Genetic Analysis of Heading Date of Japonica Rice Cultivars in Southwest China

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1 Rice Science, 2011, 18(4): Copyright 2011, China National Rice Research Institute Published by Elsevier BV. All rights reserved Genetic Analysis of Heading Date of Japonica Rice Cultivars in Southwest China ZHOU Zhen-ling 1, #, WEI Xiang-jin 1, #, JIANG Ling 1, LIU Kai 1, XU Da-yong 2, ZHAI Hu-qu 3, WAN Jian-min 1, 3 ( 1 State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing , China; 2 Lianyungang Academy of Agricultural Sciences, Lianyungang , China; 3 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing , China; # These authors contributed equally to this paper) Abstract: Heading date of 26 native japonica rice cultivars in southwest China was investigated, and their basic vegetative growth (BVG), photoperiod-sensitivity (PS) and temperature-sensitivity (TS) were analyzed under artificial short-day and natural long-day conditions in Nanjing, as well as artificial high-temperature and natural low-temperature conditions in winter in Hainan. The results showed that the PS and TS varied among different cultivars. The BVG of all the japonica cultivars was well situated, but differed within cultivars. Regression analysis showed a significant correlation between heading date and PS, indicating that PS was the main factor affecting heading date of japonica cultivars in southwest China. Genetic analysis was conducted on these 26 cultivars using a set of heading date near isogenic lines as test lines. All the japonica cultivars carried the dominant early-heading gene Ef-1 or Ef-1 t, and most of these cultivars carried the dominant photoperiod sensitivity allele E 1 or E t 1, the PS of which was slightly weaker than E 1. For the Se-1 locus, these cultivars mainly carried recessive photoperiod insensitivity gene Se-1 e. In addition, the PS of 22 japonica cultivars could be repressed or weakened by the recessive allele hd2, inhibiting the expression of E 1 and Se-1. These results indicated that the genotypes of heading date determined different PS and well situated BVG in japonica rice cultivars in southwest China. Key words: rice; heading date; genetic analysis; photoperiod sensitivity; temperature sensitivity; basic vegetative growth; southwest China Heading date (HD, days from sowing to heading) is one of the most important traits for the adaptation of rice to different cultivation areas and cropping seasons. It is essentially determined by three factors, i.e., the basic vegetative growth (BVG), photoperiod-sensitivity (PS) and temperature-sensitivity (TS). PS is found to be responsible for HD of cultivars with strong PS and it is mainly controlled by Se and E genes. Se-1, Se-2, Se-3(t), Se-4, Se-5, Se-6, Se-7, Se-9(t), E 1, E 2 and E 3 have been identified as major genes for HD of rice (Okumoto et al, 1992a, 1997; Yokoo et al, 1993; Ohshima et al, 1993; Kinoshita, 1995; Maheswaran et al, 1995). Among these genes, Se-1 and E 1 located on chromosomes 6 and 7, respectively, have proven to be the most ubiquitous alleles controlling HD (Ohshima et al, 1994; Okumoto et al, 1996; Ichitani et al, 1997). For cultivars with the same PS and TS, HD is usually determined by BVG. Ef-1, as a major gene controlling Received: 6 January 2011; Accepted: 20 May 2011 Corresponding author: WAN Jian-min (wanjm@njau.edu.cn) the duration of BVG, can accelerate floral initiation under both short-day and long-day conditions, and can also partially counteract the effects of E 1 gene under long-day conditions (Tsai 1986; Okumoto et al, 1996; Nishida et al, 2002). In addition, the recessive inhibitor i-se-1 confers early heading even under long-day conditions due to complete inhibition of Se-1 (Ohshima et al, 1993, 1994). Otherwise, a number of quantitative trait loci (QTLs) for HD have been identified by genetic mapping using different segregating populations (Yano et al, 1997; Lin et al, 1998; Maheswaran et al, 2000), and several major QTLs have been isolated by map-based cloning (Yano et al, 2000; Takahashi et al, 2001; Kojima et al, 2002; Doi et al, 2004; Xue et al, 2008). The growth region of cultivated rice in China is widely distributed. Rice types and farming systems in different ecological regions show significant differences. Thus, there is a rich diversity in the HD of the cultivated rice. Recently, studies on HD of japonica

2 288 Rice Science, Vol. 18, No. 4, 2011 rice cultivars from northeast, northwest, north, and lower region of the Yangtze River valley as well as indica rice have provided guidance for rice breeding (Xu et al, 2007; Wei et al, 2008, 2009a, b). However, systemic research of HD among cultivars from southwest China has not been reported. Southwest China comprises Yunnan, Guizhou and Sichuan provinces and other regions. The topography and ecological environments are complex, especially in Yunnan Province, which is low-latitude tropical and subtropical highlands. The regional differences and vertical changes of the climate are obvious in southwest China. The altitude of japonica cultivar planting area ranges from l 700 to m, including cold highland japonica rice region, cool-warm japonica rice region and warm japonica rice region (Jiang, 1995). The climatic characteristics of high altitude rice cropping region are so atrocious that most cultivars can not grow well when introduced from other provinces or other regions. Therefore, in this study, BVG, PS and TS of japonica cultivars grown in southwest China were analyzed under artificial short-day and natural long-day conditions, as well as artificial high-temperature and natural low-temperature conditions. Genetic analysis was conducted on these cultivars using a set of heading date near isogenic lines EG0, EG1, ER, LR, T65m and T65E b m, and NIL (Hd1) and NIL (Hd2) as test lines. The results will be helpful in elucidating the genetic mechanism of rice varieties adaptive to the environment and expanding new japonica cultivars with well adaptability in southwest China. Rice materials MATERIALS AND METHODS The following rice materials were used: (1) a set of test lines, comprising the HD near-isogenic lines EG0, EG1, ER, LR, T65m and T65E b m (Yamagata et al, 1986; Ichitani et al, 1997 and 1998; Inoue et al, 1998); the HD QTL near-isogenic lines NIL (Hd1) and NIL (Hd2), which have been constructed in a Nipponbare background (Yamamoto et al, 1998; Lin et al, 2000); (2) a panel of 26 japonica rice cultivars grown in southwest China, including Dianjingyou 4, Dianchao 4, Yundao 1, Dianchao 6, Hexi 41, Dianchao 8, Chujing 8, Chujing 9, Chujing 14, Chujing 13, Jingjingyou 1 and Dianjingyou 6 grown at warm japonica rice region in central Yunnan with an altitude of m; Dianjingyou 5, Dianjingyou 1, Yinguang, Dianxi 4, Yunzikang 21 and Hexi 40 grown at cool-warm japonica rice region in north and northeast Yunnan with an altitude of m; Fengdao 23, Fengdao 17 and Jianjing 6 grown at cold highland japonica rice region in northwest Yunnan with an altitude of m; and the landraces Honggu, Zaojiugu, Xiaojiugu, Zhuyajiugu and Yunzikang 6361 of southwest China; and (3) the F 1 hybrids and F 2 populations derived from a set of crosses between the tested cultivars and genetic stocks. Heading date investigation The heading was recorded for each plant when the first developing panicle emerged about 1 cm above the leaf sheath of the flag leaf, and monitored every two days. For the parents and F 1 hybrids, the mean days from sowing to heading of individuals were taken as heading date. Photoperiod-sensitivity and basic vegetative growth analysis The 26 tested cultivars were grown under both artificial short-day (9 h light/15 h dark) and natural long-day (about 14 h light/10 h dark) conditions in the field of Tuqiao site of Nanjing Agricultural University (32 N) in Mean temperatures experienced by the plants grown under artificial short-day (SD) and natural long-day (LD) conditions were almost the same (Fig. 1). Initial sowing was carried out on 20 May, and ten seedlings of each entry were transplanted into the field on 15 June, with a spacing of 13.3 cm 26.7 cm. Crop management followed commercial practices. The PS of each entry was calculated from the difference in HD between SD and LD. The BVG of one cultivar was represented by the HD under SD conditions. Temperature-sensitivity analysis The 26 tested cultivars were grown both in greenhouse (high temperature, HT) and under natural low-temperature conditions (LT) during the winter in Lingshui county of Hainan Province (18 29 N).

3 ZHOU Zhen-ling, et al. Genetic Analysis of Heading Date of Japonica Rice Cultivars in Southwest China 289 Fig. 1. Daily mean temperatures from seeding to heading in the greenhouse (high temperature conditions) and in the field (low temperature conditions) during the winter in Hainan Province, and natural high temperatures in the summer in Nanjing, China. Sowing was completed on 25 November, 2008, and ten seedlings of each entry were transplanted on 20 December in the field and the greenhouse, with a spacing of 13.3 cm 26.7 cm. Crop management followed commercial practices. Daily mean temperatures in the HT and LT experiments are shown in Fig. 1. The TS of each entry was given by the difference in HD between HT and LT grown plants. Genotypic analysis of heading date F 1 hybrids and F 2 populations were derived from crosses between each of the 26 cultivars and each of the test lines EG0, EG1, ER, LR, T65m, T65E b m, NIL (Hd1), NIL (Hd2) and Nipponbare. The parents of each F 1 along with the corresponding F 2 population were grown in the fields at the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences in Nanjing (32 N) in Sowing and transplanting occurred on, respectively, 18 May and 15 June. Ten parental and ten F 1 plants per cross, along with about 200 F 2 individuals were transplanted into the field with a spacing of 13.3 cm 26.7 cm. Crop management followed commercial practices. Genotypes of heading date were analyzed by comparing the HD difference of F 1 and F 2 populations derived from crosses between each cultivar and each pair of test line. RESULTS Photoperiod-sensitivity varied in different cultivars from southwest China The HD of the 26 cultivars from southwest China under SD and LD conditions is shown in Fig. 2. The HD of Dianjingyou 4, Dianchao 4, Yundao 1, Hexi 41, Dianchao 8, Chujing 8, Chujing 9, Chujing 14 and Jingjingyou 1 from warm japonica rice region in central Yunnan was shortened markedly under the SD conditions, showing strong PS. Dianjingyou 5, Dianjingyou 1, Yinguang, Dianxi 4, Yunzikang 21 Fig. 2. Heading date of 26 cultivars adapted to southwest China under natural long-day and artificial short-day conditions in Nanjing, China.

4 290 Rice Science, Vol. 18, No. 4, 2011 and Hexi 40 from north and northeast Yunnan cool-warm japonica rice region showed moderate PS. However, the HD of Fengdao 23, Fengdao 17 and Jianjing 6 grown at cold highland japonica rice region in northwest Yunnan did not show significant difference under SD and LD conditions (2.0 to 6.4 d). Landraces in southwest China, Honggu and Yunzikang 6361, were insensitive to photoperiod, whereas Zaojiugu, Xiaojiugu and Zhuyajiugu showed moderate PS. The BVG of all the japonica cultivars was well situated, but differed markedly within cultivars ( d). Japonica cultivars from southwest China were sensitive to temperature The HD of the 26 cultivars from southwest China under LT and HT conditions is shown in Fig. 3. TS also varied in different cultivars. Dianchao 4, Yundao 1, Dianchao 6, Hexi 41, Dianchao 8, Chujing 8, Chujing 13 and Chujing 14 from central Yunnan warm japonica rice region, as well as Honggu and Zaojiugu all showed strong TS, whereas Dianjingyou 5, Dianjingyou 1, Dianjingyou 6, Jingjingyou 1, Dianxi 4, Hexi 40, Fengdao 17 and Zhuyajiugu showed weaker TS ( d). All other cultivars had moderate TS. Relationships between HD and BVG, PS and TS Compared with the BVG and TS between cultivars from different rice cropping regions, PS gradually became weaker as the altitude of rice cultivation areas increased, but there was no significant variation law for BVG and TS (Fig. 2 and Fig. 3). A significant linear correlation (R 2 = 0.83) was detected between HD and PS (Fig. 4-A), suggesting that the HD of these japonica cultivars was strongly influenced by their PS. However, HD had nonlinear correlation with either BVG or TS (Fig. 4-B, C). Fig. 3. Heading date of 26 cultivars adapted to southwest China under field (low temperature) and greenhouse (high temperature) conditions in Hainan Province, China. Fig. 4. Linear regressions between HD and PS, TS, BVG of japonica cultivars adapted to southwest China.

5 ZHOU Zhen-ling, et al. Genetic Analysis of Heading Date of Japonica Rice Cultivars in Southwest China 291 Genotypes for major HD genes of japonica cultivars from southwest China Allelic relationships at the E 1 locus The near isogenic lines EG0 and EG1 both carry a PS allele Se-1 n at the Se-1 locus and a dominant early-heading gene Ef-1, which only differs at the E 1 locus within the two lines. The genotypes at the E 1 locus are e 1 e 1 in EG0, but E 1 E 1 in EG1 (Ichitani et al, 1997, 1998; Nishida et al, 2001) (Table 1). The PS allele E 1 was dominant over the e 1 (Okumoto et al, 1992a, 1996). The HD of EG1 was 19.2 d longer than EG0 in Nanjing (Table 1). The F 1 hybrids of EG1 crossed with Dianjingyou 4, Dianchao 4, Yundao 1, Hexi 41, Dianchao 8, Chujing 8, Dianxi 4, Dianjingyou 5, Dianjingyou 6, Yinguang, Fengdao 23, Yunzikang 6361, Honggu and Zhuyajiugu exhibited similar HD to F 1 hybrids of EG0 crossed with these cultivars (Table 2), suggesting that all these cultivars carry the PS allele E 1 (Table 3). However, the HD of the F 1 hybrids of EG1 crossed with Dianchao 6, Chujing 9, Chujing 13, Chujing 14, Yunzikang 21, Jianjing 6, Dianjingyou 1, Hexi 40 and Zaojiugu was about d longer than that of the F 1 between EG0 and them (Table 2). It is implied that these cultivars carry a slightly weaker PS allele than E 1, which was tentatively named as E 1 t (Table 3). The HD of the F 1 plants from the crosses of EG1 with Jingjingyou 1 and Table 1. Genotypes and heading date of the near isogenic lines grown under natural long-day conditions in Nanjing, China. Near isogenic line Locus E 1 Se-1 Ef-1 Hd2 Heading date (d) EG0 e 1e 1 Se-1 n Se-1 n Ef-1Ef ±0.6 EG1 E 1E 1 Se-1 n Se-1 n Ef-1Ef ±0.7 ER E 1E 1 Se-1 e Se-1 e Ef-1Ef ±1.3 LR E 1E 1 Se-1 u Se-1 u Ef-1Ef ±1.0 T65E b m e 1e 1 Se-1 e Se-1 e Ef-1Ef ±1.6 T65m e 1e 1 Se-1 e Se-1 ef-1ef ±1.7 Nipponbare E 1E 1 Se-1 n Se-1 n Hd2Hd2 86.9±1.0 NIL (Hd1) E 1E 1 Se-1 e Se-1 e Hd2Hd2 77.4±1.0 NIL (Hd2) E1E1 Se-1nSe-1 hd2hd2 79.9±1.5 Table 2. Heading date (HD) of F 1 hybrids between test lines and japonica cultivars adapted to southwest China. Cultivar HD of F 1 hybrids of test lines crossed with tested cultivars (d) EG0 EG1 ER LR T65m T65E b m NIL (Hd1) NIL (Hd2) Nipponbare Dianjingyou Dianchao Yundao Dianchao Hexi Dianchao Chujing Chujing Chujing Chujing Jingjingyou Dianjingyou Dianjingyou Dianjingyou Yinguang Dianxi Yunzikang Hexi Fengdao Fengdao Jianjing Honggu Zaojiugu Zhuyajiugu Xiaojiugu Yunzikang represents the absent datum.

6 292 Rice Science, Vol. 18, No. 4, 2011 Table 3. Genotypes, heading date (HD), photoperiod-sensitivity (PS), temperature sensitivity (TS) and basic vegetative growth (BVG) of japonica cultivars grown in southwest China. Cultivar Locus E 1 Se-1 Ef-1 Hd2 HD (d) PS (d) TS (d) BVG (d) Dianjingyou 4 E 1E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Dianchao 4 E 1E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Yundao 1 E 1E 1 Se-1 u Se-1 u Ef-1Ef-1 hd2hd Dianchao 6 E t t 1 E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Hexi 41 E 1E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Dianchao 8 E 1E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Chujing 8 E 1E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Chujing 9 E t t 1 E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Chujing 14 E t t 1 E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Chujing 13 E t t 1 E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Jingjingyou 1 e 1e 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Dianjingyou 6 E 1E 1 Se-1 e Se-1 e Ef-1Ef-1 Hd2Hd Dianjingyou 5 E 1E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t Dianjingyou 1 E t t 1 E 1 Ef-1Ef-1 Hd2Hd Yinguang E 1E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Dianxi 4 E 1E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Yunzikang21 E t t 1 E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Hexi 40 E t t 1 E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Fengdao 23 E 1E 1 Se-1 n Se-1 n Ef-1 t Ef-1 t Hd2Hd Fengdao 17 e 1e 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Jianjing 6 E t t 1 E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd Honggu E 1E 1 Se-1 n Se-1 n Ef-1Ef-1 hd2hd Zaojiugu E t t 1 E 1 Se-1 n Se-1 n Ef-1Ef-1 hd2hd Zhuyajiugu E 1E 1 Se-1 n Se-1 n Ef-1Ef-1 hd2hd , Xiaojiugu E t t 1 E 1 Se-1 e Se-1 e Ef-1Ef-1 hd2hd Yunzikang 6361 E 1E 1 Se-1 e Se-1 e Ef-1 t Ef-1 t hd2hd represents the absent datum. Fengdao 17 was about 19.2 d longer than that from the crosses of EG0 with these two cultivars. Therefore, they were likely to carry a recessive photoperiod insensitivity allele e 1 (Table 3). Allelic relationships at the Se-1 locus Se-1 u and Se-1 n are both dominant over Se-1 e, and the PS imposed by Se-1 u is greater than that imposed by Se-1 n (Yokoo, 1977; Ichitani, 1998). The difference of the near isogenic lines ER and LR lies at the Se-1 locus. LR harbors the PS allele Se-1 u, but ER carries the photoperiod insensitivity allele Se-1 e ; Similarly, Nipponbare harbors the PS allele Se-1 n, whereas NIL (Hd1) carries hd1 (synonymous with Se-1 e ) introgressed from the indica rice Kasalath into a Nipponbare genetic background (Yano et al, 1997, 2000; Lin et al, 2000) (Table 1). In Nanjing, ER and NIL (Hd1) headed, respectively, 22.0 and 9.5 d earlier than LR and Nipponbare. The F 1 hybrids of ER and NIL (Hd1) crossed with 18 cultivars, such as Fengdao 17, respectively, also headed about 22.0 and 9.5 d earlier than corresponding F 1 hybrids with either LR or Nipponbare (Table 2), suggesting that these cultivars carry the photoperiod insensitivity allele Se-1 e (Table 3). The HD of the F 1 hybrid between Nipponbare and Yinguang was about 7.4 d longer than the F 1 hybrid between NIL (Hd1) and Yinguang. The HD of the F 1 hybrid between ER and Xiaojiugu was about 19.7 d longer than that of the F 1 hybrid between LR and Xiaojiugu. We can imply that Yinguang and Xiaojiugu carry Se-1 e. The HD of the F 1 hybrids of LR crossed with Zaojiugu, Honggu, Fengdao 23 and Zhuyajiugu was all significantly longer than that of the corresponding F 1 hybrids of ER, whereas their F 1 hybrids of Nipponbare had almost the same HD as the corresponding F 1 hybrids of NIL (Hd1) (Table 2). These outcomes show that Zaojiugu, Honggu, Fengdao 23 and Zhuyajiugu carry Se-1 n, an allele which provides a slightly weaker PS than Se-1 u (Table 3). The F 1 hybrid of Yundao 1 crossed with ER had almost the same HD as the corresponding F 1 hybrid of LR, suggesting that Yundao 1 carries Se-1 u at the Se-1 locus (Tables 2 and 3). The F 1 hybrid of Dianjingyou 1 crossed with Nipponbare exhibited almost the same HD as that with NIL (Hd1), but HD of F 1 hybrid between Dianjingyou 1 and ER was unknown. We can only imply that Dianjingyou 1 carries Se-1 n or Se-1 u at this locus.

7 ZHOU Zhen-ling, et al. Genetic Analysis of Heading Date of Japonica Rice Cultivars in Southwest China 293 Allelic relationships at the Ef-1 locus The test line T65E b m headed 21.3 d earlier than T65m, because these two lines differ allelically at Ef-1 (Table 1). The HD of the F 1 hybrids of T65E b m crossed with Dianjingyou 4, Yundao 1, Hexi 41, Chujing 8, Chujing 9, Dianjingyou 1, Dianjingyou 6, Yunzikang 21, Fengdao 17, Hexi 40, Honggu, Zaojiugu, Zhuyajiugu and Xiaojiugu did not differ significantly from those of the corresponding hybrids from T65m (Table 2). Thus, these cultivars carry the dominant early heading allele Ef-1 (Table 3). The HD of the F 1 hybrids between T65m and Dianchao 4, Dianchao 6, Dianchao 8, Chujing 13, Chujing 14, Jianjing 6, Fengdao 23, Dianjingyou 5, Yunzikang 6361, Jingjingyou 1, Dianxi 4 and Yinguang was about d longer than corresponding hybrids crossed with T65E b m (Table 2), suggesting that these 12 cultivars carry a dominant early-heading allele slightly weaker than Ef-1, which was tentatively named as Ef-1 t (Table 3). Allelic relationships at the Hd2 locus NIL (Hd2) carries hd2 introgressed from Kasalath in a Nipponbare genetic background (Yano et al, 1997; Lin et al, 2000). It headed earlier than Nipponbare under long-day conditions, but later than Nipponbare under short-day conditions, implying that hd2 could inhibit the effect of Hd1 (Se-1) (Luo et al, 2003). NIL (Hd2) headed 7.0 d earlier than Nipponbare in Nanjing (Table 1). The HD of the F 1 hybrids between NIL (Hd2) and Dianjingyou 1, Dianjingyou 6 and Fengdao 23 was almost the same as that of the corresponding F 1 hybrids from Nipponbare ( d), suggesting that these cultivars carry Hd2 (Table 3). In contrast, the F 1 hybrids between NIL (Hd2) and the other 22 cultivars headed about 7.0 d earlier than the corresponding F 1 hybrids from Nipponbare (Table 2), suggesting that all these cultivars carry hd2 (Table 3). Segregation of HD in F 2 populations derived from crosses between testers and cultivars from southwest China The frequency distributions for HD in the various F 2 populations derived from crosses between testers and cultivars are shown in Fig. 5. The HD of the cross EG1 Dianjingyou 4 had a narrower distribution than that of the cross EG0 Dianjingyou 4 (Fig. 5-A, B), confirming that Dianjingyou 4 carries E 1, just as EG1 does. The crosses ER Dianjingyou 4 and NIL (Hd1) Dianjingyou 4 produced a narrower range of HD distribution than the crosses LR Dianjingyou 4 and Nipponbare Dianjingyou 4, respectively, confirming that Dianjingyou 4 carries Se-1 e (Fig. 5-E to H). Similarly, a comparison between the HD distributions from the crosses T65E b m Dianjingyou 4 and T65m Dianjingyou 4 (Fig. 5-C, D) could be taken to further confirm that Dianjingyou 4 carries Ef-1, as T65E b m does. Most of the plants in the F 2 population from the cross NIL (Hd2) Dianjingyou 4 headed earlier, and showed moderate and narrower HD distribution than that of the cross Nipponbare Dianjingyou 4 (Fig. 5-G, I), confirming that Dianjingyou 4 carries hd2 that might inhibit the expression of E 1 and Se-1. DISCUSSION Rice is a short-day plant, however, after long-period introduction and domestication, it has wide cropping regions in China and can grow under various climatic conditions, at latitudes ranging from 18 N to 53 N, including tropics, subtropics, warm temperate, mid temperate and cool temperate zones. HD is critical for adaptation to specific cultivation condition and cropping season, which can be divided into basic vegetative phase (BVP) and photoperiod sensitivity phase (PSP). The length of the BVP is determined by temperature, but the length of PSP is determined by the photoperiod during the growing period. Various combinations between different BVG and PS make HD of rice cultivars exhibit an abundant diversity (Vergara, 1985). Previous research suggested that almost all the japonica cultivars of China were sensitive to temperature, and the PS and BVG of the cultivated rice varied with growth regions and rice types in China. The PS of the japonica cultivars increased gradually through north region to lower region of the Yangtze River valley in the northeast region, and the PS of cultivars from northwest varied greatly. The BVG of the japonica cultivars increased gradually in northeast region, through northwest and north region

8 294 Rice Science, Vol. 18, No. 4, 2011 Fig. 5. Frequency distributions of heading date in F 2 populations derived from crosses between test lines and Dianjingyou 4. A, EG0 Dianjingyou 4; B, EG1 Dianjingyou 4; C, T65m Dianjingyou 4; D, T65E b m Dianjingyou 4; E, ER Dianjingyou 4; F, LR Dianjingyou 4; G, Nipponbare Dianjingyou 4; H, NIL (Hd1) Dianjingyou 4; I, NIL (Hd2) Dianjingyou 4. to lower region of the Yangtze River valley (Wei et al, 2008, 2009a, b). Analysis of japonica rice cultivars native to southwest China showed that the levels of BVG, PS and TS of these cultivars were highly variable. PS was the main factor affecting HD of japonica cultivars from southwest China. Cultivars with stronger PS and TS mainly distribute in the low altitude regions and head later. PS decreased gradually with the increasing of altitude, whereas BVG did not show any variation trends at different altitudes of rice cropping regions. Because of the extremely cold climate on plateau surface, rice growing season is short. After long-period natural selection and artificial domestication, rice varieties are gradually insensitive to day-length, and can even head rapidly under LD, which is similar to the cultivars at the high latitude regions. The genotypes of major HD genes have specific rule for different ecotype rice cultivars in different environments. Previous studies have shown that allelic variation at Se-1 is particularly important for the climatic adaptation of Japanese rice. The insensitive allele Se-1 e predominated in the northern (41 45 N), southeastern (36 41 N) and south (21 30 N) regions of Japan, whereas the sensitive allele Se-1 n was restricted to the southwest (31 36 N) (Okumoto et al, 1992b, 1996; Ichitani et al, 1997, 1998). Cultivars from Hokkaido carry the insensitive allele e 1 (Okumoto et al, 1996), whereas those from Taiwan carry both ef-1 and Se-1 e, which ensures a long BVP and a weak PS in these cultivars (Tsai, 1985; Okumoto et al, 1992). It has been reported recently, at the locus of E 1, most of the japonica cultivars from northeast region of China carry the photoperiod insensitivity allele e 1. All the cultivars from north China carry the PS allele E 1. Most of the cultivars from northwest and lower region of the Yangtze River valley carry the PS allele E 1 or E t 1. In this study, we discover that japonica cultivars native to southwest China mainly carry the PS allele E 1 or E t 1, except for those grown at high

9 ZHOU Zhen-ling, et al. Genetic Analysis of Heading Date of Japonica Rice Cultivars in Southwest China 295 altitude that carry a recessive photoperiod insensitivity allele e 1. At Se-1, similar to the rice cultivars from other regions, most of cultivars carry the photoperiod insensitivity allele Se-1 e. At Ef-1, most cultivars from different regions of China carry the dominant earlyheading allele Ef-1, whereas a large proportion cultivars of southwest China carry the slightly weaker effect allele Ef-1 t (Xu et al, 2007; Wei et al, 2008, 2009a, b). At Hd2, the japonica cultivars from southwest region mainly carry the recessive allele hd2, inhibiting the expression of E 1 and Se-1. These results revealed the diversity of HD genes and main reason for different PS, TS and BVG of japonica cultivars from southwest region. However, the molecular genetic pathway of flowering in rice is complex. Some other genes are also important for rice flowering, besides the major HD genes E 1, Se-1, Ef-1 and Hd2. Therefore, in order to well understand the molecular mechanisms that give rise to the diversity of flowering time in cultivated rice, we need to investigate more HD related genes in future. Southwest China is a mountainous and hilly region. With the complex terrain and circulation factor, it has formed a special climate, with water and heat resources vertically distributed. Varieties suited to each zone has significant limitations, therefore, japonica varieties of southwest region must be adapted to local conditions and rationally distributed. Different degrees of PS and TS of southwest varieties are beneficial to take full advantage of natural resources and also have better adaptability to low temperature to meet the growth environment. The BVG, PS, TS and HD genotypes of japonica cultivars from different rice cropping regions of southwest China were investigated in the study. The results will provide practical and theoretical basis for rice breeding and distribution of the japonica cultivars from low altitude to high altitude region in southwest China. ACKNOWLEDGMENTS We are grateful to Drs. Ichitani K and Yano M for provision of the testers EG0, EG1, ER, LR and QTL NILs, to Dr. Tsai Kuo Hai for the isogenic lines T65m and T65E b m. This work was supported by the National Natural Science Foundation of China (Grant No ), the National High Technology Research and Development Program of China (Grant No. 2009AA101101), the Program of Introducing Talents of Discipline to University (Grant No. B08025). REFERENCES Doi K, Izawa T, Fuse T, Yamanouchi U, Kubo T, Shimatani Z, Yano M, Yoshimura A Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1. Genes Dev, 118(8): Ichitani K, Okumoto Y, Tanisaka T Photoperiod sensitivity gene of Se-1 locus found in photoperiod insensitive rice cultivars of the northern limit region of rice cultivation. Breeding Sci, 47: Ichitani K, Okumoto Y, Tanisaka T Genetic analyses of low photoperiod sensitivity of rice cultivars from the northern most regions of Japan. Plant Breeding, 117: Inoue I, Nishida H, Okumoto Y, Tanisaka T Identification of an early heading time gene found in the Taiwanese rice cultivar Taichung 65. Breeding Sci, 48: Jiang Z N Yunnan Rice Cropping. Kunming: Yunnan Provincial Science and Technology Press. (in Chinese) Kinoshita T Report of committee on gene symbolization, nomenclature and linkage groups. Rice Genet Newsl, 12: Kojima S, Takahashi Y, Kobayashi Y Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions. Plant Cell Physiol, 43(10): Lin H X, Yamamoto T, Sasaki T, Yano M Characterization and detection of epistatic interaction three QTLs, Hd-1, Hd-2 and Hd-3, controlling heading date of rice using nearly isogenic lines. Theor Appl Genet, 101: Lin S Y, Sasaki T, Yano M Mapping quantitative trait loci controlling seed dormancy and heading date in rice, Oryza sativa L., using backcross inbred lines. Theor Appl Genet, 96: Luo L G, Xu J F, Wan J M Analysis of photoperiod sensitivity genes in Minghui 63, a restorer line of indica rice (Oryza sativa L.). Acta Genet Sin, 30(9): (in Chinese with English abstract) Maheswaran M, Huang N, Mackill D J, Sreerangasamy S R, McCouch S R Identification of RAPD markers linked to Se-3(t), a gene enhancing the level of photoperiod sensitivity in rice. Rice Genet Newsl, 12: Maheswaran M, Huang N, Sreerangasamy S R, McCouch S R Mapping quantitative trait loci associated with days to flowering and photoperiod sensitivity in rice (Oryza sativa L.). Mol Breeding, 6: Nishida H, Inoue H, Okumoto Y, Tanisaka T Analysis of

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