benton and Sasanishiki. Therefore, we confirmed here that variations in root growth angle were not related to those in the number of crown roots.

Among the nine accessions with the IR64 genetic back-grounds, three (IR64-Pup1-H, IR64-Pup1-M, and YTH183) had higher TRN values than the others (Fig. 2). Two NILs (IR64-Pup1-H and IR64-Pup1-M) were developed to intro-duce a QTL, Pup1, to increase phosphorus uptake (Wissuwa et al. 2002) into the IR64 genetic background by backcross breeding. Gamuyao et al. (2012) reported that a Pup1- specific protein kinase gene, PSTOL1, increased the number of crown roots. We confirmed the high numbers of crown roots in the two abovementioned NILs in comparison with IR64 (Fig. 2). YTH183 was developed as an INL from BC3 progeny by introducing chromosomal segments of an NPT cultivar, IR69093-41-2-3-2, into the IR64 genetic back-ground (Fujita et al. 2009, 2010). Kano-Nakata et al. (2013) found that YTH183 had significantly more roots than IR64. The introgressed chromosomal segments may include ge-netic factors that increase the number of crown roots and originate from the NPT cultivar. YTH183 harbors nine seg-ments introgressed on chromosomes 1, 2, 4 (2), 5 (2), 6, 8 and 10 (Fujita et al. 2009, 2010, Obara et al. 2014). These locations of the chromosomal segments differed from that of Pup1 on chromosome 11 (Wissuwa et al. 2002). Thus the large number of crown roots in YTH183 is controlled by


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genetic factors different from Pup1. These results suggested that the variations in number of crown roots were controlled by several genetic factors.

We found no relationship between variation in growth angle and number of crown roots in rice, and we hypothesize that these characteristics are controlled by different genetic mechanisms. We also hypothesize that root angle distribu-tion is modified by a small number of genetic factors. By using hybrid populations derived from the accessions inves-tigated here, it should be possible to use QTL analyses to clarify the genetic factors and mechanisms determining root angle distribution. The information will be used for breed-ing of rice cultivars which adapt to various field conditions.

Acknowledgments

We would like to sincerely thank Dr. Matthias Wissuwa in JIRCAS for providing near isogeneic lines; IR64-Pup1-H and IR64-Pup1-M. This study was performed under the Japan International Research Center for Agricultural Sciences Research Projects “Rice Innovation for Environmentally Sustainable Production Systems” (2011 to 2015) and “Envi-ronmental Stress-Tolerant Crops” (2016 onward).

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Genetic variation of root angle distribution in rice