Breeding Science 56 : 405–413 (2006)

Note

Development of Isogenic Lines of Koshihikari with Early and Late Heading by Marker-assisted Selection

Yoshinobu Takeuchi1,4), Takeshi Ebitani2,5), Toshio Yamamoto1,2), Hiroyuki Sato3), Hisatoshi Ohta3), Hideyuki Hirabayashi3), Hiroshi Kato3), Ikuo Ando3), Hiroshi Nemoto3), Tokio Imbe3) and Masahiro Yano*2)

1) Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, 2) National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 3) National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan 4) Present address: National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan 5) Present address: Toyama Agricultural Research Center, 1124-1 Yoshioka, Toyama, Toyama 939-8153, Japan

Key Words: L., heading date, quantitative trait locus, pleiotropic effect, linkage drag, isogenic line, backcross breeding.

The cultivar Koshihikari is cultivated in (Yamamoto et al. 2001, Ebitani et al. 2005). western and central Japan (630,000 ha in 2004, or about These four QTLs have been mapped as single Mende- 37.1% of rice cultivation area) (The Ministry of Agriculture lian factors by using populations derived from crosses be- Forestry and Fisheries of Japan 2005). It has been character- tween Nipponbare and Kasalath (Yamamoto et al. 1998, ized by good eating quality and cool-temperature tolerance Yamamoto et al. 2000, Lin et al. 2002, 2003). Among them, at the booting stage. In addition, Koshihikari in the market Hd1, Hd6, and Hd5 have been isolated by map-based clon- has been accepted security of a stable supply, brand recogni- ing (Yano et al. 2000, Takahashi et al. 2001, Yamanouchi et tion by consumers, and advantageous prices. Koshihikari al. 2005). Those studies provide information about the pre- has been a leading cultivar for the last three decades, and it cise position of target QTLs for heading date and their tight- has been extensively used as a parental line to develop new ly linked DNA markers. This has allowed us to embark on rice because of its favorable qualities. However, developing isogenic lines (ILs) of Koshihikari with modified because of its heading and maturity date, commercial culti- heading date. vation of Koshihikari has been limited to western and central In this study, to facilitate the selection of plants in Japan. Modification of heading date toward early and late which recombination occurred near the target heading date heading is one of breeding objectives to enhance the crop- QTLs, we developed several PCR-based DNA markers. Us- ping potential of Koshihikari. ing these markers and marker-assisted selection (MAS), we Heading date is a typical quantitative trait in rice. Many developed four isogenic lines (ILs) of Koshihikari with dif- quantitative trait loci (QTLs) for heading date have been ferent heading dates, Wakei 367 (Hd6), Kanto IL1 (Hd1), identified (Li et al. 1995, Xiao et al. 1995, 1996, Lin et al. Wakei 370 (Hd4), and Wakei 371 (Hd5). In addition, we 1996, Yano et al. 1997, Lin et al. 1998, Yano et al. 2001, compared yield and several morphological and physiologi- Hittalmani et al. 2003, Mei et al. 2005). In previous studies, cal characteristics, such as culm length, panicle length, and three QTLs—qDTH3, qDTH6, and qDTH8—were identi- panicle number, grain and eating quality, cool-temperature fied on chromosomes 3, 6, and 8, respectively, in cross of tolerance, and field resistance to leaf blast, which are the Koshihikari and Kasalath (Yamamoto et al. 2001). Recently, most important characteristics in rice breeding in Japan, of an additional putative QTL for heading date, tentatively des- these ILs with the isogenic control, Koshihikari. ignated qDTH7, was identified on chromosome 7 using chromosome segment substitution lines (Ebitani et al. 2005). Development of ILs Their chromosomal locations suggest that those QTLs corre- The scheme for developing ILs for heading date is shown spond with QTLs Hd6, Hd1, Hd5, and Hd4, which have in Figure 1. A japonica cultivar Koshihikari was crossed been detected in crosses of Nipponbare and Kasalath with an indica cultivar Kasalath, and the resulting F1 plants were backcrossed to Koshihikari to obtain BC1F1 plants. Communicated by K. Okuno These plants were self-pollinated to develop 187 BC1F3 lines Received June 23, 2006. Accepted August 22, 2006. by the single-seed-descent method (Yamamoto et al. 2001). *Corresponding author (e-mail: [email protected]) To develop ILs for each QTL, we selected four suitable 406 Takeuchi, Ebitani, Yamamoto, Sato, Ohta, Hirabayashi, Kato, Ando, Nemoto, Imbe and Yano

site (STS), cleaved amplified polymorphic sequence (CAPS), derived CAPS (dCAPS), and simple sequence re- peat (SSR) markers—were used to determine the genotype of each plant. RFLP analysis was performed according to the methods of Kurata et al. (1994). Total DNA of plants was extracted from leaves by the CTAB method (Murray and Thompson 1980). A total of 116 RFLP markers covering the 12 rice chromosomes were selected from a high-density ge- netic map (Harushima et al. 1998). STS, CAPS, and dCAPS analysis were performed ac- cording to the methods of Lin et al. (2002). In the case of STS markers, amplified product was electrophoresed in 3.0% agarose gel to determine genotype classes. In the cases of CAPS and dCAPS markers, the amplified products were digested with an appropriate restriction enzyme and then electrophoresed in 2.5% or 4.0% agarose gel. The primer se- quences and restriction enzymes used in PCR analysis are shown in Table 1. For SSR analysis, the reaction mixture consisted of 1 µL template DNA, 0.7 µL 10× PCR buffer (Promega, Madi- son, WI, USA), 0.4 µL 25 mM MgCl2, 0.7 µL 2 mM each dNTP (Boehringer Mannheim), 0.1 µL 5 U Taq DNA poly- merase (Promega), 0.3 µL 20-pM solutions of each primer, and 2.8 µL H2O in a 6.0-µL volume. Amplification was per- formed for 30 cycles of 94°C (1 min), 55°C (2 min), and 72°C (3 min), and a final cycle of 72°C for 7 min. Amplified DNA product was electrophoresed in 3.5% agarose gel. We used five SSR markers (previously developed) located near Fig. 1. Scheme for the development of isogenic lines of Koshihikari. target QTL regions (Temnykh et al. 2001, McCouch et al. 2002; Table 1). In addition, four SSR markers (P0456CT1, P0456GC1, OS0040CT1, and OS1590CCT1) were newly BC1F3 lines in which a recombination had occurred on one developed from sequence data of three PAC clones: side of the target QTL according to genotypes of 116 restric- P0456E06 on chromosome 6, OSJNBa0040E01 on chromo- tion fragment length polymorphisms (RFLPs) (Yamamoto some 3, and OJ1590E05 on chromosome 8, respectively et al. 2001). We successively backcrossed these plants with (Table 1). Sequence data were obtained from the IRGSP data Koshihikari and used MAS to select desirable plants in of the Rice Genome Research Program (2005; http:// which recurrent chromosome segments could be recovered rgp.dna.affrc.go.jp/IRGSP/). as possible as they were (negative selection) and the chro- mosomal region including the target QTL was maintained as Genotype survey for ILs selected heterozygous (positive selection). Four resulting secondary Using 28 PCR-based DNA markers, such as STS, backcross progeny (SBC1F1) were self-pollinated to obtain CAPS, dCAPS and SSR (Table 1), recombination points four SBC1F2. MAS was conducted to select appropriate were precisely determined. Recombination were occurred plants for further development of ILs. One of selected between markers C10915 and Y4836L and between SBC1F2 plants was self-pollinated for develop Kanto IL1 P0456CT1 and P0456GC1 in Kanto IL1 (Fig. 2B); between (Hd1). On the other hand, additional backcrossing with OS0040CT1 and RM416 and between cnt13I and G1015 in Koshihikari was performed on other three SBC1F2 plants to Wakei 367 (Fig. 2C); between E4071 and R46 and between produce three SBC2F1 lines. We selected plants in which RM6449 and RM5481-1 in Wakei 370 (Fig. 2D); and be- recombination occurred in the flanking region (other side tween RM8266 and OS1590CCT1 and between R902 and of first recombination) of the target QTLs. As results, one RM1111 in Wakei 371 (Fig. 2E). If we assume that recombi- SBC1F2 (with Hd1) and three SBC2F2 (with Hd6, Hd4, and nation occurred in the middle of each marker interval, then Hd5, respectively) plants containing a minimum chromo- the substituted genomic fragments was approximately 560 somal segment containing each target QTL were selected. kb in Kanto IL1, 170 kb in Wakei 367, 7960 kb in Wakei Additional self-pollinations were required to select ILs. 370, and 625 kb in Wakei 371, respectively. In order to determine the genotype of background ge- DNA marker analysis nome in each candidate IL, we used 116 RFLP markers Five kinds of DNA markers—RFLP, sequence tagged (Harushima et al. 1998). As results, no introgression of Isogenic lines of Koshihikari with early and late heading 407 (2001) (2001) (2002) (2002) (2002) (2002) (2002) (2001) et al. et al. et al. et al. et al. et al. et al. et al. et al. 3) (2002) (2003) (2003) (2003) et al. et al. et al. et al. Wu Lin McCouch Temnykh Takahashi Takahashi unpublished data Takahashi Lin McCouch McCouch McCouch Lin McCouch ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ AAG TGC ATA ACC A 3 T AGA TGC CAT TC 3 STS and SSR markers. CAA AAT GAC AA 3 TAT CAG TT 3 TCA TAT ACG A 3 C TAA GCA AG 3 CA CTG CTT CG 3 primer sequence Reference GTA GTT GGG CTG ATG GCC TTT CAC CTG AC 3 TAC GCT GAC GAT CAA GTA GAG TGA CAC CTC GCT ACT TCT G 3 CTC TCG CTG ACA GGT GGG AC 3 AGT AGC ACA ACG CAT TC 3 AAC TTG GGT ATC TTT ATG CAG 3 TGC TAC CTC AAC TAA TGG AA 3 GAC CCT TTC TGG AAC AG 3 GGC GAC CGG CGA ATT CAA AC 3 TCC AGT TTC A TCG GCC ATC CGC TGA TTT GG 3 CTT GCA ATT GG AGT CAC TTT CAA ATG ACG TAC CAA AAT ATC GG 3 CAG TTC ACA AAG CCA TCA AT 3 TTA TCC AAA CGT CCG AAG GG 3 GCG TAG GGA CTA GAG GG 3 CTT TCT TAA TGA AGC TCC AA 3 GCT CCA TCA CAA AGG GTA GT 3 GGT GAA GCA TCC AT 3 ACA AGT AAC GGG TGC TTA 3 CAG CTG GCC GTC GCG GAC 3 ACA GAC ATA ACG AAA TGC CC 3 TTC CTT CAA TTA TG CTT ATC CAC TTG CCC TCT CG 3 TAT CAA TCC CTT GGC TGG AA 3 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 . No digestion is needed for ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ GAA GTT GC 3 T CAT CCA TC 3 between Koshihikari and Kasalath obtained at http://rgp.dna.affrc.go.jp. primer sequence 3 CTC ATC TAG CTT TCT GCC T 3 TGG AGT GAA GGT ACT GAT CCC CCG ATG GTG TTC CG 3 GGA CTG AGA TGG AAT GTG CT 3 GAC TCG CAT ATA ATC CCT CG 3 CTT TAA TGC GCG CGG GGA CG 3 CTA CAA GAG TAC GCA AAG CG 3 GCC AGG GAA CTT TCA TCT C 3 CAA GGC TAC CCA AGG AAC AA 3 GTG CTC CGT AGG ATC TC 3 TGT GTT GCA CGC ATG CGA GC 3 GGG AGT TAG GGT TTT GGA GC 3 TCG GTG GAA CGA ACA AGT GC 3 CTG CAA GTT GCC GAT CA 3 CTC TAA GCC GGT ACA GGG 3 GCA GGG ATT AGC AGG ATG TC 3 TGA GAG AAA AAG GAA CAG GC 3 TCG TTT CAG TTC TCT TGC AC 3 CAA GAG CAG CTT GAC AG 3 TGT TAT GTG AGC CAT AAT GA 3 AGT AAG ATT GGT GAC GGA AG 3 CTG GTG AAG GAG AGG TCG TC 3 AAT AAG AGT CCA CAC ATG 3 GGC GTA CTT GCG CAT GGT C 3 CAA CCC AAA GA TCA CGA AAG AAC ACA TTA TTT ATC C 3 CCT GTC GGA TCT GGT AG 3 ATG ACA TCC ATC ACT GG 3 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 were revealed by STS, CAPS, dCAPS or SSR. Amplified loping ILs of the heading QTLs ithout particular reference can be band size (bp) in Koshihikari I3000 2) Mva I2000 I2000 I550 I1300 III 2000 I1500 RI 4300 81I 120 I700 I180 I2000 I100 I1500 I, Mvo Eco Mbo Sca Eco Aci Pst Rsa Hae Kpn Afa Pst Xba Kpn Enzyme 1) Type of marker PCR-based markers used for deve 1.

Polymorphism between Koshihikari and Kasalath Restriction enzymes used for CAPS and dCAPS to detect polymorphism Information for PCR-based markers w 1) 2) 3) Table C10915S2539 STSP0456GC1R2123 CAPS SSR CAPS R2462RM5995 220 CAPS RM416 SSR 150 SSRE4071 250 C492 STS 150 CAPS RM8266R2976 SSR 230 R902 STS dCAPS 270 1000 Y4836LP0456CT1 dCAPS SSROS0040CT1 SSR 200 170 RM6449RM5481-1R646 SSR SSR STSOS1590CCT1 SSRRM1111 250 S1461 230 SSR 600 CAPS 150 230 cnt13IG1015 CAPS CAPS C39 CAPS R2401 CAPS R2171 CAPS C944 CAPS S21656 CAPS Marker Marker used for developing Wakei 370 Marker used for developing Kanto IL1 Marker used for developing Wakei 367 Marker used for developing Wakei 371 408 Takeuchi, Ebitani, Yamamoto, Sato, Ohta, Hirabayashi, Kato, Ando, Nemoto, Imbe and Yano

Fig. 2. Graphical representation of the genotypes of Kanto IL1, Wakei 370, Wakei 367, and Wakei 371. A: QTLs for heading date detected in previous studies (Yamamoto et al. 2000, Ebitani et al. 2005) are indicated by triangles. B–E: Graphical representations of each genotype. In each IL, white and black blocks indicate chromosomal regions derived from Koshihikari and Kasalath, respectively. RFLP, STS, CAPS, dCAPS, and SSR markers on the linkage map and physical map are shown on the right. PCR-based STS, CAPS, dCAPS, and SSR markers are indicated in bold letters. The positions of markers were derived from a high-density RFLP linkage map (Harushima et al. 1998). chromosome segment from donor variety, Kasalath, was de- Evaluation of ILs for heading date and other traits with ag- tected at marker points. Although we cannot rule out the ronomic value possibility that a tiny Kasalath chromosome segment was Kanto IL1, Wakei 367, Wakei 370, and Wakei 371 integrated between the RFLP markers, introgression of were cultivated in a paddy field at the National Agricultural Kasalath chromosome segment was not likely to be occurred Research Center, Yawara, Tsukuba, Ibaraki, Japan, in 2003 in non-target regions. and 2004. These lines were sown on 18 April 2003 and 22 Isogenic lines of Koshihikari with early and late heading 409

April 2004 and transplanted on 14 and 18 May, respectively. We also investigated the eating quality of the four ILs We raised 128 plants per plot with two replications. For over two years. The eating quality of Kanto IL1 was inferior heading date, maturity date, culm length, panicle length, to that of Koshihikari in 2003 but not in 2004 (Table 2). In number of panicles per plant, grain weight, yield of brown other three ILs, no clear difference between three ILs and rice, grain quality, and eating quality, evaluation methods Koshihikari was observed for eating quality in both years. and criteria for classification followed Yamamoto et al. Field resistance to leaf blast was assessed in an upland (1996). The date when 50% of all panicles of each line had field at the National Agricultural Research Center, Kannon- emerged from the flag leaf sheath was recorded as heading dai, Tsukuba, Ibaraki, in 2003 and 2004. Seeds of four ILs date. Days-to-heading (DTH) was measured as the number and three reference cultivars—Koshihikari (susceptible), of days from sowing to heading date. Maturity was recorded Koganenishiki (resistant), and Nipponbare (susceptible)— as the date when the most of seeds in all panicles had be- were sown in blast nursery beds on 5 June 2003 and 10 June come brown. Culm length, panicle length, and number of 2004, respectively. The ILs and reference cultivars were panicles were also measured 10 randomly selected plants in grown with two replications; blast severity was determined each IL and reference cultivars. Grain weight, yield of by eye observation and scored from 0 (no lesions) to 10 , grain quality, and eating quality were measured (leaves totally dead) (Yamamoto et al. 1996). Field resis- in bulks of 50 plants. Trait data were measured in both plots tance to leaf blast in four ILs was almost the same as that of of each line, and their means were compared with that of Koshihikari (Table 3). Therefore, the ILs appears to possess Koshihikari by t-test. These traits were also compared with no major gene for resistance to leaf blast. those of japonica cultivars Koshihikari, Akitakomachi, Cool-temperature tolerance of ILs at booting stage was Asanohikari, and Nipponbare. also tested in running cold-water paddy fields at the Miyagi DTH of Koshihikari was 107 days. That of Kanto IL1 Prefecture Furukawa Agricultural Experiment Station, Furu- was 95 days, 12 days earlier than that of Koshihikari; that of kawa, Miyagi, Japan and the Nagano Prefecture Haramura Wakei 367 was 117 days, 10 days later; and that of Wakei Agricultural Experiment Station, Suwa, Nagano, Japan in 371 was 118 days, 11 days later (Fig. 3 and Table 2). Al- 2003. ILs and four reference cultivars—Chubo 35 (highly though there is no significant difference in mean value, in tolerant), Mutsunishiki (moderately tolerant), Koshihikari both years 2003 and 2004, DTH of Wakei 370 was consis- (highly tolerant), and Hourei (tolerant)—were sown on 17 tently increased by 2 and 3 days than that of Koshhikari, re- April 2003 and 16 April 2003 and transplanted in running spectively. cold-water paddy field on 15 May 2003 and 27 May 2003, Most of morphological traits of Kanto IL1, including respectively. ILs and four cultivars were irrigated with cool ripening period, panicle number, 1000-grain weight, appear- water (about 19°C) for about 2 months from early July ance grain quality, and lodging degree, were the same as (young panicle formation stage of the early lines) to early those of Koshihikari (Table 2). However, culm length was September (heading of the late lines). At maturity, the de- shorter than that of Koshihikari, which may be resulted in gree of cool-temperature tolerance of each line was mea- slight reduction of yield in brown rice. Among the other sured by floret sterility of nine (in Miyagi) or six panicles (in three lines, all traits tested, including culm length, were al- Nagano) from three plants (three or two panicles per plant) most the same as those of Koshihikari. (Yamamoto et al. 1996). The floret sterility of each IL was compared with those of reference cultivars with different heading date. Cool-temperature tolerance at booting stage of Kanto IL1 was similar to that of Mutsunishiki and was infe- rior to that of Chubo 35 (Table 4). This result suggests that cool-temperature tolerance of Kanto IL1 is inferior to that of Koshihikari. Cool-temperature tolerance of the other ILs was similar to that of Hourei and Koshihikari.

Association between early heading date and other traits Phenotypic performances of ILs developed in this study were almost as same as those of Koshihikari. However, culm length and cool-temperature tolerance of Kanto IL1 were modified as well as DTH (Table 2 and Table 4). A shorter culm length may improve lodging resistance, but may be as- sociated with less potential yield and shorter growth dura- tion. Although we could not define the genetic basis of such modifications, we can propose two possibilities. First, the Fig. 3. Four ILs and their isogenic control, Koshihikari. A: Kanto IL1 Kasalath Hd1 allele affected not only DTH, but also other (Hd1); B: Koshihikari; C: Wakei 370 (Hd4); D: Wakei 367 traits, such as culm and panicle length, through pleiotropy. (Hd6); E: Wakei 371 (Hd5). Second, other tightly linked genes might be involved in the 410 Takeuchi, Ebitani, Yamamoto, Sato, Ohta, Hirabayashi, Kato, Ando, Nemoto, Imbe and Yano 3) 0.35 1.48** 0.41 0.91** 0.04 0.17 0.17 0.68 0.09 1.26** − − − − − − − − − − Eating quality 2) degree Lodging variety. ** showed signifi- 1) grain quality Appearance i was used as the standard weight (g) weight 1,000-grain good to 9: especially bad). (kg/a) Yield of brown rice ) 2 No. of No. panicles (No./m grade (1: excellent 5: especially bad). Koshihikar − Panicle length (cm) classified into nine 8** 18.7 367** 44.6 21.2** 3.0** 0.0** (cm) nd their isogenic control, Koshihikari ee (5: excellent good to Culm length Ripening period (days) ed into eleven degr Maturing date (m.d) Wakei 370, 367, 371, a mparison with ordinary rice varieties and (days) heading Days-to- degree (0: standing to 9: lodged). uation and was classifi Heading date (m.d) e 1% level, respectively. 2004Average 7.25 7.28 94 99 9.08 9.08 46 42 90.3 84.2 19.2 18.4 408** 388 59.6 53.5 21.4 20.7 4.3 3.8 8.0 4.0 0.09 2004Average 8.01 8.06 101 107 9.09 9.14 39 40 100.8 94.8 18.7 18.6 498 413 64.3 56.6 21.0 20.6 4.9 4.5 9.0 5.5 0.09 2004Average 8.04 8.082004Average 104 110 8.12 8.152004 9.15 9.19Average 112** 117 8.11 8.16 42 42 9.23 9.26 111** 118 95.4 91.2 43 43 9.22 9.27 19.7 18.9 99.9 96.9 42 42 426 388 19.5 102.0 18.9 95.7 62.3 57.3 430 18.9 389 18.8 21.4** 21.2 60.7 432 57.3 6.0 399 5.0 20.5 60.8 20.8 9.0 57.4 6.1** 5.5 21.4 5.3 21.1 0.48** 8.0 0.16 5.3 5.3 4.5 0.30 9.0 0.20 6.0 0.22 0.13 Average 7.24 95 8.28 35 81.6 16.8 416 54.1 21.1 4.9 5.0 2004 7.20 89** 8.25 36 88.8** 17.0** 484 60.4** 21.6 5.7 9.0 Agronomic characteristics of Kanto IL1, 2.

Appearance grain quality was estimated in co Lodging degree was classified into ten Eating quality show the aggregate eval cant differences at th 1) 2) 3) Line Year Nipponbare 2004 8.12 112** 9.22 41 82.1 17.8 420 59.0 22.4 3.7 0.0** Asanohikari 2003 8.13 117 9.20 39 71. Akitakomachi 2003 7.31 104** 9.07 38 78.5** 17.5 367** 47.3 20.0 3.3** 0.0** Wakei 367 2003Wakei 371 8.18 2003 122**Koshihikari 8.21 2003 9.29 125** 8.10 43 10.02 114 93.9 42 9.18 18.4 89.4 40 349 18.6 54.0** 88.7 367** 21.0** 18.5 54.1 4.4 328 20.8** 2.5 3.6 48.8 0.09 3.0 20.1 0.04 4.1 2.0 Wakei 370 2003 8.12 116 9.23 42 87.0 18.1 350 52.4 21.0 4.0 2.0 Table Kanto IL1 2003 7.28 101** 8.31 34 74.4** 16.7 348 47.8 20.6 4.0 1.0 Isogenic lines of Koshihikari with early and late heading 411 modification of those traits, through linkage drag. Kawai Table 4. Cool-temperature tolerance of Kanto IL1, Wakei 370, and Sato (1969) reported that most early heading mutant Wakei 367, Wakei 371, and their isogenic control, strains had short culms. In addition, Li et al. (1995) and Yu Koshihikari et al. (2002) found a significant positive correlation between Miyagi Pref. Furukawa Nagano Pref. Haramura heading date and plant height. Using progeny of the same Agr. Exp. Sta. Agr. Sta. Line cross combination as used in this study, Yamamoto et al. Heading date Floret Heading date Floret (2001) identified a QTL controlling internode length in the (m.d) sterility (%) (m.d) sterility (%) same region as Hd1. Thus, it is likely that an early heading Kanto IL1 8.10 89.2 8.21 84.5 gene at Hd1 has pleiotropic effects on culm length. Even Wakei 370 9.06 60.2 9.01 55.3 though we tried to minimize the length of the introgressed Wakei 367 9.09 46.1 8.30 53.7 chromosome segment from the donor parent in the target Wakei 371 9.15 69.9 9.09 32.0 QTL region, a region of about 560 kb was substituted in Chubo 35 8.05 61.8 8.21 44.8 Kanto IL1. This small segment contains about 40 genes Mutsunishiki 8.07 94.7 (GRAMENE 2006). Thus, it is difficult to rule out the possi- Koshihikari 9.02 52.3 8.28 56.3 bility of two tightly linked genes. Further analysis such as Hourei 9.07 56.2 fine mapping will be required to clarify the allelic relation- ship between heading date and culm length. In general, eating quality of rice is largely affected by In general, the degree of cool-temperature tolerance of environmental conditions, in particular, temperature during particular cultivars or lines should be evaluated by compari- ripening period (Matsue et al. 1991). Thus, modification of son with cultivars with the same heading date. Thus, it is dif- heading date may affect on eating quality. Kanto IL1 was in- ficult to compare directly the cool-temperature tolerance of ferior in eating quality compared with that of Koshihikari in Koshihikari with that of early-heading Kanto IL1. However, 2003, but not in 2004 (Table 2). In fact, the average temper- in fact, it was very clear that the cool-temperature tolerance ature of the ripening period was significantly cooler in 2003 of Kanto IL1 was inferior to that of Koshihikari (Table 4). than in 2004, and the temperature was abnormally low from There are three possibilities for this association between the end of July to the middle of August 2003. This low tem- DTH and cool-temperature tolerance; (1) pleiotropic effect perature might be resulted in the lower eating quality of of the Kasalath allele at Hd1, (2) effect of other gene, that is Kanto IL1. Thus, it will be necessary to perform additional linked to Hd1, for cool-temperature tolerance (linkage drag) investigations at different sites and under different condi- and (3) effect of other genes in background genome. Graph- tions to define the eating quality of Kanto IL1. ical genotype of each IL was determined by using 116 RFLP markers covering the 12 rice chromosomes. Based on the re- Potential utility of ILs developed sults, we recognized that only one heading QTL region was IL is developed to introduce a small chromosomal seg- substituted with Kasalath in the genetic background of ment containing a favorable gene from a donor cultivar into Koshihikari in each IL. However, it is difficult to exclude the an elite cultivar. Since the development of DNA markers as possibility that tiny chromosome segments of Kasalath were tools for mapping major genes, such as those for disease and substituted in non-target regions. Further analysis, such as insect resistance, and QTLs, MAS has been used to develop QTL analysis and further fine mapping will be required us- new cultivars with particular traits of interest (Tanksley et ing progeny derived from a cross between Kanto IL1 and al. 1989). However, few examples of MAS have been re- Koshihikari, to verify three possibilities above mentioned. ported in rice so far, being limited to bacterial blight resis- tance, blast resistance, plant height, and seed number per panicle (Singh et al. 2001, Hayashi et al. 2004, Sugiura et al. Table 3. Field resistance to leaf blast in Kanto IL1, Wakei 370, 2004, Ashikari et al. 2005, Wang et al. 2005). Wakei 367, Wakei 371, and their isogenic control, In this study, to enhance the utility of Koshihikari by Koshihikari modifying heading date to extend its cultivation area, we Severity Score1) used MAS to develop ILs with early and late heading dates. Line 2003 2004 As results, we successfully developed three ILs—Kanto IL1, Wakei 367, and Wakei 371—with a very small Kasalath Kanto IL1 4.8 4.7 chromosome segment (170–625 kb) including a heading- Wakei 370 4.5 4.0 Wakei 367 4.2 6.0 date QTL in the genetic background of Koshihikari by MAS. Wakei 371 4.8 6.3 Wakei 370, had a larger integrated chromosome segment of Koshihikari 4.0 5.5 Kasalath than the other ILs. Because Hd4 has only been rough- Koganenishiki 3.2 4.5 ly mapped on chromosome 7, we could not design any DNA Nipponbare 5.0 5.0 marker that was tightly linked to the QTL. This prevented us 1) The disease severity of 40–50 day old plants was scored using a from selecting plants with recombination near Hd4. We rating from 0 (no lesions) to 10 (leaves totally dead) based on demonstrated that DNA markers tightly linked to the QTLs the the diseased area. for heading date were very effective tools for minimizing 412 Takeuchi, Ebitani, Yamamoto, Sato, Ohta, Hirabayashi, Kato, Ando, Nemoto, Imbe and Yano the length of substituted chromosome segments. Ebitani, T., Y. Takeuchi, Y. Nonoue, T. Yamamoto, K. Takeuchi and It is very important to maintain the same phenotypic M.Yano (2005) Construction and evaluation of chromosome performance of ILs as that of Koshihikari. Independent of segment substitution lines carrying overlapping chromosome indica the size of introgressed chromosome segments, Wakei 367, segments of rice cultivar ‘Kasalath’ in a genetic back- japonica Wakei 370, and Wakei 371 showed almost the same pheno- ground of elite cultivar ‘Koshihikari’. Breed. Sci. 55: 65–73. typic performance as Koshihikari. These ILs will be very GRAMENE (2006) Oryza_sativa. URL:http://www.gramene.org/ important materials for breeding new cultivars to achieve Oryza_sativa/. several practical objectives, such as expanding the northern Harushima, Y., M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki, limit of cultivation of Koshihikari, avoiding labor concentra- T. Yamamoto, S.Y. Lin, B.A. Antonio, A. Parco et al. (1998) tion during harvest time, and avoiding high temperature A high-density rice genetic linkage map with 2275 markers damage during the ripening period. On the other hand, using a single F2 population. Genetics 148: 479–494. Kanto IL1 exhibited a lower cool temperature tolerance. It Hayashi, K., N. Hashimoto, M. Daigen and I. Ashikawa (2004) Devel- should be considered a potential risk, such as cool tempera- opment of PCR-based SNP markers for rice blast resistance ture damage, in case of the introduction of Kanto IL1into genes at the Piz locus. Theor. Appl. Genet. 108: 1212–1220. northern part of Japan. Hittalmani,S., N.Huang, B.Courtois, R.Venuprasad, H.E.Shashidhar, et al Once we develop ILs for each QTL, it becomes easier J.Y.Zhuang, K.L.Zheng, G.F.Liu, G.C.Wang, J.S.Sidhu . (2003) Identification of QTL for growth- and grain yield- to pyramid detected QTLs. We are developing new ILs in related traits in rice across nine locations of Asia. Theor. Appl. which two QTLs are combined by crossing our ILs and us- Genet. 107: 679–690. ing MAS. Combining two or more late-heading genes may Kawai, T. and H. Sato (1969) Studies on early heading mutations in enable us to develop extremely late-heading ILs. This pyra- rice. Bull. Nat. Inst. Agric. Sci. D20: 1–33. miding strategy will allow us to perform further fine-tuning Kurata, N., Y. Nagamura, K. Yamamoto, Y. Harushima, N. Sue, J. Wu, of heading date in rice. Furthermore, shifting the heading B.A. Antonio, A. Shomura, T. Shimizu, S.Y. Lin et al. (1994) date of a cultivar could avoid environmental stresses such as A 300 kilobase interval genetic map of rice including 883 ex- flood and drought. Our strategy for fine-tuning of heading pressed sequences. Nat. Genet. 8: 365–372. date and DNA markers might be used to avoid such stress Li, Z.K., S.R.M. Pinson, J.W. Stansel and W.D. Park (1995) Identifica- periods. tion of quantitative trait loci (QTLs) for heading date and plant Oryza sativa Recently, a semi-dwarfing gene, sd1, from indica culti- height in cultivated rice ( L.). Theor. Appl. Genet. 91: 374–381. var IR24 was introduced into Koshihikari by MAS (Wang et Lin, H.X., H.R. Qian, Z.M. Xiong, S.K. Min and K.L. Zheng (1996) al. 2005). Grain number 1 (Gn1), involving the number of Mapping of major genes and minor genes for heading date in spikelets per panicle, was also cloned by a map-based strat- several rice varieties (Oryza sativa L.). Chin. J. Genet. 23: egy and has been introduced into Koshihikari (Ashikari et al. 107–114. 2005). Once further Koshihikari ILs with single QTLs are Lin, H.X., M. Ashikari, U. Yamanouchi, T. Sasaki and M. Yano (2002) developed, it will be possible to combine several target Identification and characterization of a quantitative trait locus, genes by simple crossing and MAS. These ILs will be useful Hd9, controlling heading date in rice. Breed. Sci. 52: 35–41. resources for the improvement of Koshihikari. Lin, H.X., Z.W. Liang, T. Sasaki and M. Yano (2003) Fine mapping and characterization of quantitative trait loci Hd4 and Hd5 control- Acknowledgments ling heading date in rice. Breed. Sci. 53: 51–59. Lin, S.Y., T. Sasaki and M. Yano (1998) Mapping quantitative trait loci controlling seed dormancy and heading date in rice, Oryza We thank Dr. K. Eguchi, managing director of the Rice sativa L., using backcross inbred lines. Theor. Appl. Genet. Genome Research Program at the Institute of the Society for 96: 997–1003. Techno-innovation of Agriculture, Forestry and Fisheries, Matsue, Y., K. Mizuta, K. Furuno and T. Yoshida (1991) Studies on for his valuable comments on this work. We thank Drs. palatability of rice grown in northern Kyushu. I. Effects of K. Nagano and N. Nakazawa of Miyagi Prefecture Furukawa transplanting time and lodging time on palatability and physico- Agricultural Experiment Station and Nagano Prefecture chemical properties of milled rice. Jpn. J. Crop Sci. 60: 490– Haramura Agricultural Experiment Station, respectively, for 496. cool-tolerance testing of ILs. This work was supported by McCouch, S.R., L. Teytelman, Y. Xu, K.B. Lobos, K. Clare, M. Walton, a grant from the Ministry of Agriculture, Forestry and B. Fu, R. Maghirang, Z. Li, Y. Xing et al. 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