Figures
Abstract
Cold stress causes various injuries to rice seedlings in low-temperature and high-altitude areas and is therefore an important factor affecting rice production in such areas. In this study, root conductivity (RC) was used as an indicator to map quantitative trait loci (QTLs) of cold tolerance in Oryza rufipogon Griff., Dongxiang wild rice (DX), at its two-leaf stage. The correlation coefficients between RC and the plant survival rate (PSR) at the seedling and maturity stages were –0.85 and –0.9 (P = 0.01), respectively, indicating that RC is a reliable index for evaluating cold tolerance of rice. A preliminary mapping group was constructed from 151 BC2F1 plants using DX as a cold-tolerant donor and the indica variety Nanjing 11 (NJ) as a recurrent parent. A total of 113 codominant simple-sequence repeat (SSR) markers were developed, with a parental polymorphism of 17.3%. Two cold-tolerant QTLs, named qRC10-1 and qRC10-2 were detected on chromosome 10 by composite interval mapping. qRC10-1 (LOD = 3.1, RM171-RM1108) was mapped at 148.3 cM, and qRC10-2 (LOD = 6.1, RM25570-RM304) was mapped at 163.3 cM, which accounted for 9.4% and 32.1% of phenotypic variances, respectively. To fine map the major locus qRC10-2, NJ was crossed with a BC4F2 plant (L188-3), which only carried the QTL qRC10-2, to construct a large BC5F2 fine-mapping population with 13,324 progenies. Forty-five molecular markers were designed to evenly cover qRC10-2, and 10 markers showed polymorphisms between DX and NJ. As a result, qRC10-2 was delimited to a 48.5-kb region between markers qc45 and qc48. In this region, Os10g0489500 and Os10g0490100 exhibited different expression patterns between DX and NJ. Our results provide a basis for identifying the gene(s) underlying qRC10-2, and the markers developed here may be used to improve low-temperature tolerance of rice seedling and maturity stages via marker-assisted selection (MAS).
Citation: Xiao N, Huang W-n, Zhang X-x, Gao Y, Li A-h, Dai Y, et al. (2014) Fine Mapping of qRC10-2, a Quantitative Trait Locus for Cold Tolerance of Rice Roots at Seedling and Mature Stages. PLoS ONE 9(5): e96046. https://doi.org/10.1371/journal.pone.0096046
Editor: Mingliang Xu, China Agricultural University, China
Received: October 20, 2013; Accepted: April 3, 2014; Published: May 1, 2014
Copyright: © 2014 Xiao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Natural Science Foundation(BK2011426 and BK2011427) of Jiangsu Province, and the Priority Academic Program Development of Jiangsu Higher Education Institutions and International Atomic Energy Agency (12228/RO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Rice (Oryza sativa L.) is one of the three major food crops in the world and feeds about 21% of the global population in terms of per capita energy [1]. Cold stress is a common problem for rice cultivation and is a crucial factor affecting global food production [2]. A primary objective for rice breeding in low-temperature and high-altitude areas is to improve cold tolerance of rice cultivars. Therefore, mining cold-tolerant germplasms and mapping related genes would provide an opportunity to improve rice yield through marker-assisted selection (MAS) [3]–[5]. Recently, several quantitative trait loci (QTLs, e.g., Ctb-1, Ctb-2, qCTB3, qCTB7, and qCTB8) related to cold-induced rice damages to aboveground organs–including seedling mortality and growth, leaf wilting, and necrotic yellowing–were mapped [3], [6]–[11]. To date, however, only Ctb-1, which improves the seed setting rate by enhancing spikelet fertility, has been isolated and cloned and has been proven to be involved primarily in the ubiquitin-proteasome pathway [8]. QTLs contributing to cold tolerance of seedlings are located on several rice chromosomes, accounting for 4–49.3% of phenotypic variations [4], [12]–[22]. In addition, certain chromosomes also bear germination cold-tolerant QTLs [23], [24]. Cold tolerance of rice is controlled by multiple genes, and different cold-tolerance-related mechanisms take effect during the growth period of rice.
Nevertheless, cold-tolerant QTLs have not been reported in rice roots, an important underground organ that protects rice from cold stress. At low temperatures, the integrity of plant cellular membranes can be compromised by the accumulation of peroxide and oxide radicals, which increasing membrane permeability [25]–[27]. Rice roots are more sensitive to low temperature than leaves, and cold resistance at the seedling stage can be directly determined by the contribution of roots to cold resistance [28]. Therefore, root conductivity (RC) tests have been used to determine the degree of cold-induced injuries in wheat [29]. However, cold-tolerant QTLs have not been reported in rice roots based on the RC test.
Dongxiang wild rice (DX), a Chinese common wild rice (Oryza rufipogon Griff., AA genome) grown in Jiangxi Province (116°36′ E, 28°14′ N), is the northernmost wild rice known in the world [30]. It possesses extremely high tolerance to cold stress and is able to safely survive winter temperatures as low as –12.8°C [31]. In our present study, we reported fine mapping of a major QTL, qRC10-2, related to root cold tolerance using DX as a cold-tolerant donor and the cold-sensitive indica variety Nanjing 11 (NJ) as a recurrent parent. We also evaluated the effects of qRC10-2 at the seedling and maturity stages. The analysis of the QTL derived from the wild rice will provide novel alleles for improving rice cold tolerance by MAS and also genetic materials for understanding the molecular mechanism of cold tolerance in rice.
Materials and Methods
Plant Materials
The cold-tolerant variety DX was used as a donor parent to cross with the cold-sensitive NJ in the summer of 2006 (Fig. 1). Then, a primary mapping population was constructed by employing the advanced backcross approach described by Thomson et al. [32]. The F1 progeny was first backcrossed to NJ to produce 151 BC1F1 plants, which were then grown in a greenhouse, and subsequently each BC1F1 plant was individually backcrossed to the recurrent parent until BC2F1 was obtained. No cold phenotype was selected for RC during population development because phenotype evaluation is destructive and thus cannot be conducted on individual plants. All BC2F2 seeds from 151 BC2F1 plants were individually harvested, and the resulting population of 151 individual BC2F2 families was used for RC phenotypic characterization in September of 2008.
To eliminate the effect of qRC10-1, a single BC2F2 plant that contained the DX-introgressed segment at the locus qRC10-2 was selected for continuous backcrossing with NJ followed by MAS using the markers RM25570 and RM304. This yielded 14 BC4F2 backcross inbred lines (BILs). Later, 150 simple-sequence repeat (SSR) markers covering the whole genome were used to determine the genetic background recovery rates, which varied from 89.33% to 94.67% (Fig. S1). Among these BILs, L188-3 (hereafter, L188) had a background recovery rate of 94.67%, which was found to have low RC. A large BC5F2 fine-mapping population of 13,324 progenies was constructed by crossing NJ with L188 in the summer of 2012. Then, molecular markers were used to detect recombinant events covering qRC10-2. All needed recombinants were separately harvested and evaluated for RC, and the BC5F4 seeds from the BC5F3 lines were harvested to calculate the plant survival rate (PSR) at the seedling and maturity stages. All plants were grown in the Yangzhou Wantou experimental fields at the Lixiahe Agricultural Research Institute of Jiangsu Province (119°42′ E, 32°39′ N) in summer and in Sanya of Hainan Province (110°02′ E, 18°48′ N) in winter.
Evaluation of Cold Tolerance
The RC phenotypes of the BC2F1 and BC5F2 plants were measured as the average RC of the derived BC2F2 and BC5F3 plants across three replicates, respectively. Twenty seeds were randomly selected and placed in an illuminated incubator for growth to day 9. The seedlings were then given simulated diurnal alternating illumination treatment: 12 h dark at 4±1°C versus 12 h with 25,000 Lx of illumination at 4±1°C with relative humidity of 75–85%. After 48 h treatment, the 2-cm taproot section was immediately cut from each plant, and placed into an Eppendorf centrifuge tube with 2 mL deionized H2O (conductivity of 1.5 µs/cm). After soaking for 1 h, the RC of each plant was measured with a conductivity meter (Mettler Toledo 326) and data were adjusted for RC of deionized H2O.
Approximately 100 BC5F4 seeds were selected from each BC5F3 line with high or low RC to evaluate the PSR using the criteria of RC<10 and RC≥10 for cold tolerance and cold sensitivity, respectively. At day 9, the seedlings were cold-treated as described above and then returned to constant 28°C for recovery under illumination at 25,000 Lx for 72 h. The plants were divided into a sensitive group (with completely-withered leaves) and a tolerant group (normal growth). PSR was estimated as the proportion of the plants showing a tolerant phenotype to all the plants by percentage (0–100%). The PSR of each BC5F3 line was measured in three replicates.
Root cold tolerance at maturity was evaluated as follows: The BC5F4 seedlings obtained from the BC5F3 lines were planted in the field on May 10, 2013. Uniform seedlings were then transplanted into a hydroponic greenhouse on June 10 at Yangzhou University. The hydroponic pool was 880 cm long, 130 cm wide, and 50 cm deep. The cement anchoring the rice plants was 135 cm long, 16.7 cm wide, and 2.5 cm thick, and each had a plate of 14 holes that were 4.0 cm in diameter and away from each other by 10 cm. A single seedling was planted in each hole, which was fixed by cork and sponges. Each group (low RC vs. high RC) contained 126 plants in three replicates. The hydroponic solution (pH 5.8) contained 10 p.p.m. of nitrogen (NH4NO3), phosphorus (NaH2PO4), potassium (K2SO4), calcium (CaCl2), and magnesium (MgSO4) each, 2 p.p.m. of iron (Fe(III)-EDTA), and 0.5 p.p.m. of manganese (MnCl2), and the solution was changed every other day. An oxygen pump was used to provide a steady flow of hydroponic nutrients, which maintained the nutrient concentrations and pH conditions in the pool during plant growth from seedling to maturity. These concentrations were elevated to twice the starting concentrations 60 days later. After 15-day post-heading, the cold hydroponic solution at 5±1°C was circulated around the roots for 24 h. After recovery at 28°C for two days, the PSR was measured.
Linkage Mapping and QTL Analysis
A genetic linkage map was constructed with Mapmaker/Exp 3.0 [33]. The segregation data of each marker were statistically analyzed to measure the segregation distortion from Mendelian expectations. Table S1 lists the SSR markers. QTL analysis was conducted by composite interval mapping using QTL Cartographer 2.5 [34]–[36]. The logarithm of odds (LOD) threshold of 2.5 was used to identify QTLs while avoiding false positives.
DNA Extraction and Molecular Marker Analysis
DNA was extracted from leaves following the cetyltrimethylammonium bromide (CTAB) method [37]. All leaves were stored at –80°C before extraction. Additional SSR markers between RM25570 and RM304 were chosen from the Gramene database (http://www.gramene.org). The sequences of indica cv. 93-11 and japonica cv. Nipponbare were downloaded from IRGSP1.0 (http://rapdb.dna.affrc.go.jp/) and used to design insertion/deletion (indel) markers and real-time polymerase chain reaction (PCR) markers using Premier 5.0 (Tables S2 and S3).
PCR and electrophoresis were performed following the method of Panaud et al. [38]. PCR was performed in a 20 µL volume containing 20 ng of template DNA, 0.15 µL of 10 mM dNTPs, 2 U of Taq DNA polymerase, 2 µL of 10×PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, and 0.01% gelatin), and 1.5 µL of 2 µM forward and reverse primers each. The cycling conditions were 5 min at 94°C, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min. The PCR products were subjected to electrophoresis on 6% denaturing polyacrylamide gels, which were then stained with silver [39].
Data Analysis
Differences between parents and BILs for cold-related measurement were compared via t-test using SPSS 11.0 for Windows (SPSS Inc., 2002). The correlation coefficient between RC and the PSR was calculated using the SAS software (SAS Institute, 2000).
Gene Expression Analysis
At the two-leaf stage after cold treatment for 0, 6, 12, or 24 h, the total RNA was extracted from 100 mg roots and 100 mg leaves using the RNeasy extraction kit (Invitrogen, Carlsbad, CA, USA), and any remaining genomic DNA was removed by DNaseI treatment. Total RNA (3 µg) was used as a template for cDNA synthesis using M-MLV transcriptase (TaKaRa Biotechnology, Dalian, China) with oligo (dT)18 primer. On an ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA), real-time PCR was performed in a 20 µL volume containing 2 µL of first-strand cDNA, 10 µL of 2×SYBR Premix Ex Taq (TaKaRa, Shiga, Japan), 0.4 µL of 50×ROX reference dye, and 2 µL of each primer (2.5 µM). The used amplification protocol was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Rice Actin 1 was used as an internal control, and each experiment was repeated at least three times.
Results
RC is a Reliable Indicator of Cold Resistance in Rice Seedlings
RC was significantly different for DX and NJ at day 8 (P = 0.01, Table 1). The stem root was quite long and had excessive root hairs after 9 days, making it difficult to measure RC in an Eppendorf tube. Therefore the cold-treatment period was determined to be 9 days. The difference in RC between cold-sensitive and cold-tolerant plants was apparent for 1 h cold treatment (Fig. 2A). The RC for cold-tolerant seedlings increased to 8.1 at 24 h, but at 48 h the RC for DX decreased to 4.4 whereas that for NJ increased to 12.9. In addition, NJ had high RC of 16.2 at 72 h whereas the cold-tolerant DX had low RC of 5.3. The high RC in NJ can be explained by the observation that more intracellular liquids leak into the environment per unit time (Fig. 2B).
A. RC measured as a function of cold treatment duration. B. RC measured as a function of soaking time. An asterisk (*) indicates that the RC between NJ and DX was significantly different (P<0.01).
QTLs for Cold Tolerance
After recovery at 28°C, DX grew normally, whereas NJ showed cold-induced injuries (Fig. 3A). The RC was 2.9 for the cold-tolerant DX and 12.1 for NJ, and the distribution of the RC phenotypes among the BC2F1 population was continuous and skewed towards tolerance (Fig. 3B).
A. Cold-induced injury phenotype of DX and NJ after exposure to 4°C for 48 h. B. Frequency distribution of the RC values among BC2F1 population. Arrows indicate the mean scores of DX and NJ.
A total of 653 SSR markers were used to screen polymorphisms between the two parents. Only 113 markers were codominant, with the polymorphism of only 17.3%; all the 113 developed markers were mapped essentially and uniformly to the 12 chromosomes. Based on this, two seedling cold-tolerant QTLs, named qRC10-1 and qRC10-2 (Fig. 4A), were detected on chromosome 10 via composite interval mapping. qRC10-1 (LOD = 3.1) was mapped at 148.3 cM between RM171 and RM1108, and qRC10-2 (LOD = 6.1) was mapped at 163.3 cM between RM25570 and RM304, which accounted for 9.4% and 32.1% of phenotypic variances, respectively (Table 2).
A. Location of qRC10-2 on rice chromosome 10. B. The linkage map shows six indels and a SSR marker. C. Progeny test of homozygous recombinants delimited qRC10-2 to the region between the markers RM25570 and RM304. The number of recombinants in each group and the phenotypic difference of each group from controls are shown. D. Further fine mapping of qRC10-2. E. Six genes in the candidate region. aSignificantly different from P1 at P = 0.01; bsignificantly different from P2 at P = 0.01. P1: DX; P2: NJ.
Fine Genetic and Physical Mapping of qRC10-2
To obtain a fine map of qRC10-2, 45 molecular markers were developed between RM25570 and RM304. As shown in Table S3, 10 of these markers showed codominant polymorphisms between DX and NJ, consisting of 2 SSR markers (RM25681 and RM25628) and 8 indel markers (qc8, qc18, qc19, qc25, qc27, qc45, qc48, and qc55). The arrangement of these markers is shown in Fig. 4B and 4D.
Using the markers RM25570 and RM304, a total of 211 BC5F2 BIL recombinants were identified. Among them, 64 showed the homozygous genotype for DX or NJ alleles and were divided into six groups (LR1, LR2, HR1, HR2, R1 and R2) based on the genotypes of qc8, qc18, qc19, qc25, qc27, RM25681 and qc55 (Fig. 4C). Group LR1 (RC = 4.1) had DX alleles to the left of qc55, and group LR2 (RC = 3.5) had DX alleles to the right of RM25681, indicating that qRC10-2 was located upstream of qc55. Groups R1 and R2 had recombinant loci between RM25681 and qc55, and there was a large range in RC for these groups. Based on this, we deduced that qRC10-2 was located between RM25681 and qc55 (Fig. 4C).
Based on the genotypes of the three polymorphic markers qc45, qc48 and RM25628 (Fig. 4D), groups R1 and R2 were further divided into four subgroups: R1-1, R2-1, R1-2 and R2-2. Then, according to the RC data, the candidate region was narrowed to 68.7 kb between qc45 and RM25628, and qc45, qc48 and RM25628 were used to rescreen all mapped plants. Finally, we obtained four BC5F2 plants with high RC of 11.4 (named group R3) heterozygous in the qc48 allele. Therefore, qRC10-2 between qc45 and qc48 was restricted to 48.5 kb based on the Nipponbare sequence, in which a bacterial artificial chromosome clone (AC146481.2) contained six candidate genes, as shown in Fig. 4D and 4E.
Effects of qRC10-2 on Cold Tolerance at the Seedling and Maturity Stages
Subgroups R1-1, R2-1, R1-2 and R2-2 were used to determine PSRs. The PSRs for R1-1 and R2-1 with high RC values were 25–44% in seedlings and 21–35% in mature plants, whereas those for the subgroups R1-2 and R2-2 with low RC values were 52–60% in seedlings and 48–57% in mature plants. The correlation coefficients between RC and the PSR at the seedling and maturity stages were –0.85 and –0.9 (P = 0.01), respectively (Table 3), indicating that a lower RC value was correlated with a higher PSR. Lines with qRC10-2 grew normally after 24 h under 5±1°C treatment, and lines lacking qRC10-2 showed necrosis or death at the maturity stage (Fig. 5). These results indicated that qRC10-2 contributed to rice cold tolerance at both stages.
Identification of Cold-inducible Genes in the Candidate Region
O. rufipogon Griff., common wild rice, is the ancestor of cultivated rice [40]–[42]. The collinearity of cold-tolerant genes between different species and the conservation in the cold-responsive pathway has been reported [30], [43], [44]. Therefore, Nipponbare (japonica) was selected as the reference genome to screen candidate genes. Table 4 lists the predicted functions of the six candidate genes in IRGSP1.0 (http://rapdb.dna.affrc.go.jp/).
To further identify genes related to cold tolerance, real-time PCR was used to assess gene expression in the leaf and root tissues after 0, 6, 12, and 24 h of cold treatment (Fig. 6). The results suggested that Os10g0489448, Os10g0489650, Os10g0489800, and Os10g0489900 shared a similar gene expression profile in DX, L188 and NJ. Os10g0489500 was induced under cold stress and showed high levels of inducible expression in the leaves of NJ at 6 h and 12 h, whereas no significant variation was observed in DX. This pattern was different from that in the roots, as it was highly expressed in the roots of DX at 6 h but not up-regulated in NJ until 24 h. Os10g0490100 in the roots of NJ showed higher expression than in DX before and after cold treatment. Therefore, Os10g0489500 and Os10g0490100 were considered as the genes associated with cold tolerance in the qRC10-2 region.
Discussion
Cold stress is one of the major environmental concerns in rice cultivation, especially in low-temperature and high-altitude areas [45]. The rice subspecies indica and japonica showed cold injury symptoms below 18°C and 15°C, respectively [46]. Under low-temperature stress, the root cell plasma membrane might become damaged and less fluid, resulting in functionality loss, which in turn caused increased cell membrane permeability and intracellular material’s extravasation into the external environment [47], [48], [49]. In our present study, RC was used as a cold phenotype indicator reflecting the integrity and permeability of root membrane at low temperatures. With prolonged treatment time, the RC values of DX and NJ increased gradually, indicating cumulative cold injuries in both parents. Besides, more serious damages accumulated in the NJ root membrane. During the recovery period, high-RC plants showed wilting leaves and retarded growth, but low-RC plants maintained green leaves. Such a cold-tolerant phenotype had a consistent effect on plants at the maturity stage with qRC10-2. Moreover, the correlation coefficients (–0.85 and –0.9) between RC and the PSR were significant in seedlings and mature plants (P = 0.01), indicating that RC could be used as a reliable index for evaluating cold tolerance in rice.
In previous studies, most cold-tolerant QTLs at the seedling stage were detected using recombinant inbred lines derived from indica/japonica cultivars [12], [18], [50], [51]. However, QTL analysis on a wider range of variations from wild species could be equally important. Koseki et al. identified the cold-tolerant QTL qCtss10 (LOD = 3.0) from W1943 (O. rufipogon) on chromosome 10, which accounted for 9.8% of phenotypic variations [17]. In our study, two QTLs qRC10-1 and qRC10-2 were identified on the same chromosome in DX. A QTL for cold tolerance (qCTSS-10) was also identified in seedlings by Yang et al. [52]. qCTSS-10 for 7°C cold treatment was mapped by high-throughput sequencing in the F3 mapping population from a cross between a cold-tolerant japonica rice variety (Nipponbare) and a cold-sensitive indica variety. The interval of qCTSS-10 (15.48–21.06 Mb) overlapped that of qRC10-1 and qRC10-2, indicating that these QTLs may be controlled by the same cold-tolerant gene. Liu et al. also identified the same QTL (qCST10, LOD = 2.4) with 12% of the phenotypic contribution from the cold-tolerant line IL112 [53]; all early seedlings of the line IL112 survived normally for 9 days at 4–5°C. A molecular marker tightly linked to qCST10 was OSR33, which was the same marker as RM171 linked to qRC10-1 and qRC10-2 according to the Gramene marker data (http://www.gramene.org/). As described above, the same cold-tolerant QTL was identified on chromosome 10 among different parents and cold-treatment conditions based on a different cold tolerance index, suggesting that qRC10-2 was a stable and reliable cold-tolerant QTL for the rice seedling stage. Further studies on the candidate gene(s) in this region will aid in elucidating the molecular mechanism underlying rice cold tolerance.
In the candidate region, Os10g0489500 and Os10g0490100 were differentially expressed between cold-tolerant and -sensitive plants. Os10g0489500 encoded a terpene synthase that was localized to the cytoplasm. In our study, Os10g0489500 was up-regulated in response to cold stress in the leaves of NJ. In the roots, however, its expression in DX and L188 was ∼18-fold higher than that in NJ, suggesting that the root may be a key organ for the Os10g0489500 activity. Yang et al. also identified Os10g0489500 as a gene up-regulated by low temperatures in a cold-tolerant variety [52]. After analyzing differentially-expressed transcripts in indica (K354) and japonica (C418) under controlled conditions (25°C) and cold stress (4°C) for 48 h, Zhang et al. identified two terpene synthase genes (Os02g36140 and Os08g07100) that were associated with signal transduction cascades from K354 during cold-stress responses in seedlings [44]. Another gene differentially-expressed between cold-tolerant and -sensitive plants was Os10g0490100, which encoded virulence factors that degraded the pectin components of the plant cell wall [54], [55]. Pectin functioned as an adhesive in cell wall formation, and the pectin content directly influenced cell wall integrity, which was related to rice cold tolerance as reported previously [56]–[58]. Solecka et al. found that cold acclimation induced an increase in the pectin content in the cell wall and increased tolerance to freezing [59]. A mutation in QUASIMODO1, a gene encoding the glycosyl transferase required for pectin synthesis, caused reduced pectin content, leading to less cell adhesion and faster water loss during cold stress in common rice plants compared with the wild type [58]. Using Zhonghua 10 plants, Liu et al. overexpressed OsBURP16, a polygalacturonase 1β that degraded pectin, and showed that an increase in the polygalacturonase activity led to a decrease in the pectin content in the cell wall, resulting in more severely curled leaves in the transgenic plants [53]. When the transgenic seedlings were exposed to 4°C for 72 h and allowed to recover for 7 days under normal conditions, the PSR of Zhonghua 10 plants was 42%, and that of the transgenic plants OE3 and OE4 was only 28% and 4%, respectively [60]. The above studies suggested that pectin degradation in the cell wall affected cell wall integrity and decreased tolerance to cold stress. In our present study, Os10g0490100, a pectin lyase fold family protein that participated in pectin degradation, was more highly expressed in NJ roots than in DX and L188 roots before and after cold treatment, indicating that fewer pectin was maintained in cold-sensitive plants.
In conclusion, our data provided useful information for cultivating cold-tolerant rice varieties by pyramiding qRC10-2 based on other QTLs and also offered a molecular basis for rice cold tolerance mechanisms at the seedling and maturity stages.
Supporting Information
Figure S1.
Genetic background recovery rates determined among the BC4F2 plants.
https://doi.org/10.1371/journal.pone.0096046.s001
(JPG)
Table S2.
Real-time PCR primer sequences for candidate genes.
https://doi.org/10.1371/journal.pone.0096046.s003
(DOCX)
Table S3.
Primer sequences and sizes of polymorphic markers developed for fine mapping locus qRC10-2.
https://doi.org/10.1371/journal.pone.0096046.s004
(DOCX)
Author Contributions
Conceived and designed the experiments: NX ZYD JMC. Performed the experiments: YG XXZ YHL. Analyzed the data: WNH LY. Contributed reagents/materials/analysis tools: GQL CHP. Wrote the paper: NX JMC. DNA extraction and molecular marker identification: AHL YD.
References
- 1.
Maclean JL, Dawe DC, Hardy B, Hettel GP (2002) Rice Almanac: source book for the most important economic activity on earth. In. 3rd edn. Int. Rice Res. Inst., pp6–8.
- 2. Nakagahra M, Okuno K, Vaughan D (1997) Rice genetic resources: history, conservation, investigative characterization and use in Japan. Plant Mol Biol 35 (1–2): 69–77
- 3. Kuroki M, Saito K, Matsuba S, Yokogami N, Shimizu H, et al. (2007) A quantitative trait locus for cold tolerance at the booting stage on rice chromosome 8. Theor Appl Genet 115 (5): 593–600
- 4. Jiang L, Xun M, Wang J, Wan J (2008) QTL analysis of cold tolerance at seedling stage in rice (Oryza sativa L.) using recombination inbred lines. Journal of Cereal Science 48 (1): 173–179
- 5. Suh JP, Jeung JU, Lee JI, Choi YH, Yea JD, et al. (2010) Identification and analysis of QTLs controlling cold tolerance at the reproductive stage and validation of effective QTLs in cold-tolerant genotypes of rice (Oryza sativa L.). Theor Appl Genet 120 (5): 985–995
- 6. Saito K, Miura K, Nagano K, Hayano-Saito Y, Araki H, et al. (2001) Identification of two closely linked quantitative trait loci for cold tolerance on chromosome 4 of rice and their association with anther length. Theor Appl Genet 103 (6–7): 862–868
- 7. Saito K, Hayano-Saito Y, Maruyama-Funatsuki W, Sato Y, et al. (2004) Physical mapping and putative candidate gene identification of a quantitative trait locus Ctb1 for cold tolerance at the booting stage of rice. Theor Appl Genet 109 (3): 515–522
- 8. Saito K, Hayano-Saito Y, Kuroki M, Sato Y (2010) Map-based cloning of the rice cold tolerance gene Ctb1. Plant science 179 (1): 97–102
- 9. Zhou L, Zeng Y, Zheng W, Tang B, Yang S, et al. (2010) Fine mapping a QTL qCTB7 for cold tolerance at the booting stage on rice chromosome 7 using a near-isogenic line. Theor Appl Genet 121 (5): 895–905
- 10. Shirasawa S, Endo T, Nakagomi K, Yamaguchi M, Nishio T (2012) Delimitation of a QTL region controlling cold tolerance at booting stage of a cultivar, ‘Lijiangxintuanheigu’, in rice, Oryza sativa L. Theor Appl Genet 124. (5): 937–946
- 11. Dai L, Lin X, Ye C, Ise K, Saito K, et al. (2004) Identification of quantitative trait loci controlling cold tolerance at the reproductive stage in Yunnan landrace of rice, Kunmingxiaobaigu. Breeding Science 54 (3): 253–258
- 12. Qian Q, Zeng D, He P, Zheng X, Chen Y, et al. (2000) QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid between indica and japonica rice. Chin Sci Bull 45 (5): 448–453
- 13. Miura K, Lin SY, Yano M, Nagamine T (2001) Mapping quantitative trait loci controlling low temperature germinability in rice (Oryza sativa L.). Breeding Science 51 (4): 293–299
- 14. Teng S, Zeng D, Qian Q, Kunihifo Y, Huang D, et al. (2001) QTL analysis of rice low temperature germinability. Chin Sci Bull 46 (21): 1800–1803
- 15. Andaya VC, Thomas HT (2007) Fine mapping of the qCTS4 locus associated with seedling cold tolerance in rice (Oryza sativa L.). Mol Breeding 20 (4): 349–358
- 16. Ji SL, Jiang L, Wang YH, Zhang WW, Liu X, et al. (2009) Quantitative trait loci mapping and stability for low temperature germination ability of rice. Plant Breeding 128 (4): 387–392
- 17. Koseki M, Kitazawa N, Yonebayashi S, Maehara Y, Wang ZX, et al. (2010) Identification and fine mapping of a major quantitative trait locus originating from wild rice, controlling cold tolerance at the seedling stage. Mol Genet Genomics 284 (1): 45–54
- 18. Suh JP, Lee CK, Lee JH, Kim JJ, Kim SM, et al. (2012) Identification of quantitative trait loci for seedling cold tolerance using RILs derived from a cross between japonica and tropical japonica rice cultivars. Euphytica 184 (1): 101–108
- 19. Andaya VC, Tai TH (2006) Fine mapping of the qCTS12 locus, a major QTL for seedling cold tolerance in rice. Theor Appl Genet 113 (3): 467–475
- 20. Cai W, Morishima H (2002) QTL clusters reflect character associations in wild and cultivated rice. Theor Appl Genet 104 (8): 1217–1228
- 21. Zhang ZH, Su L, Li W, Chen W, Zhu YG (2005) A major QTL conferring cold tolerance at the early seedling stage using recombinant inbred lines of rice (Oryza sativa L.). Plant Science 168 (2): 527–534
- 22. Lou Q, Chen L, Sun Z, Xing Y, Li J, et al. (2007) A major QTL associated with cold tolerance at seedling stage in rice (Oryza sativa L.). Euphytica 158 (1–2): 87–94
- 23. Jiang L, Liu SJ, Hou MY, Tang JY, Chen LM, et al. (2006) Analysis of QTLs for seed low temperature germinability and anoxia germinability in rice (Oryza sativa L.). Field Crops Research 98(1): 68–75
- 24. Wang ZF, Wang FH, Zhou R, Wang JF, Zhang HS (2011) Identification of quantitative trait loci for cold tolerance during the germination and seedling stages in rice (Oryza sativa L.). Euphytica 183 (3): 405–413
- 25. Saruyama H, Tanida M (1995) Effect of chilling on activated oxygen-scavenging enzymes in low temperature-sensitive and -tolerant cultivars of rice (Oryza sativa L.). Plant Science 109 (2): 105–113
- 26. Murata N, Ishizaki-Nishizawa O, Higashi S, Hayashi S, Tasaka Y, et al. (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356 (6371): 710–713
- 27. Murata N, Wada H (1995) Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria. Biochem J 308 (Pt 1): 1–8.
- 28. Saltveit ME (2001) Chilling injury is reduced in cucumber and rice seedlings and in tomato pericarp discs by heat-shocks applied after chilling. Postharvest Biology and Technology 21 (2): 169–177
- 29. Yu J, Zhang L, Cui H, Zhang YX, Cang J, et al. (2008) Physiological and biochemical characteristics of Dongnongdongmai 1 before wintering in high-cold area. Acta Agronomica Sinica 34 (11): 2019–2025
- 30. Tian F, Zhu ZF, Fu YC, Wang XK, Sun CQ (2006) Construction of introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits. Theor Appl Genet 112 (3): 570–580.
- 31. Li F, Guo S, Zhao Y, Chen D, Chong K, et al. (2010) Overexpression of a homopeptide repeat-containing bHLH protein gene (OrbHLH001) from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis. Plant Cell Rep 29 (9): 977–986
- 32. Thomson MJ, Tai TH, McClung AM, Lai XH, Hinga ME, et al. (2003) Mapping quantitative trait loci for yield, yield components, and morphological traits in an advanced backcross population between Oryza rufipogon and the Oryza sativa cultivar Jefferson. Theor Appl Genet 107: 479–493.
- 33. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, et al. (1987) MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174–181.
- 34. Lander ES, Botstein D (1989) Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121: 185–199.
- 35.
Lincoln S, Daly M, Lander ES (1992) Mapping genes controlling quantitative traits with MAPMAKER/QTL 1.1. Whitehead Institute technical report, 2nd edn. Massachusetts.
- 36.
Wang S, Basten CJ, Zeng ZB (2007) Windows QTL Cartographer 2.5. Department of Statistics. North Carolina State University, Raleigh, NC. http://statgen.ncsu.edu/qtlcart/WQTLCart.htm.
- 37. Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5 (2): 69–76
- 38. Panaud O, Chen X, McCouch SR (1996) Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Mol Gen Genet 252 (5): 597–607
- 39. Xiao N, Sun GL, Hong Y, Xia RX, Zhang C, et al. (2011) Cloning of genome-specific repetitive DNA sequences in wild rice (O. rufipogon Griff.), and the development of Ty3-gypsy retrotransposon-based SSAP marker for distinguishing rice (O. sativa L.) indica and japonica subspecies. Genet Resour Crop Evol 58 (8): 1177–1186
- 40.
Oka HI (1988) Origin of cultivated rice. Developments in crop science, vol 14. Elsevier, Amsterdam.
- 41. Wang ZY, Second G, Tanksley SD (1992) Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by analysis of nuclear RFLPs. Theor Appl Genet 83: 565–581.
- 42. Second G (1982) Origin of the genetic diversity of cultivated rice (Oryza spp.), study of the polymorphism scored at 40 isozyme loci. Jpn J Genet 57: 25–57.
- 43. Mao DH, Chen CY (2012) Colinearity and Similar Expression Pattern of Rice DREB1s Reveal Their Functional Conservation in the Cold-Responsive Pathway. Plos one 7(10): e47275.
- 44. Zhang F, Huang LY, Wang WS, Zhao XQ, Zhu LH, et al. (2012) Genome-wide gene expression profiling of introgressed indica rice alleles associated with seedling cold tolerance improvement in a japonica rice background. BMC Genomics 13 (1): 461
- 45. Dai LY, Lin XH, Ye CR, Ise K, Saito K, et al. (2004) Identification of quantitative trait loci controlling cold tolerance at the reproductive stage in Yunnan landrace of rice. Breed Sci 54: 253–258.
- 46. Lyons JM (1973) Chilling injury in plants. Annu Rev Plant Physiol 24: 445–451.
- 47.
Yoshida H, Kato A (1994) Cold–induced accumulation of RNAs and cloning of cDNAs related to chilling injury in rice. Breed Sci 44, 361–365.
- 48. Lee TM, Lur HS, Chu C (1995) Abscisic acid and putrescine accumulation in chilling–tolerant rice cultivars. Crop Science 35: 502–508.
- 49. Orvar BL, Sangwan V, Omann F, Dhindsa RS (2000) Early steps in cold sensing by plant cells: The role of actin cytoskeleton and membrane fluidity. Plant J. 23(6): 785–794.
- 50. Andaya VC, Mackill DJ (2003a) Mapping of QTLs associated with cold tolerance during the vegetative stage in rice. J Exp Bot 54 (392): 2579–2585
- 51. Andaya VC, Mackill DJ (2003b) QTLs conferring cold tolerance at the booting stage of rice using recombinant inbred lines from a japonica x indica cross. Theor Appl Genet 106 (6): 1084–1090
- 52. Yang ZM, Huang DQ, Tang WQ, Zheng Y, Liang KJ, et al. (2013) Mapping of Quantitative Trait Loci Underlying Cold Tolerance in rice seedlings via high-throughput sequencing of pooled extremes. Plos one 8(7): e68433.
- 53. Liu F, Xu W, Song Q, Tan L, Liu J, et al. (2013) Microarray-assisted fine-mapping of quantitative trait loci for cold tolerance in rice. Mol Plant, 6 (3): 757–67
- 54. Barras F, Van GF, Chatterjee AK (1994) Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopathol 32 (1): 201–234
- 55. De LG, Cervone F, Hahn MG, Darvill A, Albersheim P (1991) Bacterial endopectate lyase: evidence that plant cell wall pH prevents tissue maceration and increases the half-life of elicitor-active oligogalacturonides. Physiol Mol Plant Pathol 39 (5): 335–344
- 56. Cao J (2012) The pectin lyases in Arabidopsis thaliana: evolution, selection and expression profiles. PLoS ONE 7 (10): e46944
- 57. Lee H, Guo Y, Ohta M, Xiong L, Stevenson B, et al. (2002) LOS2, a genetic locus required for cold-responsive gene transcription encodes a bi-functional enolase. EMBO J 21 (11): 2692–2702
- 58. Bouton S, Leboeuf E, Mouille G, Leydecker M, Talbotec J, et al. (2002) QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. The Plant Cell 14, 2577–2590.
- 59. Solecka D, Zebrowski J, Kacperska A (2008) Are pectins involved in cold acclimation and de-acclimation of winter oil-seed rape plants. Ann Bot 101 (4): 521–530.
- 60. Liu HH, Ma Y, Chen N, Guo SY, Liu HL, et al. (2013) Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice. Plant Cell Environ,