ISSN : 2287-5174(Online)
QTLs Analysis of Agronomic Traits Based on Cultivation of Low and High Altitude Locations in Rice (Oryza sativa L.)
The need for high yielding rice varieties has become more conspicuous due to its potential demands in raw processing material market of Korea. However, expansion of cultivation area for high yielding varieties is very limited due to its susceptibility to low temperature (MAF 1983). So far, it is well understood that limited screening methods (site) and germplasms for cold stress screening are main obstacles together with many genes involved in different environmental conditions (Kaw & Khush 1986, Xu et al. 2008). However, the symptoms of cold stress vary depending on plant growth phases. Among them, reduced panicle emergence is considered as one of most important criterion of cold tolerance (Lee 1979, Hamdani 1979). Kwon (1984) also reported complicated symptoms of cold stress where low content and activity of chlorophyll delayed growth in young seedlings and yield decreased in late growth.
Recently, introduction and progress of molecular marker has prompted the conduct of QTL analysis in breeding and gene expression (Kang et al. 2008, Oh et al. 2004, Zhang et al. 2005). And diverse sets of QTLs are reported in QTL studies from wide varieties of species, accessions and environments (Xiong et al. 1999, Cai & Morishima 2002, Koinange et al. 1996). Among them, similar results such as clustered QTLs on chromosomal block (Oh et al. 2004), significant skewing of marker distribution and coincidence of QTLs in multiple environments (Thomson et al. 2003) were also discussed across studies. Many kinds of cold stress were evaluated for identification of QTLs associated with spikelet fertility. Five days of low temperature (12℃) treatment two weeks before booting (Andaya & Mackill 2003) and cold water (17-18℃) irrigation from tillering to grain maturity (Suh et al. 2010) were evaluated. In previous studies related to cold stress, three putative QTLs associated with germination ability at low temperature were reported (Suh et al. 1999). On the other hand, Oh et al. (2004) reported that several QTLs associated with cold tolerance were clustered in a few chromosomal blocks and about 78.6% of the opposite QTLs were derived from weedy rice. Recently, protein analysis associated with cold stress was introduced. Kim (2007) reported 15 different proteins induced by low temperature through two dimension electrophoresis where 12 of them were increased while the other three were decreased under low temperature stress.
It is well known that growth responses are multiplicity depending on varieties, degree and kind of stress, and locations. And the degrees of damage or injury are more serious in Tongil type and indica varieties due to high sensitivity to temperature (Glaszmann et al. 1990, Nahar et al. 2009). Thus, this study was carried out to identify QTLs of agronomic traits based on cultivation in low and high altitude areas where cold stress may cause different responses for growth of rice plant.
MATERIALS AND METHODS
For the QTL analysis associated with agronomically important traits based on high and low altitude conditions, Tongil type cultivar Gayabyeo showing high yielding performance as well as high cross affinity was crossed with japonica cultivar Chhomrong which is preferred by farmers in high altitude area and originated from Nepal. Backcross was performed using Gayabyeo as recurrent in 2005 summer season. From BC1F2 individual plants, single seed descent (SSD) method was adopted up to BC1 F4 generation in field and greenhouse conditions during 2006 and 2007. A total of 181 lines of BC1 F5 RILs (Gayabyeo*2/Chhomrong) were developed and cultivated separately in Milyang, Korea and Khumaltar, Nepal in 2008 for genetic map construction and QTLs analysis.
Genetic map construction
Young leaves were collected from 181 lines of BC1 F5 RILs. CTAB (cetyltrimethyl ammonium bromide) buffer was used for DNA extraction following Chen & Ronald (1999) methods. A total of 666 simple sequence repeat (SSR) markers were selected to detect polymorphisms and map construction. PCR reaction was carried out using modified methods of McCouch et al. (2002). The PCR products were analyzed for polymorphisms using 3% agarose gel electrophoresis. For genetic map construction, Macintosh MapManager program (Manly 1993) was used for data input and management. The framework map was obtained using “group” (LOD>3.0), “order” and “ripple” commands of Mapmaker v. 2.0 (Lander et al. 1987). Finally, the “try” (LOD>2.0) was used to anchor all markers. Haldane function was adopted for calculation of genetic distance (map units, cM).
Data collection and QTLs analysis
For data collection and QTL analysis of agronomic traits, a total of 181 BC1 F5 RILs (Gayabyeo*2/Chhomrong) were separately cultivated and evaluated at low altitude location of Milyang, Korea (latitude: 35°30´ N, altitude: 12m) and high altitude location of Khumaltar, Nepal (latitude: 27°30´ N, altitude: 1,400m) in 2008. Seeding date, planting distance and fertilization (N-P2O5-K2O) of Milyang to Khumaltar were April 25 to May 1, 30 × 15 to 20 × 15 (cm), and 15-4.5-5.7 to 8-3-3 (kg/10a), respectively. Transplanting was practiced after one month of seeding. The weather conditions during plant growing in both sites of Milyang, Korea and Khumaltar, Nepal were presented in Table 1.
Table. 1.Weather conditions during plant growing of Milyang, Korea and Khumaltar, Nepal in 2008.
Days to flowering, culm length, panicle length, number of panicles per hill, panicle exertion and spikelet ripening ratio were investigated with the same parameters in both sites of Milyang and Khumaltar. For panicle exertion, representative three panicles per hill were selected and measured from node of flag leaf sheath to panicle node using digimatic caliper (CD-20CPX, Mitutoyo, Japan). The other characters were measured from the standard evaluation manual of RDA (RDA 1983). Correlation analysis between traits was performed using the SAS program (SAS 9.2 TS Level 2M2). For QTL analysis, simple interval mapping (LOD >3.0) of Macintosh QGENE program (Nelson 1997) was used. Genetic parameters such as explained variation (R2) and genetic effect of each QTL were estimated. The identified QTLs were named following the nomenclature of McCouch et al. (1997).
Response of agronomic traits based on different altitude location
The growth of parental lines was quite different between low and high altitude cultivations (Table 2). In high altitude of Khumaltar Nepal, 5 days of delayed flowering was observed in Gayabyeo while Chhomrong showed 26 days advanced flowering compared to that of Milyang location. And Gayabyeo showed 5 cm longer culm length while Chhomrong showed 25 cm shorter culm length in Khumaltar location. Furthermore, Chhomrong showed high spikelet ripening ratio of 95.6% in Khumaltar, Nepal compared to that of 50.7% in Milyang, Korea while Gayabyeo showed similar results between two locations. For the other traits such as panicle length, number of panicles per hill, and panicle exertion except spikelet ripening ratio showed contracted growth under high altitude location of Khumaltar in parental lines as well as 181 lines. Especially, dramatic contraction in panicle exertion was observed in high altitude location of Khumaltar, Nepal.
Table. 2.Responses of 6 traits in Gayabyeo*2/Chhomrong BC1F5 181 lines under low and high altitude location of Milyang, Korea and Khumaltar, Nepal.
As shown in Table 3, complicated correlation coefficients were observed among traits and locations. Except relatively high correlation coefficient of 0.81 in culm length of two locations, mostly low and negative correlation coefficients were obtained among traits and locations. The frequency distributions of six traits are shown in Fig. 1. Except panicle exertion in Khumaltar, all traits showed a normal distribution patterns indicating that those traits were controlled by QTLs. Moreover, the distribution patterns of low altitude area of Milyang were tend to bias to higher values compared to that of high altitude area of Khumaltar, Nepal in culm length, panicle length and No. of panicles. However, extreme contraction was also observed in panicle exertion in high altitude location of Khumaltar. These results support that kinds of stress derived from the cultivation of high altitude location had caused growth contraction throughout the whole stages.
Table. 3.Correlation coefficients among 6 traits in Gayabyeo*2/Chhomrong BC1F5 181 lines under low and high altitude location of Milyang, Korea and Khumaltar, Nepal.
Genetic map and QTLs identification based on different altitude location
A total of 157 DNA markers were mapped with an average interval of 5.8 cM through 12 chromosomes (Fig. 2). Although their parental relationships were somewhat apart from each other in terms of origin and pedigree background, relatively low polymorphism was observed in this population. Moreover, clustered marker distributions such as on chromosome 3 were observed across the chromosomes.
In the QTLs analysis based on low and high altitude cultivations, a total of 42 QTLs were identified across traits and locations (Table 4 and Fig. 2). Among them, relatively high numbers of QTLs were identified in culm length (9 QTLs), panicle length (8 QTLs) and spikelet ripening ratio (10 QTLs). For days to flowering, 3 QTLs on chromosome 3, 8, and 10 were coincided between two locations. Likewise, four QTLs in culm length, two QTLs in number of panicles, 6 QTLs in panicle length and one QTL in panicle exertion as well as in spikelet ripening ratio were coincided between both locations of Khumaltar and Milyang. In days to flowering, the QTL harboring RM14281-RM489 on chromosome 3 which was detected in both locations showed relatively high LOD/R2 values of 7.65/17.69 (Milyang) and 12.39/27.04 (Khumaltar) with 3-4 days of delayed flowering date controlled by Chhomrong allele. Other QTLs were controlled by Gayabyeo allele with 3-6 days of early flowering date. For culm length, the two QTLs on chromosome 1 and 12 controlled by Chhomrong alleles showed high LOD/R2 values (Khumaltar: 18.05/36.82, 20.1/40.03, Milyang: 9.14/ 20.75, 10.05/22.56). In panicle length, 6 QTLs on chromosome 1, 3, 5, 8, and 12 were coincided across the two locations. Among them, only the one QTL harboring RM14281- RM489 on chromosome 3 was controlled by Chhomrong allele with 1.2-1.4 cm long panicle length. In number of panicles, two QTLs were coincided between two locations and three QTLs were controlled by Chhomrong allele except one on chromosome 1. In panicle exertion, six QTLs were detected across 5 chromosomes. Interestingly, the five QTLs for panicle exertion except one on chromosome 5 were detected only in high altitude location of Khumaltar, Nepal indicating that those QTLs were highly affected under high altitude conditions. Moreover, LOD and R2 values of the panicle exertion QTLs on chromosome 5 showed high additive effect in panicle exertion compared to other QTLs. In spikelet ripening ratio, many QTLs were detected across chromosomes. However, most of QTLs except one QTL on chromosome 2 were not coincided across locations. This result indicates that spikelet ripening ratio is environmental dependent trait.
Table. 4.Identification of putative QTLs associated with agronomic traits in Gayabyeo*2/Chhomrong BC1F5 181 lines.
Table. 4.Identification of putative QTLs associated with agronomic traits in Gayabyeo*2/Chhomrong BC1F5 181 lines.
Generally, delayed growth, spikelet sterility and defective panicle exertion traits are well recognized in previous cold stress studies. In this study, agronomically important traits were studied based on cultivation of low and high altitude conditions (from 12 m to 1,400 m above sea level of Milyang, Korea to Khumaltar, Nepal) for QTLs analysis. As shown in Table 2 and Fig. 1, all traits were greatly affected under high altitude location of Khumaltar, Nepal compared to that of low altitude location of Milyang, Korea. Especially both parental lines showed definitely different result between two locations in days to flowering. The flowering date of Gayabyeo showed 5 days of delayed flowering while that of Chhomrong showed 26 days of advanced flowering in high altitude of Khumaltar, Nepal. Mackill & Lei (1997) reported that cold stress usually caused a delayed flowering date, stunted growth, and sterility. In this study, Chhomrong showed shorter flowering days under high altitude condition of Khumaltar, Nepal. This result indicates that Chhomrong is sensitive to photoperiod (short day length) resulted in early flowering date and subsequently reduced culm length in Khumaltar, Nepal.
Consequent of different response to photoperiod, quite different results were obtained in spikelet ripening ratio. Chhomrong showed 44.9% of increased spikelet ripening ratio under high altitude location of Khumaltar, Nepal while Gayabyeo showed similar spikelet ripening ratio in both sites of Korea and Nepal (Table 2). This result may support that low numbers of spikelet per panicle caused by short panicle length helped to increase spikelet ripening ratio. The other traits such as panicle length, number of panicles per hill, and panicle exertion showed stunted growth under high altitude of Khumaltar, Nepal. Thus, these results obtained in this study may explained by the relatively low temperature of July and August during panicle formation and flowering stages rather than other reasons under high altitude location of Khumaltar, Nepal as shown in Table 1.
In this study, as shown on chromosome 3 and 8, clustered marker distributions were observed across the chromosomes (Fig. 2). These marker distortion were assumed to be caused by wide cross of Gayabyeo (grouped into Tongil) and Chhomrong (grouped into japonica). Similar phenomena caused by wide crosses were reported in previous studies (Chao et al. 1994, Causse et al.1994, Xu et al. 1997). In Table 3, unlike other traits and locations, high correlation coefficient of 0.81 was observed in culm length indicating that major QTLs are involved in culm length (Table 4). However, mostly negative or low correlation coefficients were obtained among locations and traits. This result suggests that complicated environmental factors were involved through the whole stages of early to late growth and consequently QTLs were independent to locations.
Fig. 2.Putative QTLs associated with agronomic traits in Gayabyeo*2/Chhomrong BC1F5 181 lines. QTLs detected in both Milyang and Khumaltar at p <0.05 (LOD > 2.84) are boxed.
In QTLs analysis of agronomical traits based on cultivation of low and high altitude conditions, a total of 42 QTLs were identified across both locations (Table 4 and Fig. 2). Among them 16 QTLs were attributed in Khumaltar and 9 QTLs were in Milyang, respectively. And 17 QTLs of 42 QTLs (40.5%) were coincided across both locations of Milyang and Khumaltar. In previous studies, many QTLs has been reported on chromosome 6 related to flowering date (Dung et al. 1998, Yano et al. 1995). However in this study, no QTL was detected on chromosome 6 related to days to flowering. This indicates that the parental lines used in this study may not share the same allele reported before due to different ecotype or strong dominant allele effects on the other chromosomes. Furthermore, this may imply that the basic vegetative growth must be inhibited by some genetic factors. Many studies were reported on culm length, especially on sd-1 gene (Suh & Heu 1978, Cho et al. 1994, Hideo et al. 1997). Hiroaki et al. (2000) reported that the QTL associated with culm length controlled by Kinuhikari with 70% of phenotypic variation was linked to RFLP marker C86 and C386 on chromosome 1. In this study, the QTL qCL1-2 on chromosome 1 apparently coincided with other reports was believed to share the sd-1 allele (Causse et al. 1994, Maeda et al. 1997). Panicle exertion is one of the most important traits in cold tolerance study (Hamdani 1979, Lee 1979). In this study, five of six QTLs were detected in high altitude location of Khumaltar, Nepal related to panicle exertion. This result explains that those Khumaltar specific five QTLs related to panicle exertion were largely affected by relatively low temperature stress of July and August under high altitude conditions before flowering (Table 1). It is also well understood that spikelet ripening ratio together with sterility is one of the main obstacles caused by cold stress (Lafitte et al. 2004). Suh et al. (2010) reported 3 QTLs on chromosome 3, 7, and 9 related to seed set after cold water irrigation. Andaya and Mackill (2003) also reported 8 QTLs on chromosome 1, 2, 3, 5, 6, 7, 9 and 12 to confer cold tolerance at the booting stage. However in this study, 10 QTLs were identified associated with spikelet ripening ratio and none of the QTLs were coincided between two locations except the QTLs on chromosome 2 indicating environmental effects rather than gene dependent. Moreover, considering QTLs numbers, the phenotypic variations explained by each QTL were relatively low compared to other traits.
Among the QTLs, days to flowering and panicle length on chromosome 3, culm length, number of panicles and panicle exertion on chromosome 1, and spikelet ripening ratio on chromosome 12 were coincided across the locations. Likewise, 40.5% of QTLs (17 of 42 QTLs) were coincided across chromosomes and locations indicating that those QTLs were environmental depending genes and mainly controlled by major genes. However, 38.1% of QTLs (16 of 42 QTLs) in high altitude location of Khumaltar, Nepal and 21.4% of QTLs (9 of 42 QTLs) in low altitude location of Milyang, Korea are assumed to be controlled by environmental factors such as temperatures and photoperiod. Unfortunately, direct comparison of QTLs between this study and previous studies were not possible because of remitted marker sets although some of them were apparently coincided. However, together with major QTLs, environmental or location specific QTLs such as panicle exertion as revealed in this study also should be carefully considered in MAS programs for development of cold tolerance lines in indica and Tongil type breeding. Furthermore, combined efforts such as chemical and enzyme study (Kwon 1984, Li & Komatsu 2000) should be accompanied in cold stress study for more informative research and breeding programs.
This study was supported by a grant of Technology Cooperation Bureau (Joint project of Korea and Nepal, Project title: Development of cold-tolerant parental lines and analysis of cold tolerance gene, 2006~2008) of the RDA, Republic of Korea.
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