Identification of QTLs for Cadmium Tolerance During Seedling Stage and Validatio

Ding Shilin, Liu Chaolei, Shang Lianguang, Yang Shenglong, Zhang Anpeng, Jiang Hongzhen, Ruan Banpu, Fang Guonan, Tian Biao, Ye Guoyou, Guo Longbiao, Qian Qian, Gao Zhenyu

Research Paper

Identification of QTLs for Cadmium Tolerance During Seedling Stage and Validation ofin Rice

Ding Shilin1, 2, #, Liu Chaolei2, #, Shang Lianguang3, Yang Shenglong2, Zhang Anpeng2, Jiang Hongzhen2, Ruan Banpu2, Fang Guonan2, Tian Biao2, Ye Guoyou4, Guo Longbiao2, Qian Qian2, Gao Zhenyu2

(Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China; State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China;Agricultural Genomics Institute, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China; Strategic Innovation Platform, International Rice Research Institute, DAPO Box 7777, Metro Manila, the Philippines; These authors contributed equally to this work)

Cadmium (Cd) is a non-essential toxic metal that is harmful to plants. To investigate the genetic mechanism of Cd tolerance in rice, quantitative trait loci (QTLs) associated with Cd tolerance at the seedling stage were analyzed using a recombinant inbred line (RIL) population derived from a cross between PA64s and 93-11. A total of 36 QTLs associated with shoot length, root length, shoot dry weight, root dry weight and total dry weight were detected in Hangzhou and Lingshui of China. Among them, 15 QTLs were identified under the control condition and 15 QTLs were identified under the Cd stress condition, and 6 QTLs for Cd tolerant coefficient were detected on chromosomes 1, 3, 7 and 9. Theandwere identified in Hangzhou and Lingshui, respectively, and had overlapping intervals on chromosome 1. To further confirm the effects ofand, we developed a chromosome segment substitution line (CSSL), CSSL, in 93-11 background harboring/from PA64s. Compared to 93-11, CSSLhad increased shoot length under the Cd stress condition. These results pave the way for further isolation of those genes controlling Cd tolerance in rice and marker-assistant selection of rice elite varieties with Cd tolerance.

cadmium tolerance; recombinant inbred line;; rice; quantitative trait locus

Cadmium (Cd) is a highly toxic heavy metal element to both plants and humans. In recent years, due to unreasonable industrial discharge, poor treatment of solid waste, sewage irrigation and application of fertilizers containing heavy metal elements, Cd content in the soil has increased sharply and its pollution has become increasingly serious (Clemens et al, 2013). Cd enters human body mainly through the food chain and has a half-life period of 10‒35 years in human body (Clemens et al, 2013). Cd is mainly accumulated in the kidneys after entering the human body, which will be harmful to human bones and respiratory system, thus causing a series of diseases (Jarup and Akesson, 2009; Nawrot et al, 2010).

Rice is one of the most important crops in China. Cd pollution not only deteriorates soil quality of paddy rice fields, but also damages rice growth and development. Meanwhile, it can be accumulated in rice grains through absorption and transportation in rice plants, which has become an important source for endangering human health (Yao et al, 2003). Genetic variation in Cd tolerance of rice indicates that it is possible to select cultivars with strong resistance to Cd and low Cd concentration in grains. Therefore, the study of genetic mechanism underlies Cd tolerance in rice is helpful to develop Cd-tolerant rice cultivars.

For complex traits controlled by multiple genes, quantitative trait locus (QTL) mapping has become a powerful means to identify the number, location and effects of genetic factors, and also an important step to understand molecular genetic mechanism for complicated phenotype. So far, many QTLs and genes related to Cd accumulation have been identified and isolated in rice, including(Agrawal et al, 2002),(Agrawal et al, 2003),(Agrawal et al, 2003),(Mukhopadhyay et al, 2004),(Koike et al, 2004),(Nakanishi et al, 2006),(Nakanishi et al, 2006),(Lee et al, 2007),(Kuramata et al, 2009),(Shim et al, 2009),(Shimo et al, 2011),(Oda et al, 2011),(Miyadate et al, 2011),(Uraguchi et al, 2011),(Takahashi et al, 2011),(Ishimaru et al, 2011),(Takahashi et al, 2012),(Yuan et al, 2012),(Ishikawa et al, 2012),(Ramegowda et al, 2013),(Lim et al, 2014),(Yu et al, 2015),(Wang et al, 2015),(Wang et al, 2016),(Das et al, 2017),(Tan et al, 2017),(Luo et al, 2018) and(Yan et al, 2019). Although genetic studies were mainly focused on Cd accumulation in rice, genetic analysis of QTLs related to Cd tolerance in shoots and roots of rice was relatively fewer.

To identify QTLs for Cd toxicity tolerance in rice at the seedling stage, we treated 118 recombinant inbred lines (RILs) derived from a cross between parents 93-11 and PA64s with 50 µmol/L CdCl2under controlled conditions. With a high-density SNP linkage map, a total of 36 QTLs associated with 5 important traits were detected in Hangzhou and Lingshui of China. A novel QTL,for shoot length under Cd stress was validated by a chromosome segment substitution line CSSLin 93-11 background harboringfrom PA64s, and candidate genes were predicted, which will be helpful for further cloning of the QTL and understanding of the genetic basis for rice Cd tolerance.

Table 1. Phenotypic values of parents under different concentrations of Cd.

Data are Mean ± SD (= 6). * and ** represent 5% and 1% significant differences, respectively, according to the Student’s-test.

RESULTS

Phenotypic variance in RIL population of 93-11 and PA64s in Hangzhou and Lingshui

The traits of root length, shoot length, root dry weight, shoot dry weight and total dry weight were used to evaluate the Cd stress tolerance of the parents 93-11 and PA64s, and their RIL population in Hangzhou and Lingshui. To determine the suitable Cd concentration for treatment, gradient Cd concentrations (0, 10, 20, 30, 40, 50, 100 and 150 μmol/L) were applied on the two parents (93-11 and PA64s) in 2018, and 50 μmol/L Cd concentration was selected for Cd treatment (Table 1). The seeds of parents (93-11 and PA64s) and RILs harvested in 2019 were treated with 50 μmol/L CdCl2. Except for root length, root dry weight in Hangzhou and Lingshui, and total dry weight in Lingshui of PA64s, the values of traits were reduced in Cd stress compared to the control for two parents (Table 2). Cd tolerance coefficients of all the measured traits were higher in PA64s than 93-11 both in Hangzhou and Lingshui, indicating that PA64s is relatively tolerant to Cd compared with 93-11.

The mean, range, skewness and kurtosis of each examined trait in RIL population were summarized in Table 2. Except for root dry weight, the phenotypic values of these traits in both locations under the Cd stress were lower than those under the control condition without Cd. And the segregation of these traits was continuously distributed among all the RILs (Figs. S1 and S2). Additionally, transgressive segregation in both directions was observed for all the traits, suggesting that both parents transmitted favorable alleles for each trait. All the traits of 118 RIL lines showed approximately normal distribution, which reflected polygenic segregation and satisfied the request of QTL analysis. In addition, all the tested traits exhibited a significantly positive correlation under the control and Cd stress conditions in Hangzhou and Lingshui, respectively (Table S1).

Table 2. Phenotypic values of recombinant inbred line (RIL) population and their parents under control and Cd stress.

CTC, Cd tolerance coefficient.Data are Mean ± SD (= 6). * and ** indicate 5% and 1% significant differences between the two parents, respectively, according to the Student’s-test.

QTL analysis of five traits associated with cadmium tolerance

To identify QTLs for the five traits associated with cadmium tolerance under the control and Cd stress, a total of 36 putative QTLs were detected on rice chromosomes 1, 2, 3, 4, 5, 7, 9, 10 and 11 (Table 3 and Fig. 1). Among them, 15 QTLs were identified under the control condition with each QTL accounted for 7.8%‒18.1% of phenotypic variation, 15 QTLs were detected under the Cd stress condition with each QTL accounted for 0.9%‒12.8% of phenotypic variation, and 6 QTLs for Cd tolerance coefficient were also mapped on chromosomes 1, 3, 7 and 9 with each QTL accounted for 6.2%‒15.4% of phenotypic variation.

Under the control and Cd stress conditions in Hangzhou and Lingshui, most of QTLs had positive allele coming from PA64s, indicating that most alleles from PA64s increase Cd tolerance. Total of seven clusters of QTLs were found, including two QTLs(and) mapped on the same region of chromosome 1, three QTLs(,and) on the same or overlapping region of chromosome 1, four QTLs (,,and) on the same or overlapping region of chromosome 2, three QTLs(,and) on the same or overlapping region of chromosome 4, three QTLs(,and) on the same or overlapping region of chromosome 5, four QTLs (,,and) and two QTLs(and) on the same or overlapping region of chromosome 11, respectively. These clusters of QTLs were all involved in dry matter accumulation under the control and Cd stress conditions. Onlyandwere detected both in Lingshui and Hangzhou, indicating the QTLs are genetically stable and independent on the two different environments (Table 3 and Fig. 1).

Table 3. Putative QTLs with LOD > 2.0 detected in rice recombinant inbred line population.

Individual QTL is designated with the italicized abbreviation of the character and the chromosome number. When more than one QTL affecting a character is identified on the same chromosome, they are distinguished using decimal numbers.Maximum-likelihood LOD score for the QTL calculated by MultiQTL package.The positive or negative value indicates that allele from 93-11 or PA64s increases the trait score, respectively. CTC, Cd tolerance coefficient; RL, Root length; SL, Shoot length; RDW, Root dry weight; SDW, Shoot dry weight; TDW, Total dry weight; Chr, Chromosome; Add, Additive effect; Var, Variation explained by the putative QTL.

Fig. 1. Chromosomal locations of all QTLs for Cd tolerance in rice recombinant inbred line population at seedling stage.

The genetic distance of marker (cM) is annotated on the left of each chromosome. Chr, Chromosome; CTC, Cd tolerance coefficient; RL, Root length; SL, Shoot length; RDW, Root dry weight; SDW, Shoot dry weight; TDW, Total dry weight.

Validation of qCDSL1 for shoot length under Cd stress and determination of candidate genes

To validate the physiological role ofin Cd stress response, we developed CSSL(Fig. 2-A). This line harbored the PA64s-derivedallele in 93-11 genetic background. To investigate whetherregulates the response to Cd stress, we exposed the 7-day-old plants of 93-11 and CSSLto 0 and 50 μmol/L Cd for 10 d in a hydroponic experiment. The shoot length of CSSLwas significantly shorter than that of 93-11 under the control condition and significantly longer than that of 93-11 under the Cd stress condition (Fig. 2-B and -C). There were 127 annotated genes in the 873.7 kb region for. Besides 13 transposon protein genes, 16 retrotransposon protein genes, 7 hypothetical protein genes and 25 expressed protein genes, 66 functionally annotated genes were included in the region (Table S2). Previous studies showed that thegene plays an important role in response to Cd stress and it is also located in theregion(Yu et al, 2015). Sequencing of thegene revealed six SNPs in the promoter ofbetween 93-11 and PA64s (Fig. 2-D). The expression level ofin CSSLwas significantly lower than that of 93-11 under the control condition and significantly higher than that of 93-11 under the Cd stress condition (Fig. 2-E), which was consisted with previous observation thatexpression increased5-fold in roots when treated with Cd (Yu et al, 2015).

DISCUSSION

Rice tolerance to Cd is a quantitative trait with complex genetic basis (Xue et al, 2009). To study the genetic basis of rice tolerance to Cd stress, it is necessary to select appropriate specific traits. Rice seedlings and seed germination are sensitive to heavy metal stress. It was found that with the increase of Cd concentration, the seedling length, root length, fresh weight and dry weight were all decreased (Yang et al, 2017). Different concentrations of Cd showed inhibition on seedling growth with different degrees (Li et al, 2019). Thus, it is assumed that the suitable Cd level should be selected in order to detect QTLs with large effects on Cd tolerance. Here, when the concentration of Cd was 50 µmol/L, the differences of shoot length, shoot dry weight, root dry weight and total dry weight between the two parents were the most significant (Table 1). Therefore, 50 µmol/L of Cd was chosen to treat RIL seedlings.

The detection of QTLs is affected by population size, threshold value, marker number, heritability, genetic background and experimental conditions. In previous studies, Xue et al (2009) detected 22 QTLs for 6 traits under the control and Cd stress conditions, including shoot height, root length, shoot dry weight, root dry weight, total dry weight and chlorophyll content, and 6 QTLs related to Cd tolerance coefficient at the seedling stage. Among them,for total dry weight were mapped in the region of G249‒G164, where thelocated for relative root dry weight in our study. Li et al (2019) recently detected six QTLs for Cd tolerance coefficient on chromosomes 1, 4, 7, 8 and 10, among which thefor tolerance index of root fresh weight was also located in the region overlapped withhere, suggesting a major QTL controlling Cd tolerance on chromosome 7 in rice seedlings. A total of 34 new QTLs detected in the study indicated that Cd stress has different effects on various organs of rice seedlings and the genetic complexity of Cd tolerance in rice.

Up to now, a few studies have been performed on mapping of QTLs for Cd stress tolerance at the rice seedling stage. However, no reports have been published on cloning of QTLs/genes for Cd tolerance at the rice seedling stage. After searching for genes related to Cd metabolism,was found to be located inorregion,was found to be located inandregion, indicating thatandare likely to control the expression of QTLs for these traits under the Cd condition. After annotation analysis of genes within theregion, thegene was found to be associated with Cd stress responses (Yu et al, 2015). Sequencing and expression analyses suggestedwas most probably responsible for regulating tolerance to Cd stress. Further fine mapping and functional confirmation will be conducted with F2population by crossing the CSSL with 93-11. Pyramiding of major QTLs is a powerful strategy in rice breeding. Therefore, QTLs related to high Cd resistance identified here and QTLs for low Cd accumulation in grains can also be used to develop novel elite rice varieties by QTL pyramid.

METHODS

Rice materials

93-11 is anvariety, while PA64s is an-like variety with maternal origin of. The RIL population was generated from an advanced self-fertilization population of 93-11/PA64s F1plants. A total of 118 lines were obtained, and the seeds were harvested at the China National Rice Research Institute in Hangzhou and Lingshui of China with normal conventional field cultivation. The chromosomal segment substitution line, CSSL, was selected from the advanced backcross population (BC4F2) derived from a cross of the recurrent parent 93-11 and the donor parent PA64s (Zhang et al, 2019).

Fig. 2. Validation of.

A, Schematic graph of chromosome 1 of CSSL, the parents 93-11 and PA64s. The white and black bars represent 93-11 and PA64s alleles, respectively. B, Comparison of seedling growth morphology of 93-11 and CSSLunder the control and Cd stress conditions. Scale bar, 2 cm.C, Shoot lengths of 93-11 and CSSLunder the control and Cd stress conditions. Data are Mean ± SD (= 6). * and ** indicate 5% and 1% significant levels compared to 93-11 under the control and Cd stress conditions, respectively, according to the Student’s-test.D, Gene structure and sequence differences of() between PA64s and 93-11.E, Relative expression level ofin shoots of 93-11 and CSSLunder the control and Cd stress conditions. Data are Mean ± SD (= 3). ** indicates 1% significant level compared to 93-11 under the control and Cd stress conditions, respectively, according to the Student’s-test.

Culture conditions

The uniform seeds of the two parents and RILs were surface sterilized in 3% H2O2solution for 10 min, and then rinsed five times with deionized water. The seeds were then soaked in deionized water in the dark at 28 ºC for 2 d, and then transferred to a net floating on deionized water for further 5 d in a controlled chamber with a photoperiod of 16 h light/8 h dark. After 5 d, rice seedlings with similar size were selected and transplanted into 40 L plastic containers containing nutrient solution (96 seedlings per container) and cultivated in the hydroponic form. The light/dark temperatures were set at 32 ºC/ 28 ºC, and relative humidity was kept at 80%. The seedlings were cultured in a half-strength Kimura B nutrient solution (pH 5.4) with the following composition: 90 μmol/L KH2PO4, 270 μmol/L MgSO4, 180 μmol/L (NH4)2SO4, 90 μmol/L KNO3, 180 μmol/L Ca(NO3)2, 3 μmol/L H3BO3, 0.5 μmol/L MnCl2, 1 μmol/L (NH4)6Mo7O24, 0.4 μmol/L ZnSO4and 20 μmol/L Fe3+-EDTA. The solution was renewed every 2 d. At 3 d after transplanting to the basic solution, two treatments were established: control, in which nine plants grew in the nutrient solution without Cd addition; and cadmium stress, CdCl2was added to the solution in equal increments every day, and after 5 d, the final Cd concentration was 50 µmol/L.

Trait measurements

At the 10 d after Cd treatment, shoot length, root length, shoot dry weight, root dry weight and total dry weight of each treatment were measured. Shoot length was measured from the coleoptile node to the tip of the longest leaf, and root length was measured from the coleoptile node to the tip of the longest root. The mean values were calculated from measured 4–6 plants. The sampled plants were separated into roots and shoots, dried at 70 ºC for 3 d in an oven, and then weighted root and shoot dry weights. Total dry weight was calculated according to shoot and root dry weights. Cd tolerance coefficient (CTC) of relative root length, relative shoot length, relative shoot dry weight, relative root dry weight and relative total dry weight were calculated using the following formula: CTC = The value in Cd stress treatment / The value in the control.

Data analysis and QTL mapping

Statistical analysis was conducted with the SAS software (version 9.0). By resequencing parents 93-11 and PA64s and 118 RILs, we obtained 2 622 single nucleotide polymorphism (SNP) markers with high quality and polymorphism, and constructed a high-density SNP linkage map. The SNP linkage map covered a total of 1 381.9 cM of the rice genome, with an average linkage map spacing of 0.392 cM (Gao et al, 2013). QTL analysis was performed with the MultiQTL package (www.mutiqtl.com) using the maximum likelihood interval mapping approach for the RILs. QTL was determined with threshold< 0.005 and the threshold of LOD > 2.0 was chosen for claiming a putative QTL. The genetic parameters, additive effects and explained variation of each QTL were also estimated. We followed the rules for the QTL nomenclature by McCouch et al (1997).

Expression analysis at transcript level

To analyze the transcriptional expression of candidate genes, the 7-day-old plants of 93-11 and CSSLwere exposed to 0 or 50 μmol/L CdCl2for 10 d, and then the shoots were excised for RNA extraction. Total RNA was extracted using the RNA Extraction kit (Axygen, New York, America). DNase I- treated RNA (1 μg) was used to synthesize the first-strand cDNAs by using a ReverTra®Ace qPCR-RT kit (TOYOBO, Osaka, Japan). The cDNA products and the 2× SYBR Green PCR Master Mix (TOYOBO, Osaka, Japan) were used for real-time PCR analysis.was used as an internal control. Data were collected in accordance with the ABI PRISM 7900HT Sequence Detector system. Primers forandare listed in Table S3.

ACKNOWLEDGEMENTS

This research was supported by the National Natural Science Foundation of China (Grant No. 31671761) and the Agricultural Science and Technology Innovation Program, Shenzhen Science and Technology Program (Grant No. 2017050414212249).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/journal/ 16726308; http://www.ricescience.org.

Fig. S1. Frequency distribution of Cd tolerance at the seedling stage in RIL population with seeds from Hangzhou under control and Cd stress conditions.

Fig. S2.Frequency distribution of Cd tolerance at the seedling stage in RIL population with seeds from Lingshui under control and Cd stress conditions.

Table S1. Correlation coefficients between five traits under control and Cd stress conditions.

Table S2. Functionally annotated genes in the 873.7 kb region for.

Table S3. Primers used for qRT-PCR in the study.

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