Rice Science, 2013, 20(6): −                                                                        

Copyright © 2013, China National Rice Research Institute Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(13)60150-X

Mapping of QTLs of Germination Characteristics under Non-stress and Drought Stress in Rice
Zahra MARDANI1, Babak RABIEI1, Hossein SABOURI2, Atefeh SABOURI1
(1Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of Guilan, Rasht 41635-1314, Iran; 2 Department of Plant Production, Faculty of Agriculture and Natural Resources, Gonbad University, Gonbad 4971799151, Iran)

Abstract: Identification of genetic factors controlling traits associated with seed germination under drought stress conditions, leads to identification and production of drought tolerance cultivars. Present study by using a population of F2:4 derived from a cross between a drought tolerant variety, Gharib (indica) and a drought sensitive variety, Sepidroud (indica), is to identify and compare QTLs associated with traits germination under drought stress and non-stress. Through QTL analysis, using composite interval mapping, regarding traits such as germination rate, germination percentage, radicle length, plumule length, coleorhiza length and coleoptile length, totally 13 QTLs was detected under pole drought stress (-8 MPa poly ethylene glycol 6000) and 9 QTLs under non-stress conditions. Of the QTLs identified in non-stress conditions, QTLs associated with the coleorhiza length (qCOL-5) and germination rate (qGR) explained 21.82% and 19.73% of the total phenotypic variation, respectively. Under drought stress conditions, QTLs associated with coleorhiza length (qCOL-3) and plumule length (qPL-5) explained 18.34% and 18.22% of the total phenotypic variation, respectively. A few droughttolerance-related QTLs identified in previous studies were found to be near the QTLs detected here, and several QTLs are novel alleles. The results showed that the major QTLs like qGR-1, qGP-4, qRL-12 and qCL-4 that were identified in both conditions of stress for traits such as germination rate, germination percentage, radicle length and coleoptile length respectively, should be considered by breeders as the important and stable trait-controlling QTLs in rice seed germination. Those major or minor QTLs could be used to significantly improve drought tolerance by marker-assisted selection in rice. Key words: rice; drought stress; seed germination; quantitative trait locus

Drought stress is a serious limiting factor to rice production and yield stability in worldwide (Dey and Upadhyaya, 1996). As a result, understanding the mechanisms of drought tolerance and breeding for drought-resistant crop plants has been the major goal of plant biologists and crop breeders (Xiong et al, 2006). Furthermore, standard assays used to measure drought tolerance must be enhanced since they are important to success in genetic improvement (Lanceras et al, 2004). Moreover, genotype × environment (GE) interaction is a challenge to plant breeders and has been shown to reduce the progress of the quantitative traits from selection. Because of the GE interaction, quantitative trait loci (QTLs) that are important in one environment may not be as important as in another environment in determining the phenotype (Tanksley, 1993). For this reason, QTLs with little GE interaction across a set of environments would be desirable in
Received: 24 March 2013; Accepted: 10 June 2013 Corresponding author: Zahra MARDANI (zahra.mardani64@gmail.com)

marker-assisted breeding programs (Zhang et al, 2005). QTL analysis of seed germination has been reported in Arabidopsis, rice, beans, lettuce and soybean (Mano and Takeda, 1997; Teng et al, 2001; Price et al, 2002; Fujino et al, 2004; Miura et al, 2004; Cheng et al, 2008; Hayashi et al, 2008; Ikeda et al, 2009; Ji et al, 2009; Oh et al, 2009; Park et al, 2009; Yano et al, 2009; Wang et al, 2011; Sasaki et al, 2013). Moreover, QTLs conferring stress tolerance in rice were identified mainly at the seedling stage, but few reports on rice seed germination stage (Wang et al, 2011). Cheng et al (2008) found two main-effect QTLs associated with tolerance to alkali damage on chromosomes 3 and 7, using by 120 double haploid (DH) lines derived from the cross between TN1 (Oryza sativa L. subsp. indica) and Chunjiang 06 (O. sativa L. subsp. japonica). Also, a few efforts have been made on comparative studies of QTLs for germination traits in rice under salt stress and non-stress conditions. For example, Foolad et al (1996) detected several QTLs for seed


Further. Mapping of QTLs of Germination Characteristics in Rice germination under cold stress. 25 randomly selected seedlings were used to measure the length radicle. An F2 population derived from a cross of these parents was used in order to construct a genetic linkage map. This study focused on comparative investigation of QTLs for drought tolerance trait in a F2:4 population grown under drought stress and non-stress conditions. using BC1S1 lines. Two replications were conducted and the mean value was used for data analysis. University of Guilan. and the results showed that the -8 Mpa was suitable for drought treatment in this study. each derived from bagged seeds of a single F2 plant. the different PEG concentrations were applied among two parents. Wang et al (2011) detected one QTL qIR-4 for seed imbibition under salt stress and non-stress conditions on chromosome 4 with the recombinant inbred line (RIL) population derived from IR26/Jiucaiqing.48 μL of MgCl2 (50 mmol/L). and then rinsed three times with sterile distilled water.4 μL of forward and reverse primers each of 10 pmol concentration. 0.org) and the linkage map was constructed following McCouch et al (2002) based on their map location. The seeds were incubated in a growth chamber at (30 ± 1) °C for 10 d. The 40 seeds were then placed in Petri dishes (diameter is 9 cm) with two sheets of filter paper to which 10 mL of solution was added.1% hydrargyrum chloratum solution for 15 min.gramene. and the seeds germinability was observed each day to estimate the total germination percentage (GP). Then. 0. The experiments were conducted in the Faculty of Agricultural Sciences Laboratory. Phenotypic evaluation The 148 F4 families and their parental varieties were   . Han et al (2006) mapped several QTLs associated with agronomic traits in rice under different growing environments. to disclose the relationship of QTLs detected in both conditions. Srividhya et al (2011) find five stable QTLs among 24 detected for drought related traits at the seedling stage under the poly ethylene glycol (PEG) induced stress and control conditions in rice. to further identify specific QTLs associated with drought tolerance in Iranian rice at the germination stage.6 μL of dNTPs (2 mmol/L). which will be useful in marker-assisted breeding for drought resistance in rice. All seeds were surface-sterilized with 0. with two replications (Each petri dish contained 40 seeds for each replication).Zahra MARDANI. salt stress and nonstress conditions. length coleoptile and length coleorhizae. In total. Polymerase chain reaction (PCR) was carried out in a total volume of 10 μL per reaction containing 2 μL of template DNA. To determine the suitable PEG 6000 concentration of treatment. In addition. et al. Moreover.12 μL of Taq polymerase (5 U/μL). the evaluation of seed germination among 148 families and parents were conducted under -8 Mpa and control (distilled water) conditions. 0. final count. length plumule. The genetic material used in phenotype evaluations involved 148 F4 families. GR = X1 / Y1 + (X2 – X1) / Y2 +···+ (Xn – Xn-1) / Yn. Iran from 2009 to 2011. Sepidroud is a drought susceptible variety. A total of 575 SSR markers of known chromosomal position were used to survey the parents for polymorphism. germination rate (GR) and germination percentage (GP) were calculated by the following formulae (Maguire. Also. 1 μL of 10 × MATERIALS AND METHODS Rice materials The rice material was F2:4 population consisting of 148 families each derived from a cross between two Iranian rice varieties Gharib and Sepidroud. Kamoshita et al (2002) could find only two stable QTLs among 31 detected for seven traits associated with root morphology in two experiments with different sowing dates under anaerobic conditions. used to evaluate drought tolerance. DNA extraction was performed by using the CTAB as described by Saghai Maroof et al (1994). respectively. MacMillan et al (2006) mapped six QTLs for root traits in four different environments in rice and most QTLs for an identical trait in the four environments were different. Genotyping DNA was extracted from fresh leaves of each F2 plant and the parental lines. Where Xn is the number of germinated seeds on the nth days and Yn is the number of days from the first day experiment. 1962): GP = Number of germinated seeds / Total number of seeds planted × 100%. Simple sequence repeat (SSR) markers on 12 rice chromosomes were selected from the Gramene database (http://www. Polymorphic markers were used to detect the genotype of 148 F2 plants. Gong et al (2001) reported two QTLs on chromosome 4 for HD (heading date) and the other on chromosome 6 for GPP (grains number per panicle) under salt stress and non-stress conditions. Gharib is a drought tolerant variety and other parental line. 0.

PCR products were separated on 6% polyacrylamide gels (19 acrylamide : 1 bisacrylamide). 1997). radicle length and plumule length. with minor modifications. coleoptile length. 3. Nine AFLP primer combinations (EcoRI primer and MseI primer) showing polymorphisms between Gharib and Sepidroud were used for genotype analysis of the F2 population. 2007). respectively. The AFLP protocol developed by Vos et al (1995) was followed. 105 produced polymorphic bands between two parents and 131 polymorphic markers. also of 9 primer pair combination AFLP. coleorhizae length. Vol. The distributions of the six germination traits among the F4 population showed continuous and Fig. The thermal cycle protocol includes 94 °C for 5 min (initial denaturation). An automatic cofactor selection using a forward/backward regression. The LOD values above 3.2 and 3. 1. No. 2013 PCR buffer and 5 μL of sterile nanopure H2O. followed by 35 cycles of 94 °C for 30 s (denaturation).5 (Basten et al. 6. Genetic linkage map construction and QTL analysis A genetic map was constructed with AFLP and SSR markers.8. germination percentage.3. Germany).Rice Science. RESULTS Phenotypic variation Fig. The linkage map was established using the program QTXB17 Map Manager (Wang et al.1. Phenotypic distribution of six traits related to drought tolerance in 148 F4 families under drought stress. The QTLs were mapped using the composite interval mapping (CIM) function of the WinQTL Cartographer v2. PCR amplification was performed in a thermal cycler (Applied Biosystems.4 were considered to indicate the presence of QTLs for germination rate. 3. 1 shows the frequency distribution for drought tolerance at germination of the F4 families of Gharib/ Sepidrud. 72 °C for 2 min (extension) and at least 72 °C for 5 min (final extension). distortion was detected using the χ2 test for goodness of fit to expected allelic frequency for SSR of 1:2:1 and for AFLP of 1:3. 20. PCR products were separated on 6% denaturing poly acrylamide gels and visualized by the silver staining method. 3. 3.   . From 575 SSR markers.0. 131 markers displayed polymorphism. and then stored at 4 °C. Parental surveys were conducted to identify polymorphism between the parents using amplified fragment length polymorphism (AFLP) markers. All the 131 polymorphic marker loci involved in construction of the genetic map tested for segregation distortion. 55 °C for 30 s (primer annealing with most of the primers while some were adjusted).

QTLs mapping under non-stress and drought stress   Table 1.46 ± 0.55 ± 0. was phenotypically more tolerant than the male parent.83) × 10-3 3.14) × 10-3 **.04 Coleoptile length (cm) 0. The mean values and standard deviation (SD) of each trait in the parents and F4 lines together with ttest for evaluating the significant differences between parents are shown in Table 1. The markers were distributed on each chromosome based on the MacCouch’s map ( McCouch et al. the population showed typical normal distribution for all the six traits investigated (Fig.29 ± 0. for all evaluated traits.76** 39.Zahra MARDANI. For the six traits.03 ± 0. therefore all traits evaluated in both environments could be analyzed for QTL mapping. Phenotypic distribution of six traits related to drought tolerance in 148 F4 families under non-stress.67 ± 0. Phenotypic analysis of physiological traits related to drought tolerance in parents and F4 families.52 ± 0.01 (0.12) × 10-3 7. Indicate significant differences between the two parental lines at P < 0. significant transgressive segregation with values either larger or smaller than those of the parents. Gharib had greater values than Sepidrud. et al. Mapping of QTLs of Germination Characteristics in Rice Fig.01 1.01.600) × 10-3 T-value (P1 – P2) 27. Sepidroud. differences between parents for all the studied traits were significant (Table 1).08 1.031 ± 0.75 ± 0. The F4 families showed significant variability for the six drought tolerance parameters examined in the present study.03** 26.31** 72.48 cM between marker loci (Fig. Trait F4 (Mean ± SD) 5.02 Coleorhiza length (cm) 0.81 ± 0.92** -75.16 ± 0.00 ± 0. Also.001 (0.48 ± 0.7 cM of the rice genome with an average interval of 10.14 Germination percentage 86.011 ± 0. The female parent. Gharib.13 (94.01 4. suggesting involvement of multiple genes with quantitative inheritance. 2002 ). Parent Sepidroud (Mean ± SD) Gharib (Mean ± SD) Germination rate (3.041 ± 0.35) × 10-3 4.94 2.61 ± 0. 2. The linkage map covered 12 chromosomes and spanned 2475.20** 29.41 Radicle length (cm) (0.06 Plumule length (cm) 1.10 ± 0.43**   . All the parameters showed transgressive segregation. 2). Linkage map There are 12 linkage groups.20 98. 3).58 ± 0.62 ± 0. A linkage map of 12 rice chromosomes was constructed from genotypic data of 148 F2 plants with 131 SSR and 105 AFLP polymorphic markers. In the nonstress environment.

respectively. Germination percentage (GP) Three QTLs for GP were identified on three chromosomes under drought condition. the QTL detected by the same marker interval across environments indicates that QTL for this trait is stable and not essentially affected by environmental factors. the QTL on chromosome 1 was located between the two markers RM237–RM246 and determined 19.73% of the total variation.80% of the total phenotypic variation. Two QTLs qGR-1 and qGR-4.19 and 3. Moreover. No. The increased allele effect of these QTLs was contributed by Gharib.40% and 17. one QTL were identified for GR. The results of QTL mapping under drought stress and non-stress are shown in (Table 2).64% and 18.03%. respectively. showed large effects on GR and explained 15. The alleles from Gharib at two QTLs had positive effects.) are shown at the top and the Kosambi values (cM) and markers are indicated at the right and left sides of the chromosomes. Linkage map and QTLs related to drought tolerance in the F2:4 population from the cross Gharib × Sepidroud. The QTLs for six studied traits are signed on the chromosomes under drought and non stress. respectively.74 and phenotypic variation explaining 17. In non-stress. 2013 Fig 3. 5 and 7 under non-   . Three QTLs for germination percentage were identified on chromosomes 4. 20. Vol. Of these. Germination rate (GR) Two QTLs were detected for GR on chromosomes 1 and 4 under drought stress condition. two QTLs (qGP-4 and qGP-7) mapped on chromosomes 4 and 7 with LOD values of 4. The numbers of chromosomes (Chr. Major QTL (qGR-1) mapped to the same region of chromosome 1 in both conditions.Rice Science. 6.

Coleoptile length.67 0.44 SPD qCOL-5 RM6320–E38-M61-11 5 5. Chr.03 GHB qGP-9-b RM3903–RM160 9 4.10 -0.1 4.14 1.48% and 18.98 GHB qGP-7 RM70–RM3608 7 5. Radicle length. Chromosome.12 0.01 0.76 1.19 0.24 0.74 0. GP.12 GHB qGP-5 RM480–E36-M59-9 5 1.86 0.02 0.17 0.10 10.1 3.87 -1.9 3.22% of the total phenotypic variation. Moreover.80 GHB GP qGP-4 E37-M60-3–RM8213 4 4.10 4.76 GHB qCL-4 RM8213–E37-M60-3 4 1. Coleoptile length (CL) Under drought conditions.65 GHB PL qPL-8 E36-M59-7–E38-M61-1 8 3. one QTL was mapped for length plumule on chromosome 8.6 4.03 -2. while the Sepidrud alleles of QTL on chromosome 5 decreased PL under severe drought stress.2 3.9 4.18 0.58 5.45 0. PL.01 -0.19 0. under non-stress.03 2.39 0.19 0.33 SPD GR.28 16. putative QTL showed over dominance effects. Besides. two QTLs were identified   .7 3. The Gharib alleles of QTL on chromosomes 1 increased PL. QTL qRL-12 was detected in vicinity of E36-M61-9–RM101 on chromosome 12 and explaining 15. Plumule length (PL) Two QTLs. Under non-stress.25 0.3 4.04 0.13 0. In all the three QTLs.40 GHB qGR-4 RM252–E37-M61-6 4 1. the QTL qGP-7 was as well detected under non-stress with main effect. QTL qCOL-3 was commonly detected in both conditions. d DPE. b QTL position from the nearest flanking marker (cM).51 15.13 -9. one each on chromosome 1 (RM212– RM1268) and chromosome 5 (E36M59-10–RM440) were detected for PL under drought stress conditions and explained 15.86 -0.15 17. Germination percentage. respectively.3 4.09 9.78 2.3 3.41 18. RL.45 -1.49 0.45 1.12 -1.6 3. under drought conditions. This QTL showed positive effects on the PL and explained 15.11 0.78 17.28 GHB CL qCL-4 RM8213–E37-M60-3 4 1.81 1. Furthermore. putative QTL showed over dominance effects. Plumule length.28 -2.24 -0.73 GHB GP qGP-4 E37-M60-3–RM8213 4 4.7 3.1 3.38 1. a Underlined markers are more closer to QTL. Putative QTLs for germination traits in F2:4 population under drought stress and non-stress condition.10 0.6 3. respectively.98 GHB PL qPL-1 RM212–RM1268 1 1. one QTL associated with radicle length was found on chromosome 12.9 3.04 0.43 15.56 0.2 4. Further.48 GHB qPL-5 E36-M59-10–RM440 5 3.7 3..23 2.73 16. Germination rate.23 0. Radicle length (RL) Under drought conditions.07 0.59 10. which identified on chromosome 3 located at the interval RM7–RM282 and comprised 18. the alleles from Gharib increased coleorhiza length and qCOL-5 exhibited partial dominance for increase this trait. Coleorhiza length (COL) Two QTLs were identified for COL on chromosomes 3 and 9 under stress conditions.  Trait QTL Markers interval a Chr.47 0.37 18.66 on average.64 GHB qGP-7 RM70–RM3608 7 5.19 15. two QTLs qGP-4 and qGP-7 were also identified under both conditions.18 21.40 17. The major QTL (qCOL-5) was detected only under non-stress and explained 21. Direction of phenotypic effect.19 0.1 4.12% of the total phenotypic variance.28 18.49 11.28% of the total phenotypic variance. GHB and SPD indicate Gharib and Sepidroud.47 13.5 3.24 SPD Non-stress  GR qGR-1 RM237–RM246 1 0. COL.11 GHB CL qCL-1 RM1287–RM237 1 0.22 1.11 0. The QTL detected by the same marker interval across environments indicates that QTL for this trait is stable and not essentially affected by environmental factors. The QTL qGP-4 showed the largest effect on germination percentage and explained 16.17 0.01 0. et al. stress. the alleles from Gharib increased germination percentage by 0.97 9.08 0. One of the QTLs.20 1.91 3.32 0.7 3.93% of the total phenotypic variance. Position (cM) b LOD Additive effect (A) Dominance effect (D) D/|A| (R2) c DPE d Drought stress  GR qGR-1 RM237–RM246 1 0.80 GHB qRL-12 E36-M61-9–RM101 12 11.37 1. Coleorhiza length.98 5.05 15.23 1.93 GHB COL qCOL-3 RM7–RM282 3 3.84 0.66 19.63 -0.7 4.33 0.20 15.34% of phenotypic variation also allele inherited from Gharib parent increased COL. two QTLs were identified for RL on chromosomes 5 and 12. Mapping of QTLs of Germination Characteristics in Rice Table 2.05 0.75 GHB RL qRL-5 RM421–E37-M61-5 5 5. c Phenotypic variance explained by each QTL. Two QTLs were identified for coleorhiza length on chromosomes 3 and 5 under nonstress.27 0.34 GHB qCOL-9 RM434–E36-M59-11 9 3. CL.20 GHB RL qRL-12 E36-M61-9–RM101 12 11.3 4.Zahra MARDANI.04 -2.12 1.91 16.65 % of total variance.12 -0.22 GHB COL qCOL-3 RM7–RM282 3 3.10 0.

7 and 10. No. Comparing two treatments. which might play essential roles in germination percentage. qGP-4. Wen et al (2008) detected one QTL for seed dormancy on chromosome 1. 1996. putative QTL showed over dominance effects. This result differed for the locations of the QTLs detected for radicle length by other researchers (Redona and Mackill. The QTLs detected for this trait during our study is similar to the results reported by Angaji et al (2008) and Zahng et al (2005). In the present study. The region and variance phenotypic QTL (qGP-4) was also similar to QTL detected by Teng et al (2001).33% of the phenotypic variation was determined by this QTL under drought stress and non-stress. showed a range of partial to over dominance effects. qPL-5. other factors affect seedling establishment. six major QTLs (qGR-1. qGR-4. 9 and 12 under non-stress conditions. It is obvious because they studied different trait as well as the ways of phenotyping were different. Also.24 and 15. this study reports for the first time the detection of QTL for this trait on chromosomes 3. qCL-1 and qCL-4) were located on chromosomes 1. but different to the findings of Liang et al (2006). qRL-12. suggesting the   . Wang et al (2011) detected seven QTLs for this trait by using RIL (F2:9) population. One QTL for plumule length on chromosome 8 under non-stress conditions was found in similar region reported by Zhang et al (2005). 5. Price et al (2002). In fact.6 that explained 9% of the total phenotypic variance. In this study. and some of them are being reported for the first time. They were on chromosomes 4. the region RM212 on chromosome 1 found in the present study to harbor one QTL for PL trait. degree of dominance (D/|A|) was high. 2005). Vol. we found two QTLs for CL on chromosomes 1 and 4 under both conditions against the chromosomes reported by Redona and Mackill (1996). qRL-12. Under the drought stress. In addition. qGP-7. For instance. Most of the QTLs identified in the current study. qCL-4 was in a similar position to that found under non-stress and showed greater effect. Coleoptile growth. 7 and 12 associated with respectively. 8 and 12. Lafitte et al (2002) and Wang et al (2005) have reported QTLs for drought tolerant in the same region. 3. 4. 5 (two QTLs). of which 13 QTLs were detected in drought stress environment and 9 QTLs under nonstress conditions.1) for GR detected in this study. only six QTLs (qGR-1. respectively. 20. 5. 4. One major QTL for GR was found on chromosome 1 under both conditions. qGP-7 and qRL-12) were detected in stress and non-stress environments and they were located on chromosomes 1. QTL qGP-4 for GP was found under both conditions. 6. 4. In addition. In most of the cases. but putative QTLs were found in agreement with chromosomes reported by Hu et al (2007). qCOL-3. Sabouri and Sabouri (2008) detected two QTLs for shoot length on chromosomes 3 and 10. DISCUSSION This study compares the QTLs identified and discusses the genetic relationships between drought tolerance traits at the germination stage under two conditions. Furthermore. indicating complexity of the traits under consideration (Table 2). qCOL-3. and additional studies are needed to clarify the allelic relationship of these QTLs. the locus (qCL-1) mapped on chromosome 1 in this study could be approximately the same locus reported by Hu et al (2007). Totally of 22 QTLs were detected under both conditions. 5 and 9. three QTLs were detected associated with COL. Putative QTLs for this trait are stable and not essentially affected by environmental factors. 6 and 7. Thus. 10 main effect QTLs (qGR-1. this QTL was similar to QTL in this study. four QTLs related to RL were detected on chromosomes 3. this region appears to be a good candidate for breeding for drought tolerance through markerassisted selection as well as for fine mapping and positional cloning of the underlying genes. 3. 3. In this research. Similar QTL was found in interval RM1287–RM237 and 16. In this study. which has been reported by many earlier workers. qPL-1. qPL-8. qGP-4 with LOD = 2. They were placed close to the marker RM8213 on chromosome 4 on both conditions. qCL-4. 5. which were very close to the QTL (qGR. 2013 for CL on chromosomes 1 and 4. qGP-4. Nine QTLs associated with coleoptile length were detected on chromosomes 1 (two QTLs). 7 and 12.Rice Science. 2 (two QTLs). It is hard to precisely compare the chromosomal location of these QTLs because of different genetic materials and lack of common markers used for mapping. The QTLs did not have the same map location and there was no similarity with results of previous researchers. qGP-4. As many as 22 QTLs were detected to be associated with drought tolerant. qCOL-5 and qCL-4) were detected on chromosomes 1. Zhang et al. While under non-stress conditions.

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