NAL1 allele from a rice landrace greatly increases yield

in modern indica cultivars
Daisuke Fujitaa,b,c,1, Kurniawan Rudi Trijatmikoa,2, Analiza Grubanzo Taglea, Maria Veronica Sapasapa, Yohei Koidea,b,3,
Kazuhiro Sasakia,b, Nikolaos Tsakirpalogloua, Ritchel Bueno Gannabana, Takeshi Nishimurad, Seiji Yanagiharab,
Yoshimichi Fukutab, Tomokazu Koshibad, Inez Hortense Slamet-Loedina, Tsutomu Ishimarua,b,
and Nobuya Kobayashia,b,c,4
Plant Breeding, Genetics, and Biotechnology, International Rice Research Institute, Metro Manila, Philippines; bJapan International Research Center for
Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan; cNational Agriculture and Food Research Organization, Institute of Crop Science, 2-1-18
Kannondai, Tsukuba, Ibaraki 305-8518, Japan; and dDepartment of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan

Increasing crop production is essential for securing the future food
supply in developing countries in Asia and Africa as economies and
populations grow. However, although the Green Revolution led to
increased grain production in the 1960s, no major advances have
been made in increasing yield potential in rice since then. In this
study, we identified a gene, SPIKELET NUMBER (SPIKE), from a
tropical japonica rice landrace that enhances the grain productivity of indica cultivars through pleiotropic effects on plant architecture. Map-based cloning revealed that SPIKE was identical to
NARROW LEAF1 (NAL1), which has been reported to control vein
pattern in leaf. Phenotypic analyses of a near-isogenic line of a popular indica cultivar, IR64, and overexpressor lines revealed increases
in spikelet number, leaf size, root system, and the number of vascular bundles, indicating the enhancement of source size and
translocation capacity as well as sink size. The near-isogenic line
achieved 13–36% yield increase without any negative effect on
grain appearance. Expression analysis revealed that the gene
was expressed in all cell types: panicles, leaves, roots, and culms
supporting the pleiotropic effects on plant architecture. Furthermore, SPIKE increased grain yield by 18% in the recently released
indica cultivar IRRI146, and increased spikelet number in the genetic background of other popular indica cultivars. The use of
SPIKE in rice breeding could contribute to food security in indica-growing regions such as South and Southeast Asia.

experiments (9, 13, 15). However, no novel cloned gene has been
reported to increase grain yield in indica cultivars (17). Here, we
identified a gene in a tropical japonica landrace and used the
allele to increase the grain yield of modern indica cultivars at
the crop level through a breeding concept developed by International Rice Research Institute (IRRI) breeders more than
20 y ago.
In 1989, a breeding program for New Plant Type (NPT) rice
was launched at IRRI to increase the yields of modern indica
cultivars by using genetic material from tropical japonica landraces (18). Several Indonesian tropical japonica landraces—
which are characterized by large panicles, large leaves, a vigorous
root system, thick stems, and few unproductive tillers—have
been used in international breeding programs. However, despite
these features, the NPT cultivars yield less than modern indica
cultivars, mainly because of low grain fertility and low panicle
number (19, 20). To genetically dissect and elicit the valuable
traits of NPT cultivars, we backcrossed the NPT cultivars including YP9 (IR68522-10-2-2) against modern indica cultivar
IR64 to develop introgression lines (ILs) (Fig. S1). BC3-derived
ILs, which had favorable yield-related traits and few undesirable

| gene validation | pleiotropy | marker-assisted breeding

This work reports discovery of a unique gene important for rice
agriculture. A significant yield enhancement in rice modern
cultivar was achieved by identification of a gene, SPIKELET
NUMBER (SPIKE) in Indonesian rice landrace. The SPIKE increased grain yield of an indica cultivar IR64, which is widely
grown in the tropics, over four seasons at the field level and
improved plant architecture without changing grain quality or
growth period, which are important for regional adaptability.
These results indicate finding of SPIKE will be extremely valuable for contributing to increase grain production of indica
rice cultivars.


he world’s population is expected to surpass 9 billion in 2050
( Most of this increase
will occur in the developing countries of Asia and Africa. By
2035, a 26% increase in rice production will be essential to feed
the rising population (1, 2). Rice (Oryza sativa L.) is a staple food
of more than 3 billion people, mainly in Asia. Predominantly,
indica cultivars are grown in southern China, Southeast Asia, and
South Asia, occupying approximately 70% of the rice-producing
area in the world, whereas japonica cultivars are grown mainly in
East Asia (3, 4). Because of urbanization and industrialization,
an increase in the yield of indica cultivars is a pressing need in
Southeast and South Asia (5). Additionally, good grain quality
(influencing market value) and high yield are essential for the
adoption of new cultivars in local areas (6).
The grain yield of rice is determined by spikelet number per
panicle, panicle number per plant, grain weight, and spikelet
fertility. Although many quantitative trait loci (QTLs) for yield
components have been identified (, few have
so far been isolated. To date, at least nine genes or loci for yieldrelated traits in rice have been isolated from natural variation:
Gn1a and APO1 for number of grains (7–9); GS3, GW2, and
qSW5 for grain size (10–12); DEP1 and WFP for panicle architecture (13, 14); SCM2 for strong culm (15); and Ghd7 for late
heading and number of grains (16). APO1, SCM2, and DEP1
increased grain yield in a japonica genetic background in field

Author contributions: D.F., Y.F., I.H.S.-L., T.I., and N.K. designed research; D.F., K.R.T., A.G.T.,
M.V.S., Y.K., K.S., N.T., R.B.G., S.Y., and T.I. performed research; T.N., T.K., and I.H.S.-L.
contributed new reagents/analytic tools; D.F., K.R.T., A.G.T., and I.H.S.-L. analyzed data;
and D.F., I.H.S.-L., T.I., and N.K. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.

Present address: Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan.


Present address: Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Jl. Tentara Pelajar 3A, Bogor, West Java 16111,


Present address: The Hakubi Center for Advanced Research/Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.


To whom correspondence should be addressed. E-mail:

This article contains supporting information online at

PNAS | December 17, 2013 | vol. 110 | no. 51 | 20431–20436


Edited* by Gurdev S. Khush, University of California, Davis, CA, and approved November 4, 2013 (received for review June 11, 2013)

8 50 0 0. The average GYS of the NIL was 28% higher in the dry season and 24% higher in the wet season than that of IR64 (∼400 g/m2). significantly so in three of the four seasons (Fig. 1D). Therefore. 500 μm. B.1%. In addition to TSN. 1C).s. leaves (Fig. S3). and rate of filled grain (Fig. To confirm the effect on practical grain yield in the field. Fig. 1A). 1B). Quantitative RT-PCR (qRT-PCR) revealed that the expression of SPIKE in young panicles at various stages was consistently higher in NIL-SPIKE than in IR64. flag leaf width (FLW. NIL-SPIKE ( Expression analysis in a young panicle revealed that only Os04g52479 (Nal1: NARROW LEAF 1. 10 cm. and grain weight per m2 among two dry (DS) and wet seasons (WS) (J).05. 2C). presumably owing to a strengthening of assimilate supply to the larger number of spikelets by an increase in vascular bundle number (VBN. 20432 | www. and translocation capacity (high VBN). (C) Flag leaves. for high TSN. To validate our results and to gain insight into the function of SPIKE. 2E). S5). 1. we isolated the gene for qTSN4 through mapbased cloning to facilitate its use in breeding. root dry weight at maturity (n = 10) (G). Significant at ***0. and double that of IR64 at the 21. we conducted high-resolution linkage analysis by using 7. but had lower panicle number per plant and 1. A Expression Analysis of SPIKE. (Scale bars: A.0 kbp).0 NIL-SPIKE H Number of vascular bundles * Flag leaf width (cm) Spikelet number per panicle 200 IR64 G Chalky Normal NIL-SPIKE F Rate of grain chalkiness (%) IR64 E 36% ** 13% n. 1F). S6 A–C). Notably. S2 B and C). Fig. Additionally. Values are means. derived from tropical japonica landrace Daringan with an IR64 genetic background. number of vascular bundles in panicle neck (n = 20) (I). we generated overexpressor lines (using a constitutive promoter) and silencing lines (using artificial microRNA: amiRNA). grain weight. a nearisogenic line (NIL) for qTSN4 from YP9. and maize. the increase in GYS in the NIL without a decline in grain appearance was achieved through the enlargement of sink size (high TSN). one of them in the trypsin-like serine and cysteine protease domain (Fig. C. days-to-heading was unchanged (Fig. 5 cm. along with the rate of filled grain. 1G). 23) was expressed (Fig. we expressed the β-glucuronidase (GUS) reporter gene under the control of the native SPIKE promoter in transgenic IR64 plants.000grain weight (Fig. had more spikelets per panicle and more branches than IR64.996 BC4F3 plants evaluated for TSN. Aside from the coleoptile.. the grain yield per m2 (GYS) of the NIL was consistently higher than that of IR64 over four cropping seasons. We characterized qTSN4. The phenotypic effects of the gene were validated in transgenic plants and by expression analysis. **1%. High-Resolution Linkage Mapping and Identification of SPIKE.6 150 1. source size (broad FLW and high RDW). . the SPIKE protein shows >84% identity with proteins of Brachypodium. Characterization of yield-related traits of a NIL for SPIKE. Thus. The results suggest that the increase in SPIKE expression at the young panicle stage increased spikelet number. is the most probable candidate for SPIKE. lateral roots (Fig. qTSN4. Fig. sorghum. thus. *5%. D. we evaluated yield and related traits by using NILs with genetic backgrounds of popular indica cultivars. Among the QTLs. the pattern of GUS expression coincided with the organs enlarged in NIL-SPIKE. root dry weight (RDW. n. Using the ILs. (A) Plant morphologies. SPIKE is highly useful for improving yield without changing locally adapted traits. not significant. Results Characterization of NIL-SPIKE. 35% ** 20% * 500 400 300 200 100 0 434 591 414 467 377 509 380 454 IR64 NIL IR64 NIL IR64 NIL IR64 NIL 2010DS 2010WS 2011WS 2012DS Fig. were selected by field observation (21). S2D). Percentages above bars in J are yield increases of the NIL relative to IR64. S2A). the grain appearance was improved (Fig. 2D). Among yieldrelated traits. the suggested gene was associated with an increase in secondary branch number and leaf width (Fig. it had higher TSN (Fig. 1I).2 100 0. wheat.traits. with whiskers showing SD. In this study. vascular bundle at the panicle neck and culm.s. The candidate region lay between markers Ind4 and Ind12 (18. Histochemical analysis revealed GUS activity in the coleoptile. and high similarity in the trypsin-like serine and cysteine protease domain. 2B). (D) Cross-sections of panicle neck. (SEM in J). 2A). leaves (Fig. S4) and. (B) Panicle structures. A DNA fragment containing the cDNA of SPIKE from NIL-SPIKE fused C B D IR64 *** Root dry weight (g) 1. To identify a gene for SPIKE. 1E).to 50-mm stage (P = 0. This similarity suggests conservation of the biochemical function of the SPIKE protein family among these species. rate of chalkiness in brown rice (H). we identified 21 QTLs for yield components such as total spikelet number per panicle (TSN). Further. ref.1310790110 Fujita et al. designated here as SPIKE.) (E–J) Comparisons between IR64 and NIL-SPIKE of spikelet number per panicle (n = 8) (E). Gene Validation of SPIKE Through Transgenic Analysis. in which the Rice Genome Annotation Project database at Michigan State University predicts three genes (Fig. Analysis of the predicted amino acid sequence of SPIKE revealed three amino acid substitutions between IR64 and NIL-SPIKE. NIL-SPIKE had larger panicles (Fig. Fig. crown roots. was commonly detected on the long arm of chromosome 4 in five NPT lines derived from different tropical japonica cultivars (22). and panicle number. Consequently. 1J). 20 cm. and panicle necks than IR64 (Fig. To analyze the expression of SPIKE during plant development. by using an NIL for SPIKE. flag leaf width (n = 9) (F). Additionally.pnas. and young panicles (Fig.1073/pnas. 1H).4 IR64 NIL 0 12 IR64 NIL ** 10 8 6 4 2 0 IR64 NIL 14 I Brown rice 12 10 8 6 4 ** 2 0 IR64 NIL IR64NIL NIL-SPIKE J 700 25 20 *** 15 10 5 0 IR64 NIL 600 Grain yield (g/m2) 2. SPIKE was consistently expressed in several organs (Fig.

Expression is calibrated to the 3. In 50-mm stages of young panicle of IR64 and NIL-SPIKE.5 1. with whiskers showing SEM.1 10-. 3 G and H). 50 μm. including large panicles and broad flag leaves (Fig. 2 mm. and H. 3 C and D). 11. 3 A and B). Significant at *5%. The squares indicate an artifact of gene model prediction.5 0 IR64NIL IR64NILIR64NIL IR64 NIL Enhancing Grain Yield in indica Cultivars Through SPIKE. b. Values are means of three replications. 3.9 0 NIL 1. TSN. >21 mm Young panicle n. 20 cm. Values are means. c) differ significantly. Spikelet number per panicle (G) and flag leaf width (H) of NIL-SPIKE (n = 5) and of amiRNA1 (n = 4) and amiRNA4 transgenic plants (n = 3).5 0. 5 cm. * 2. suggesting increasing TSN and FLW along with expression of SPIKE. Map-based cloning and expression analysis of SPIKE. Recurrent backcrossing to IRRI146 and marker-assisted selection (MAS) produced the IRRI146-SPIKE NIL (Fig.0 0. Because IRRI146-SPIKE has 98% genetic identity to IRRI146.8 2. Results of Tukey–Kramer test for multiple comparisons at the 5% level are shown in C. Concomitantly. S7A). G. (B) Semiquantitative expression analysis of SPIKE in culm. and 21. D. not significant. S9).0 0.5 Flag leaf width (cm) IR64 C Spikelet number per panicle B Spikelet number per panicle A nal1 (loss-of-function) mutant Fn188 similarly showed reduced TSN and FLW relative to its wild type. (C and D) Production of GUS driven by the NIL-SPIKE promoter in cross-sections of crown roots and lateral roots (C) and young panicles (D). D. The 1. Spikelet number per panicle (C) and flag leaf width (D) of IR64 (n = 17) and Ubi:SPIKE plants carrying a single copy (n = 20) and five copies (n = 13). PNAS | December 17. B and F.s. 4 C– E NIL-SPIKE Multi-copies Ubi:SPIKE amiRNA1 IR64 F NIL-SPIKE Multi-copies Ubi:SPIKE amiRNA1 D c 250 200 a b 150 100 50 161 0 IR64 G 100 177 210 Ubi:SPIKE Single Multi a 80 60 b 40 c 20 87 0 NIL 40 19 c 2. transformation of two amiRNA precursors that targeted the first (amiRNA1) and fourth exons (amiRNA4) of SPIKE into NIL-SPIKE to down-regulate SPIKE (Fig.7 1.. (A) Morphologies of IR64 plant and Ubi:SPIKE plant in which SPIKE is overexpressed by the ubiquitin promoter.0 a b 1. 51 | 20433 AGRICULTURAL SCIENCES Fig. 20-. Plants carrying multiple copies had significantly greater TSN and FLW than those with a single copy.5 1. (Scale bars: A and E. S7B) produced transgenic plants with significantly lower TSN and narrower leaves than NIL-SPIKE (Fig.0-kbp region between Ind4 and Ind12.5 1. The candidate gene is indicated in red.4 1. GYS.6 a b b 1. Taichung 65 (Fig. .RM3423 AGT3 RM6909 220 kbp n=7996 2 mm 6 mm 14 mm C E 2. and root of IR64 and NIL-SPIKE (NIL). 2013 | vol. Transgenic analysis for SPIKE through overexpression and gene silencing. leaf 5-mm panicle stage of IR64. Numbers below the map show the number of recombinants. (E) Morphologies of NIL-SPIKE plant and transgenic plant in which SPIKE is silenced by amiRNA. 2. These results demonstrate that SPIKE (allele of Nal1 from tropical japonica) enlarges the panicle and flag leaf in correspondence with expression.s.) (E) Quantitative expression analysis of SPIKE in 3. (Scale bars: C. To evaluate the efficacy of SPIKE at increasing yield in different genetic backgrounds. with whiskers showing SD. leaf.s.2 0.s. 2. n. (B) Panicle structures of IR64 and Ubi:SPIKE. S7 C–E).5 Crown roots Ind4 Ind12 RM17486 RM17487 AGT3 RM3423 10 4 10 5 12 18 kbp ATG Root SPIKE Ubiquitin Relative expression of SPIKE RM5503 Os04g52479 SPIKE Lateral roots TGA Young panicle D IR64 NIL IR64 NIL IR64 NIL IR64 NIL 4L Leaf sheath CEN 4S Leaf B SPIKE Culm A Os04g52500 Os04g52504 Lateral roots n.8 0. IRRI146 (released as “NSIC Rc158” in the Philippines) (24). we introgressed it into a high-yielding indica cultivar. we observed no differences in indoleacetic acid (IAA) biosynthesis or transport between IR64 and NIL-SPIKE (Fig. A significant higher transcript in the event carrying multiple copies of SPIKE cDNA was observed (Fig. 4 A and B). 110 | no. and FLW of IRRI146SPIKE were significantly higher than those of IRRI146 (Fig. Plants carrying a single copy had significantly greater TSN and FLW than IR64 (Fig. n. S8).2 0 IR64 Ubi:SPIKE Single Multi H Flag leaf width (cm) with the ubiquitin promoter (Ubi:SPIKE) was introduced into IR64 by transformation. 3 E and F and Fig. Although Nal1 was reported to relate to auxin polar transport (23).0 1. (F) Panicle structures of NIL-SPIKE and transgenic plants. the pleiotropic effects of SPIKE in IRRI146 were similar to those in NIL-SPIKE. TSN and FLW of T0 Ubi:SPIKE plants increased with copy number (Fig. This result suggests the gene dosage effect of SPIKE on the plant phenotype. (A) A high-resolution map narrowed the SPIKE locus to an 18. The overexpressor transgenic plants showed a similar phenotype to NIL-SPIKE. Fujita et al.) Means labeled with different letters (a.

This improvement might be explained by increased VBN contributing to assimilate supply to spikelets on the secondary branches.abr. many genes for yield-related traits in rice have been isolated on the basis of genome sequence information. panicle neck diameter. 29). This approach opens the way to efficiently identifying genes that increase grain yield. and translocation capacity.4 0 50 0 BR11 BR11 -SPIKE 0 250 150 0. n = 10).0 IRRI146 565 IRRI146SPIKE 200 100 479 0 150 IRRI146-SPIKE IRRI146SPIKE 400 300 200 IRRI146 500 *** IRRI146SPIKE 600 D250 Spikelet number per panicle * IRRI146 Grain yield (g/m2) C700 IRRI146 Flag leaf width (cm) New plant type YP4 * 150 100 0 P=0. which was developed from IL YTH326.) (C–E) Comparison between IRRI146 and IRRI146-SPIKE of grain weight per m2 (C). In this Significant at ***0. among which the alleles from japonica or tropical japonica cultivars increased TSN (30–38). SPIKE notably increased secondary panicle branches as well as Gn1a (7). suggesting that NPT SPIKE is a major factor for increasing grain yield among the ILs (21). n = 27)— characterized in the field at IRRI. however. The introduction of SPIKE into indica elite cultivars gave the desired morphological effects. the grain fertility of NIL-SPIKE was improved compared with that of IR64. grain yield is a complex trait controlled by many genetic factors related to yield components (25. panicle number.A 1 2 3 4 5 6 7 8 9 101112 B 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112 F Popular cultivars in Southeast Asia PSBRc18 (Philippines). The plants homozygous for SPIKE had significantly higher TSN (Fig. (A and B) Gene location (blue ellipses) and plant morphology of New Plant Type cultivar YP4 (A) and IRRI146 and IRRI146-SPIKE (B). SPIKE. source size. Among components of sink size. TDK1 (from Laos. with whiskers showing SE. whereas Fujita et al. . Hitherto. including leaf size.6 0 0 P=0. Discussion During the last decade. Its effects were confirmed on the different genetic background of popular indica cultivars: PSBRc18 (IR51672-62-2-1-1-2-3) from Philippines. no gene for TSN or grain yield in this cluster has been isolated (39).0224 *** 1. n = 17). identified from a mutant line.0002 150 50 Swarna Swarna -SPIKE 50 PSBRc18 -SPIKE 50 250 200 100 Spikelet number 100 E2. To exploit genes for complex traits.0462 Ciherang -SPIKE 200 250 Ciherang * Spikelet number P=0.go. several QTLs for TSN have been detected. 26). increasing TSN. trait-specific ILs that have been developed through backcross breeding with phenotypic selection offer a powerful approach (27). spikelet number.000-grain weight and panicle number. n = 13). 4F) than the recurrent parent. RDW. TDK1 (Laos). where SPIKE was identified. The consequent increase in spikelets on secondary branches usually increases competition for assimilate supply.1073/pnas. 20434 | www. TDK1 from Laos. These morphological effects of SPIKE were mostly corresponding to ideal plant architecture in NPT breeding. Transgenic analysis revealed that SPIKE was identical to Nal1. Many QTLs for morphological traits. and flag leaf width (E) (n = 10).org/cgi/doi/10. Approximately 29% of the ILs had SPIKE. which affects vein patterning in leaves and polar auxin transport (23). BR11 from Bangladesh. E). SPIKE from YP9 was similarly introduced into five popular indica cultivars with different genetic and geographic backgrounds. SPIKE increases grain yield in indica genetic background. The 334 ILs with high yield potential had been selected from backcrossed populations and had a few segments introgressed from NPT sources. and BR11 (from Bangladesh.1%. and grain weight (7–16). BR11 (Bangladesh) 200 1. 4. occur in a cluster on the long arm of chromosome 4 (http://qtaro. is a unique allele from tropical japonica. Swarna (from India. Through selection of backcrossed progeny based on field observation. (Scale bars: 20 cm. Through high-resolution mapping.0002 *** 200 150 100 50 0 250 Spikelet number * 100 0. genetic factors underlying grain yield were effectively detected by using trait-specific ILs with an IR64 genetic background in a new approach that combines breeding selection with genetic analysis (21). However. The selected ILs. SPIKE had pleiotropic effects on several traits. NPT breeding aims at an ideal plant architecture characterized by fewer. Such genes associated with enlarging sink size have been identified by evaluating yield-related traits such as spikelet number. (F) Comparison of spikelet number per panicle between indica cultivars with and without SPIKE—PSBRc18 (from Philippines. identified from natural variation. spikelet number per panicle (D).2 *** 100 P=0.8 50 Popular cultivars in South Asia Swarna (India). Ciherang (from Indonesia. we identified SPIKE. which had large numbers of favorable genes for grain productivity. In our previous study. larger panicles and large leaves (18). stably increased grain yield in the IR64 genetic background at the crop level in the field. is a loss-of-function mutation. It has been unclear whether a single gene could increase grain yield in the genetic backgrounds of different elite cultivars. but decreasing the 1. and no genes have been reported to increase grain yield of indica rice at the crop level in the field (17). and root volume. Philippines. which influences TSN and FLW. Additionally. This study reports a gene that increases grain yield of indica rice through enlarging sink size. tended to have higher grain yields than IR64. FLW.0184 Spikelet number 250 SPIKE IRRI146 YP4 Spikelet number SPIKE Fig.affrc. and VBN. panicle branch number. n = 10).1310790110 NIL-SPIKE. whereas nal1. To our surprise. resulting in abortion (28. Ciherang (Indonesia) 200 150 PSBRc18 TDK1 TDK1 -SPIKE P=0. Ciherang from Indonesia. and Swarna from India. we eliminated unfavorable agronomic traits such as low panicle number and low grain fertility inherited from the NPT sources. The nal1 mutant was reduced in TSN compared with wild type.pnas. Values are means. *5%. **1%.

Fn188 was used for agronomic characterization to compare with the effects of SPIKE.000-grain weight. 51 | 20435 AGRICULTURAL SCIENCES the unique allele from tropical japonica in Nal1 showed increased TSN. and Ciherang) are grown in millions hectares (24. 40). We selected an IL with high TSN: YTH326 (IR84640-11-110-6-4-2-2-4-2-2-3-B). FOSS Analytical) with four replications per line. In each generation. The precursors were ligated into the binary vector pCAMBIA1300int-prUbi1-tNOS to generate the silencing vectors. and expression. and K were applied the day before transplanting. because we considered Nal1 to be same to SPIKE. total RNA of young panicle.weigelworld. TDK1 (Laos). The nal1 locus has been mapped between markers C1100 and C600 on the long arm of chromosome 4 (41). Laguna. and Swarna (India) (24). The amiRNA precursors were generated through site-directed mutagenesis by using overlapping PCR (Table S1) with plasmid pNW55 as a template. A whole-genome survey of 96 BC3F1 plants using 116 polymorphic SSR markers that covered all chromosomes was conducted. which was derived from a cross between indica cultivar Shennung 89-366 and tropical japonica landrace Daringan (Fig. Using Agrobacterium-mediated transformation. and root was extracted at the panicle initiation stage. Indonesia. has been released as “NSIC Rc158” in the Philippines (24). The transgenic plants (T0) were transplanted into pots. leaf sheath. we identified qTSN4. As a basal dressing. This vector was introduced into IR64 by Agrobacterium-mediated transformation (42). Selected 41 BC 4F3 plants that occurred with recombination between RM3423 and AGT3 were self-pollinated to generate BC4F4 lines to be used for a progeny test. IRRI146. S9) by gas chromatography– selected ion monitoring–mass spectroscopy (GC-SIM-MS) as described (45. SPIKE was a unique isolated allele for grain productivity from natural variation and located on the QTL cluster region of chromosome 4. PN. we applied 3 μM IAA to the top of coleoptile sections (1. and IRRI146SPIKE in a randomized plot with four replications per line. Using a BC4F2 population derived from a cross between IR64 and YTH326. Plants homozygous for SPIKE were selected from each BC2F2 population and evaluated for TSN in a field at IRRI. SPIKE would increase grain yields of further indica cultivars in South and Southeast Asia through MAS breeding. and evaluated for 1. the grain yield of NIL-SPIKE was increased as a To identify a candidate gene for SPIKE. MAS was conducted by using SPIKE-flanking markers RM5503 and RM6909. The panicle rachis was sectioned Fujita et al. PNAS | December 17. GYS was calculated on a 14% moisture content webapp. 30 kg/ha each of N. for the fourth exon)—were designed by using Web MicroRNA Designer 3 software (http://wmd3. NIL-SPIKE. the Philippines. and plants were dried in an oven at 70 °C for 5 d. BR11. Progeny of the cross between YP9 and each cultivar were backcrossed to the popular cultivar twice. Progeny of a cross between NPT IR65564-22-2-3 from tropical japonica Bali Ontjer and IRRI146 were backcrossed to IRRI146 three times. three plants were transplanted per hill at 21 d after sowing at 20 cm between hills and 25 cm between rows. Through backcross breeding. The genomic DNA of 1. between SSR markers RM3423 and RM17492 on the long arm of chromosome 4 (22). thus contributing to food security in these regions. To confirm effects of SPIKE in the broad area.0 m2 of rice plants (20 hills in each plot) was harvested. Twenty-two DNA markers were used for map construction (Table S1). in the genetic background of indica cultivar IR64 (21). transformation.nbrp. The fact suggested that the activity of auxin transport at panicle initiation stage might be related to TSN.8 m2. S1). . Using Agrobacterium-mediated transformation. At maturity. confirming its effectiveness in various genetic backgrounds. This IRRI146-SPIKE was compared with the recurrent parent for agronomic traits and grain yield. Additionally. homozygous plants from representative recombinants were selected and evaluated for TSN and FLW. France) between the maize ubiquitin promoter and the nopaline synthase terminator to generate the overexpression vector. Phenotypic Evaluation of SPIKE. Through increase in TSN.0 mm) for 30 min. Therefore. IRRI146 (IR77186-122-2-2-3). 46). Laos. Transformation of SPIKE. The fragment was ligated into the binary vector pCAMBIA1300int-prUbi1-tNOS (provided by Emmanuel Guiderdoni. Among the BC4F4 lines. was provided by Kyushu University under the National Bioresource Project (www. and T1 plants were transplanted in a screenhouse at 20 cm between hills and 30 cm between rows. To evaluate grain yield. Ubiquitin (Os01g22490). NIL-SPIKE was developed by self-pollination of a plant selected from the BC4F2 population and was used for evaluating agronomic traits. Expression Analysis and IAA Transport. PSBRc18. MAS was conducted by using the SPIKE-flanking markers Ind2 and RM17487. These plants were evaluated for TSN and FLW. The genomic DNA of 7996 BC4F3 plants generated from BC4F2 plants heterozygous for SPIKE was extracted from fresh leaves by a simple method. Ciherang (Indonesia). The regenerated plants were evaluated for transgene copy numbers by Southern blot analysis. for high TSN. we grew IR64. which have variation in agronomic traits inherited from NPT cultivars. Development of indica Cultivars with SPIKE. Organs of the regenerated plants were sampled to analyze GUS activity as described (44). RDW of plants that were grown in pots was measured at maturity. and VBN were counted under a stereomicroscope. culm. The improvement of grain yield of these cultivars is an important breeding objective in this region.5–3. One BC3F1 plant was selected and self-pollinated to develop a NIL for SPIKE in the IRRI146 genetic background. we introduced it into five indica cultivars popular in South and Southeast Asian countries and some of them (Swarna. Development of IRRI146-SPIKE. and TSN at maturity as described (21). for the first exon of SPIKE) and amiRNA4 (TTAATATCAAGTTCCAGACGC. leaf. Grain chalkiness was evaluated with a Grain Inspector (Cervitec 1625 Grain Inspector. In each generation.918-bp fragment upstream from the ATG codon of SPIKE by using primer pair UP6-1. The area of each plot was at least 4. A fragment encompassing the full-length coding region of SPIKE was amplified from cDNA derived from young panicles of NIL-SPIKE by using primer pair 8M17-c1. The rate of IAA biosynthesis in IR64 and NIL-SPIKE coleoptiles was investigated by measuring the amount of IAA that was transported from cut coleoptiles to an agar block (described in Fig. PCR was performed by using 1 μL of cDNA with the gene-specific primers for each candidate (Table S1). The data were normalized to the expression of a house hold gene. 1. BR11 (Bangladesh). 2013 | vol. P. we amplified a 1. SPIKE increased TSN in all of these cultivars. For gene silencing of SPIKE. qRT-PCR reactions were carried out with one-fifth cDNA mixtures by using primer pair seq8M17-56 with LightCycler 480 SYBR Green I Master Mix on a LightCycler 480 System (Roche Applied Science). as described (43). SPIKE was introgressed into five popular cultivars through backcrossing and MAS: PSBRc18 (IR51672-62-2-1-12-3) (Philippines).Materials and Methods Plant Materials. High-Resolution Linkage Map. carrying nal1. Two 21-bp amiRNA sequences—amiRNA1 (TATAAGAAGTATGCTGCGCTA. derived from NPT cultivar YP9 (IR68522-10-2-2).073 BC4F3 plants with recombination between flanking markers RM17450 and RM3836 was individually extracted from freeze-dried leaves by the cetyl trimethylammonium bromide method. Total RNA from each organ was extracted by using an RNeasy Plant Mini Kit (Qiagen). SPIKE would increase grain yield in all popular cultivars because at least TSN in these popular cultivars were increased by SPIKE. The amplified fragment was ligated into the binary vector pCAMBIA0380 (Cambia) upstream of the GUS reporter gene. In future study. RT-PCR was performed with 500 ng of total RNA by using primer pair seq8M17-56 and a ReverTra Ace qPCR RT Kit (Toyobo). Fn188 had been developed from BC3 progeny derived from a cross between an nal1 mutant as the donor parent and japonica cultivar Taichung 65 as the recurrent parent. the amiRNA approach was used (43). All plants were grown in a field at IRRI. Centre de Coopération Internationale en Recherche Agronomique pour le Développement. To generate the promoter:GUS vector. at 1 cm below the neck. and 30 kg/ha of N was applied twice as a topdressing at 2 and 4 wk after transplanting. For comparison of expression in different organs. then incubated the coleoptiles on an agar block for 10 min and measured the transported IAA by GC-SIM-MS as above. the breeding lines with SPIKE needs to be characterized at different locations and management levels for understanding increase of yield potential in field. To investigate polar IAA transport in IR64 and NIL-SPIKE coleoptiles. A high-yielding indica cultivar. 110 | no. we introduced the vector into IR64 (42). we developed 334 BC3-derived ILs. To understand the effect of SPIKE in different genetic backgrounds. FLW. multienvironment trials using NIL-SPIKE are ongoing in the Philippines.cgi). we performed RT-PCR by using 1 μg of total RNA. Montpellier. and subtropical Japan. The effect of a gene is generally influenced by both the genetic background and the environment. Los Baños. we introduced the vectors into NILSPIKE (42). Line Fn188.

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