ISSN : 2287-5174(Online)
Identification of Molecular Markers for Mesocotyl Elongation in Weedy Rice
Rice mesocotyl is the region between the coleoptile node and point of union of the culm with the root. The seedling emergence is also important in determining the actual yield in direct seeding cultivation of rice. The mesocotyl is considered as one factor, in addition to the coleoptile and early leaf, related to rice seedling emergence from soil. Before the shoots emerge from the soil, considerable elongation has usually begun underground. The elongation of coleoptile and mesocotyl can contribute to emergence from the soil and the survival of seedlings in monocots (Radosevich et al. 1997). Because elongation of the mesocotyl elevates the coleoptile tip above the soil surface, where it ruptures to allow the primary leaves to emerge. Few studies have reported the direct relation between mesocotyl elongation and seedling emergence. Turner et al. (1982) and Dilday et al. (1990) reported that drill-seeded semidwarf rice emerges more slowly than normal-height rice does. It is associated with shorter mesocotyl of semidwarf rice at all planting depths. This relation with mesocotyl elongation and seedling emergence is also considered in wild oat (Raju and Steeves 1998). Moreover, another mesocotyl role might be uptake and transport of water and ion like roots because mesocotyls are located below the soil surface and Casparian bands were observed in both the endodermis and exodermis, like roots does (Watanabe et al. 2006).
Several studies were conducted to map QTLs for mesocotyl elongation using various screening methods for mesocotyl elongation and segregating populations from interspecific or intrasubspecific crosses (Cai and Morishima 2002, Cao et al. 2002, Huang et al. 2010, Katsuta-Seki et al. 1996, Redoña and Mackill 1996). Redoña and Mackill (1996) detected five QTLs for mesocotyl length by the slant-board test. Eleven QTLs were detected using an RIL population at 10 days after incubation in dark conditions (Cai and Morishima 2002). Katsuta-Seki et al (1996) identified three QTLs related to mesocotyl length using the glass tube test. Cao et al. (2002) detected 8 QTLs using a doubled haploid population. Five QTLs for mesocotyl length were detected under water and plant hormone gibberellins (GA) germination condition (Huang et al. 2010). However, these QTL analyses were conducted under water or blotting paper conditions and not in soil and agar media conditions. This is the first report to analyze and map gene/QTL for mesocotyl elongation in weedy rice using soil and agar media conditions.
This study used the bulked segregant analysis (BSA) for finding molecular markers related mesocotyl elongation. Bulked segregant analysis (BSA) was developed as method for rapidly identifying markers linked to any specific gene or genomic region, in which a large amount of markers are screened using only the parents and two DNA bulks sampled from a segregating population (Michelmore et al. 1991). This method can reduce the cost and work load by several fold. BSA strategy was initially employed in the analysis of simply-inherited traits. However, this technique has also been successful applied to analyze complex traits allowing a faster identification of major QTLs (Lee et al. 2010, Ninamango-Cárdenas et al. 2003)
The first object of this study is to evaluate mesocotyl characterization of weedy rice ‘PBR’ compared with rice cultivars, Ilpumbyeo. The second object is to identify the
main locus controlling mesocotyl elongation of PBR.
MATERIALS AND METHODS
Seeds of the weedy rice, PBR (photoblastic rice) and the japonica rice cultivar, Ilpumbyeo were used for this study. PBR seed was obtained from the National Institute of Crop Science, Rural Development Administration (RDA), Suweon, South Korea. To identify molecular markers related to mesocotyl elongation, 150 F7 and 120 F8 lines were developed from a cross between Ilpumbyeo and PBR. These populations were derived from an F2 population with single seed descent. PBR was used as a pollen parent. Ilpumbyeo, PBR and the populations were grown in the experiment plot, Chungnam National University, South Korea. The seeds of F7 and F8 generations were sampled at about 60 days after heading. After harvesting, panicles were selected and dried at greenhouse for 40 days. Seeds collected from panicles were dried at 50℃ for 7 days to break dormancy and stored in envelopes at room condition until experiment.
Evaluation of mesocotyl length
The mesocotyl length of 150 F7 and 120 F8 lines were measured in both soil and agar conditions, respectively. In a soil condition, fulfilled seeds were selected and 20 seeds of each F7 line were buried at 4.5 cm depth in plastic pots (12 cm diameter, 10.5 cm height) using humus soil and vermiculite. The plastic pots were kept in darkness at 33℃ for one week. The water level of soil was kept 1 cm from the bottom of pots. The mesocotyl length of each seedling was measured using a ruler as the distance from the basal part of the seminal root to the coleoptilar node. In the agar condition, fulfilled seeds were selected and 12 seeds of each F8 line were sown at <0.5 cm depth 3% agar in a plant culture jar (8 cm diameter, 11 cm height), which consisted of 50ml of 0.3% agar medium. The culture was incubated in darkness at 30℃. The mesocotyl length was measured at 7 days after incubation. Mesocotyl lengths of the F8 lines were measured in three replications in agar condition.
DNA extraction and molecular analysis
To extract genomic DNA, leaves of each line were sampled at the seedling stage. DNA was extracted using a method described in the report by Causse et al. (1994) with slight modifications. Protocols for SSR marker amplification using the polymerase chain reaction (PCR) and size separation using polyacrylamide gel electrophoresis (PAGE), and marker detection using the silver staining procedure were performed as described in Panaud et al. (1996). The silver staining kits were purchased from Bioneer Corp. (Korea, http://www.bioneer.co.kr).
Bulked segregant analysis
For bulked segregant analysis, equal amounts of genomic DNA from each 10 F7 lines with short and long mesocotyl in the soil condition were used to construct two bulks, respectively. One Ilpumbyeo-type bulk (I1) having short mesocotyl was composed of lines with less than 9 mm length. One PBR-type bulk (P1) having long mesocotyl was composed of lines with longer than 12 mm.
One-way ANOVA was performed to determine the effect of each putative marker on mesocotyl length in the F8 lines. QTL was declared if the phenotype was associated with a marker locus at P<0.05. For detecting the precise location of the target QTL, a total of 120 F8 lines were subjected to linkage analysis with 5 and 6 additional SSR markers on the target regions of chromosomes 1 and 3, respectively. Published SSR maps (McCouch et al. 2002) were used as reference to establish the marker order.
Linkage analysis was performed using the Kosambi function of Mapmaker/EXP 3.0 software (Lander et al. 1987). QTL analysis was performed by composite interval mapping (CIM) using the QTL Cartographer version 2.5 software (Wang et al. 2007). CIM analysis was performed with a forward-backward stepwise regression-using model 6 with a 10 cm window size. Subsequently, the LOD value, 2.5 at p≤0.05 was used as the threshold to declare the significance of the QTLs. The QTL positions were assigned to the point of the maximum LOD score in the target regions. The percentage of the total phenotypic variance accounted for by each QTL was estimated using R2 value. QTL names were designated following the standard rice QTL nomenclature (McCouch 2008).
RESULTS AND DISCUSSION
Mesocotyl length of F7 and F8 lines
To measure mesocotyl elongation, we used the soil and agar conditions. In the soil condition, the effect of soil depth on mesocotyl length of PBR and Ilpumbyeo was investigated. At 4.5 cm soil depth, the difference of mesocotyl length between Ilpumbyeo and PBR is bigger than the other depth conditions, 1.5 and 3 cm (data not shown). Based on this result, the seeds were sown at 4.5 cm depth in soil. Mesocotyl of the weedy rice, PBR elongated significantly longer than that of Ilpumbyeo under both conditions (P<0.0001) (Fig. 1 and Fig. 2). The mesocotyl length of PBR and Ilpumbyeo was 21.8 ± 3.1 mm and 5.1 ± 3.1 mm, respectively under the soil condition (Fig. 2A). In the agar condition, mesocotyl length of PBR and Ilpumbyeo was 13.9 ± 0.4 mm and 0.3 ± 0.2 mm, respectively (Fig. 2B).
Fig. 1. Mesocotyl elongation of weedy rice, PBR (A) and Ilpumbyeo (B) in soil conditions. Arrowheads indicate mesocotyl region.
Fig. 2. Distribution of mesocotyl length of the 150 F7 lines (A) and 120 F8 lines (B) derived from a cross between Ilpumbyeo and weedy rice, PBR. The mesocotyl length of F7 and F8 lines were measured in soil and agar conditions, respectively. Arrows indicate mean values for Ilpumbyeo and PBR.
150 F7 lines were measured for mesocotyl length and the distribution was continuous ranging from 0.4 to 30.3 mm (Fig. 2A) in soil condition. The mesocotyl length of F8 lines was investigated in the agar condition with three replications. The agar condition was selected for F8 lines because of difficulty in sampling in the soil condition. In our previous study, the mesocotyl length of backcross inbred lines (BILs) derived from Nipponbare and Kasalath showed significant correlations between soil and agar conditions (Lee 2010). The average mesocotyl length was 4.5 mm, 4.6 mm and 4.3 mm in the F8 lines in three replicated experiments. The mesocotyl length of the F8 lines showed significant correlations among three experiments (r>0.92, P<0.0001) indicating that the measurement of mesocotyl length in the agar condition is reliable. The mesocotyl length of F8 lines ranged from 0.1 to 14.5 mm with a continuous variation. F8 lines were less variable than F7 (Fig. 2B). The parents also showed reduced mesocotyl elongation in the agar than in the soil. This result is consistent with previous reports that mesocotyl elongation is influenced by environmental conditions such as like light, oxygen, moisture, and temperature (Takahashi 1978, Takahashi 1984, Nick and Furuya 1993).
Bulked segregant analysis
92 polymorphic SSR markers between Ilpumbyeo and PBR were reported in the previous study (Lee et al. 2010). To identify SSR markers related to mesocotyl elongation, bulked segregant analysis was carried out with 42 markers. Two bulks with 10 lines each were selected based on mesocotyl length from 150 F7 lines: I1 and P1. The mesocotyl length of Ilpumbyeo-type bulk, I1 was 5.0 ± 0.6 mm. In PBR-type bulk, mesocotyl length was 17.9 ± 1.3 mm. Two SSR markers RM306 on chromosome 1 and RM426 on chromosome 3 showed associations with mesocotyl elongation. As shown in the Fig. 3, RM306 and RM426 showed the homozygous banding pattern in I1 bulk corresponding to Ilpumbyeo (lane 3) and in P1 bulk corresponding to PBR (lane 4). These results indicated that RM306 and RM426 are possibly linked to mesocotyl elongation.
Fig. 3. Bulked segregant analysis with SSR markers, RM306 (A) and RM426 (B). DNA band pattern amplified by the RM306 on chromosome 1 and RM426 on chromosome 3 in two bulks from F7 lines from a cross between Ilpumbyeo and weedy rice, PBR and parents. Lane 1 (I): Ilpumbyeo, lane 2 (P): weedy rice, PBR, lanes 3 (I1): one bulk with Ilpumbyeo phenotype, and lanes 4 (P1): one bulk with PBR phenotype.
To determine the location of the target QTL, additional SSR markers located near RM306 and RM426 were genotyped in 120 F8 lines. Two QTL were detected on chromosomes 1 and 3. The QTL, qMel-1 was detected between SSR markers RM306 and RM7419 and accounted for 37.3% of the phenotypic variance (Table 1 and Fig. 4). The other QTL, qMel-3 was located between RM8208 and RM8277 (Table 1 and Fig. 4). The phenotypic variance explained by qMel-3 was 6.5%. Mesocotyl increased by the PBR alleles on qMel-1 and qMel-3, was 1.8 mm and 0.8 mm, respectively. A non-significant digenic interaction between qMel-1 and qMel-3 was observed by two-way ANOVA (P= 0.45) indicating that qMel-1 and qMel-3 act additively in mesocotyl elongation. These two QTL cumulatively explained 48 % of the total phenotypic variance.
Fig. 4. Chromosomal locations of the QTL for mesocotyl length in 120 F8 lines from a cross between Ilpumbyeo and PBR. Chromosome numbers are indicated above each chromosome. Marker names are located to the right of each linkage map. Bars to the right of each linkage map represent the position of putative QTL.
This study is the first report to analyze and map QTL related to mesocotyl elongation in a weedy rice. The QTLs for mesocotyl elongation were reported in some researches (Cai and Morishima 2002, Cao et al. 2002, Katsuta-Seki et al. 1996, Redoña and Mackill 1996, Huang et al. 2010, Lee et al. 2012). Eleven QTLs for mesocotyl elongation were identified on chromosomes 1, 3, 4, 5, 6, 9 and 11 using an RIL population derived from a cross between an indica cultivar and wild rice, O. rufipogon (Cai and Morishima 2002). Eight QTLs on chromosomes 1, 3, 6, 7, 8, and 12 were detected using a doubled haploid population from a cross between IR64 and Azucena (Cao et al. 2002). Redoña and Mackill (1996) detected five QTLs for mesocotyl elongation on chromosomes 1, 3, 5 and 7 using an F3 population from a cross between japonica cultivar, Labelle and indica cultivar, Black Gora. Three QTLs were detected on chromosomes 3, 6 and 11 by using the population derived from a cross between Dao Ren Qiao and as Assam rice cultivar, Surjumkhi (Katsuta-Seki et al. 1996). In our previous study, a total of five QTLs for mesocotyl length were identified on chromosomes 1, 3, 7, 9 and 12 in backcross inbred lines from a cross between Nipponbare and Kasalath under agar condition (Lee et al. 2012). Notably, the QTLs for mesocotyl length were commonly mapped on chromosomes 1 and 3 in different parent crosses and experiment conditions. The present study also confirmed that the mesocotyl length QTL, qMel-1 and qMel-3 were located on chromosomes 1 and 3 under soil and agar conditions (Fig. 3, Fig. 4 and Table 1). These 2 QTLs, qMel-1 and qMel-3 are located in similar regions reported by other previous reports. The qMel-3 was mapped between RM8208 and RM8277, and this region overlapped with loci of QTLs reported in previous reports (Cai and Morishima 2002, Cao et al. 2002, Katsuta-Seki et al. 1996, Redoña and Mackill 1996, Huang et al. 2010, Lee et al. 2012). These results support that QTLs controlling mesocotyl length of a weedy rice, PBR also exist on chromosomes 1 and 3.
Table 1. Characteristics of QTLs for mesocotyl length detected on chromosomes 1 and 3 using 120 F8 lines from a cross between Ilpumbyeo and a weedy rice, PBR.
Of interest, the region of QTL qMel-1 includes a rice semidwarfing gene, sd-1, controlling plant height. The sd-1 was isolated by positional cloning and revealed to encode gibberellins 20-doxidase (GA20ox2), the key enzyme in the gibberellins biosynthesis pathway (Spielmeyer et al. 2002, Monna et al. 2002). The relation between mesocotyl length and plant height was reported (Dilday et al. 1990). The correlation coefficients for plant height vs. mesocotyl, coleoptiles and total length were positive and highly significant. It was reported that semidwarf rice has short mesocotyl (Dilday et al. 1990, Murai et al. 1995). However, a close relationship between plant height and mesocotyl and coleoptiles length in rice is not due to pleiotropism but to close linkage (Dilday et al. 1990). In addition, GA20ox2 gene did not exhibit a significant differential expression between Nipponbare and the near isogenic line carrying the QTLs qMel-1 and qMel-3 under the Nipponbare genetic background in microarray analysis (data not shown).
Weedy rice, PBR used in this study showed photoblastism which is defined as seed germination stimulated by lights (Chung and Paek 2003, Lee et al. 2010). We identified two QTLs, pbr1 and pbr12 controlling the photoblastism of PBR on chromosomes 1 and 12 with BSA and QTL analysis (Lee et al. 2010). The PBR alleles at these loci exerted the effect of inhibition of germination percentage only in dark condition, not under light exposure. Of interest, a region of pbr1 on RM8260-RM246 overlapped with a region of qMel-1 and a grain color gene, Rd (data not shown) (Fig. 4). The clustering of these genes, pbr1 and qMel-1 of PBR on chromosome 1 might indicate the possible linkage of genes related to the survival strategy of the weedy rice. QTL cluster for adaptive or domesticationrelated traits have been reported in wild and weedy rice (Xiong et al. 1999, Cai and Morishima 2002, Thomson et al. 2003, Gu et al. 2005).
Photoblastism and mesocotyl elongation are important for seedling emergence in weeds and in weedy rice. Germination of many weed species of germination can be promoted by light (Hart 1988, Bewley and Black 1994). Photoblastism can inhibit germination until the environment conditions are favorable for seedling growth. Mesocotyl length was related with speed of seedling emergence (Turner et al. 1982, Dilday et al. 1990, Murai et al. 1995). Therefore, these traits might be selected for survival strategies for seedling emergence. These two genes from weedy rice could be especially useful for manipulating the onset of germination and seedling emergence in direct seedling.
This study was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008136) of the Rural Development Administration, Republic of Korea.
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