Identification of QTLs and Validation of qCd-2 Associated with Grain Cadmium Concentrations in Rice

Liu Wenqiang, Pan Xiaowu, Li Yongchao, Duan Yonghong, Min Jun, Liu Sanxiong, Liu Licheng, Sheng Xinnian, Li Xiaoxiang

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Identification of QTLs and Validation ofAssociated with Grain Cadmium Concentrations in Rice

Liu Wenqiang, Pan Xiaowu, Li Yongchao, Duan Yonghong, Min Jun, Liu Sanxiong, Liu Licheng, Sheng Xinnian, Li Xiaoxiang

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Cadmium (Cd) is one of heavy metals harmful to human health. As rice is the main staple food in Asia and Cd is easily contaminated in rice, the molecular regulation of Cd accumulation should be explored. In this study, a recombinant inbred population derived from Xiang 743/Katy was grown in Cd-polluted fields and used to map the quantitative trait loci (QTLs) for Cd accumulation in rice grains. We identified seven QTLs distributed on chromosomes 2, 3, 6, 7, 8 and 10. These QTLs displayed phenotypic variances of 58.50% and 40.59% in 2014 and 2015, respectively. Two QTLs,and, were identified in both the two years.was detected on the interval of RM250–RM207 on chromosome 2, with an LOD of 2.51 and a phenotypic contribution of 13.75% in 2014, and an LOD of 3.35 and a phenotypic contribution of 14.16% in 2015.co-localized with the clonedon chromosome 7 and may represent the correct candidate. The other five QTLs were detected only in one year. To further confirm the effects ofa residual heterozygous line designated as RHL945, with a heterozygous interval of RM263–RM207 on chromosome 2, was selected from the recombinant inbred population and used to develop an F2population consisting of 155 individual plants. By incorporating further simple sequence repeat markers into the segmental linkage map of the target region,was delimited in the interval of RM5404–RM3774, with an LOD value of 4.38 and a phenotypic contribution of 15.52%. These results reflected the genetic regulation of grain Cd in rice and paved the way for the future cloning of.

cadmium; recombinant inbred line; quantitative trait locus; rice; simple sequence repeat

Cadmium (Cd) is a heavy metal element that is highly toxic to humans (Peng et al, 2018). The consumption of Cd leads to ‘Itai-Itai’ disease that causes severe pain in the spine and joints (Grant et al, 2008). Owing to modern industrial activities, many arable lands, particularly rice paddy fields, are contaminated with various levels of Cd. Rice is one of the most important food crops grown worldwide and it has thus become inevitable for absorbing Cd in polluted soil (Sun et al, 2017). However, rice appears to accumulate more Cd than other crops. Cd can therefore accumulate in the human body through the dietary intake of contaminated rice grains, posing a potential threat to human health. To minimize the human intake of Cd, it is imperative to dissect the genetic mechanism(s) for Cd accumulation in rice grains.

It is widely acknowledged that Cd concentration inrice varieties is generally higher than that inrice varieties. Large phenotypic variances have been observed among rice genotypes (Morishitaet al, 1987; Arao and Ae, 2003; Liu et al, 2005), suggesting that the concentration of Cd is controlled by multiple genes. Cd accumulation in rice is a complicated process involving in root uptake, xylem transport from the roots to the shoots, transfer from the xylem to the phloem, and transport through the phloem to grains. As such, several genes or quantitative trait loci (QTLs) are involved in the Cd regulation (Riesen and Feller, 2005). A number of QTLs/genes associated with Cd in specific tissues have been mapped. For example, the QTLs for Cd in rice shoots and/or grains (Ishikawa et al, 2005; Xue et al, 2009; Ishikawa et al, 2010; Shimo et al, 2011; Zhang et al, 2011; Abe et al, 2013), and the Cd distribution ratio of roots to shoots (Ueno et al, 2009a, b; Tezuka et al, 2010; Yanet al, 2013) have been reported.is identified as a regulator of xylem Cd loading in roots.controls the Cd translocation rates from the root to shoot and encodes a P1B-ATPase transporter. Defective P1B-ATPase activity results in higher levels of Cd translocation to shoots (Miyadate et al, 2011).is involved in the transport of Cd to grains through the phloem, as RNAi-mediated silencing ofdoes not influence xylem-mediated Cd transport (Uraguchi et al, 2011). Moreover, transporters that simultaneously transport Cd and other heavy metals essential for human metabolism have been studied. OsHMA2contributes to zinc (Zn) and Cd loading in the xylem and participates in root-to-shoot translocation of these two metals (Takahashi et al, 2012). OsNRAMP5contributes to manganese, Cd and iron transport in rice (Ishimaru et al, 2012). The OsNRAMP1 iron transporter is also involved in Cd accumulation in rice (Takahashi et al, 2011).

Cloning the genes associated with Cd accumulation can facilitate our understanding of Cd uptake in rice, but requires identification and validation of additional QTLs. Chromosomal segment substitution lines (CSSLs) or isogenic lines (ILs) provide alternatives for the dissection of complex traits, as these lines carry donor segments under the genetic background of a recipient genome, which generally masks the effects of genetic background. A number of genes have been map-based cloned using these techniques (Konishi et al, 2006; Song et al, 2007). Identifying and validating QTLs can also be performed in-part, by selecting residual heterozygous lines (RHLs) from advanced populations. RHL is heterozygous in the target region but homozygous elsewhere in the genome, which is equivalent to an F1individual plant derived from a cross between a pair of near isogenic lines (NILs). Numerous QTLs have been validated and fine mapped using these methods (Loudet et al, 2005; Dai et al, 2008).

To further investigate the genetic control of Cd uptake, a recombinant inbred line (RIL) population derived from the cross between Xiang 743 and Katy was used to map QTLs associated with grain Cd concentration (Liu et al, 2016). Of these QTLs, a putative QTL was further validated in a secondary population derived from self-pollination of an RHL selected from the RIL population. These results provided a necessary foundation for fine mapping and cloning of QTLs associated with Cd regulation in rice grains.

Materials aqnd methods

Rice materials and field experiments

An RIL population (F7) consisting of 151 lines was constructed using the single seed descent method described by Liu et al (2016). One of the parental lines, Xiang 743, is a glutinous rice landrace in Hunan, China, with relatively lower Cd content. The second parental line, Katy, is an Americanvariety with higher Cd content. Both parents and RIL populations were grown in an experimental field with soil Cd content of 0.89 mg/kg at pH 5.0 in Beishan, Hunan Province, China. The seeds were sown at the beginning of May in 2014 and 2015, respectively. After 28 d, the seedlings were transplanted. Two repeats of the procedure were performed through a randomized block design, and 24 individual plants for each line were grown in three rows, with a spacing of 19.8 cm between rows and 16.5 cm within a row. Once irrigation stopped in mid-August, the field was only rain-fed until the grains matured. At maturity, 12 plants were harvested for the determination of grain Cd. The experiment was conducted over two consecutive years. All field management protocols were followed locally practiced standards.

An RHL, designated as RHL945, was selected from the RIL population on the basis of flanking markers of. RHL945 self-pollinated and developed an F2population consisting of 155 individual plants. The F2population was grown in the same Cd-polluted experimental field, which was maintained under identical cultivation patterns.

Determination of Cd concentrations

The levels of accumulated Cd were determined according to Yao et al(2015). Briefly, rice grains were air-dried and de-hulled. After milling the grains into brown rice, 200 g samples were ground into powder and oven-dried at 80 oC to a constant weight. For wet digestion, samples were transferred to triangular flasks containing a mixture of HNO3-HClO4(5:1). Flasks were covered with funnels and left at room temperature overnight. Subsequently, digested samples were transferred to an electric stove and maintained at 180 oC until the solution turned colorless. The temperature was then increased to 220 oC until the solution fully evaporated. After cooling, residues were dissolved in 1% HNO3and filtered. Rice grain powder containing filtered solution was diluted to 10 mL. Blank digests with no sample and reference digests with control sample were prepared similarly. Finally, the Cd concentrations of the samples were measured using an inductive coupled plasma emission spectrometer (Agilent Technologies 700 Series ICP-OES, USA). Each sample was run twice to ensure the reliability of the results.

Extraction of DNA and SSR markers

DNAs from the F2population and the parental lines (Xiang 743 and Katy) were extracted using the CTAB method described by Liu et al (2015). Four original simple sequence repeat (SSR) markers (RM263, RM240, RM250 and RM207) on rice chromosome 2 were genotyped to construct a segmental map in the F2population. Six new SSR markers (RM6, RM5472, RM6030, RM5404, RM3774 and RM208) with a polymorphism between the parents were incorporated into the segmental linkage map.

SSR primers were amplified in a 10 μL reaction mixture containing 1 μL of 10× buffer, 0.5 μmol/L of each primer, 0.25 mmol/L dNTPs, 0.5 U ofDNA polymerase and 10–30 ng genomic DNA. Samples were pre-denatured at 94 oC for 2 min, followed by 30 cycles of 45 s at 94 oC, 45 s at 55 oC, 45 s at 72 oC, and a final extension at 72 oC for 8 min. Polymerase chain reaction (PCR) products were detected using silver staining on 6% non-denaturing polyacrylamide gels.

Data processing and analysis

MapMaker version 3.0b (Lincoln, 1992) was used to identify the most probable marker order in the target region of the F2population. An LOD threshold of 3.0 was used to determine segmental linkage groups. The genetic map distance (cM) was calculated using the Kosambi function.

QTL analysis was performed using composite interval mapping (CIM) in Window QTL Cartographer 2.5 (Wang et al, 2008). Multiple interval mapping (MIM) was used to confirm and test significant interactions between putative QTLs. LOD threshold (> 2.5) was used to declare the presence of a putative QTL. QTLs were named according to McCouch et al(1997).

Results

Phenotypic variance in the RIL population

Cd concentrations in the parental lines, Xiang 743 and Katy, were 0.71 and 1.65 mg/kg in 2014, 1.10 and 2.30mg/kg in 2015, respectively (Fig. 1). Cd concentrations in Katy were more than twice of those of Xiang 743 in both years. In 2014, Cd concentrations in the RIL population ranged from 0.41 to 2.21 mg/kg, with an average of 0.99 mg/kg. In 2015, Cd concentrations in the RIL population ranged from 0.56 to 3.26 mg/kg, averaging 1.75 mg/kg. The frequency distribution of Cd concentrations in the RIL population in 2014 was similar to that in 2015. Continuous distributions were observed over the two consecutive years, indicating that Cd is controlled by multiple genes. Transgressive segregations were also observed over the two consecutive years, suggesting that several genes may regulate grain Cd in both the two parents.

Construction of genetic linkage maps

A genetic linkage map of the RIL population was previously constructed and employed to identify the QTLs for grain shape (Liu et al, 2016). Briefly, the map contained 121 SSR markers that were evenly distributed on 12 chromosomes. The map spanned a total length of 1 797.8 cM, ranging from 0.6 to 39.9 cM between adjacent markers with an average of 14.8 cM. Ten SSR markers were used to construct a segmental linkage map in the F2population. The map encompassed 39.3 cM.

QTL analysis

Fig. 1.Frequency distributions of Cd concentration in Xiang 743/Katy recombinant inbred lines in 2014 (A) and 2015 (B).

Cd concentrations in the parental lines are indicated by arrows.

Table 1. QTL analysis for grain Cd concentration in Xiang 743/Katy recombinant inbred lines in 2014 and 2015.

2, Percentage of phenotypic variance explained by each QTL; DPE, Direction of phenotypic effect.The genetic effect of the putative QTL when Xiang 743 allele was replaced by Katy allele.

Fig. 2.Chromosomal location of QTLs for Cd concentration in a genetic linkage map of Xiang 743/Katy recombinant inbred lines.

The intervals where QTLs located were determined on the basis of LOD peak flanking markers.

Fig. 3.Schematic diagram depicting the genotype of 12 chromosomes of RHL945 genome.

The positions of DNA markers used to determine genotypes are indicated by thin lines.

Fig. 4.Frequency distribution (A) and QTL likelihood cure of LOD score (B) of Cd concentration in the F2population derived from RHL945.

, Additive effect. The genetic effect of the putative QTL when Xiang 743 allele was replaced by Katy allele.2, Percentage of phenotypic variance explained by each QTL.

A total of seven QTLs distributed on chromosomes 2, 3, 6, 7, 8 and 10 were detected (Table 1 and Fig. 2), explaining the phenotypic variances of 58.50% and 40.59% in 2014 and 2015, respectively. Among the QTLs,andwere repeatedly detected with consistent directions in both years.

Five QTLs distributed on chromosomes 2, 6, 7, 8 and 10 were detected in 2014. Among them, four QTLs (,,and) from Katy alleles increased grain Cd concentrations. A single QTL () from Xiang 743 allele increased grain Cd concentration.displayed the largest phenotypic variance of 16.98%, with an LOD value of 3.31 (Table 1).

Four QTLs distributed on chromosomes 2, 3, 6 and 7 were detected in 2015. Among them, three QTLs (,and) from Katy alleles increased grain Cd concentration. One QTL () from Xiang 743 allele increased grain Cd concentration.displayed the largest phenotypic variance of 14.16%, with an LOD value of 3.35 (Table 1). In addition, epistatic interaction analysis was performed between putative QTLs. The results demonstrated no significant interactions.

Validation of a putative QTL qCd-2

To further confirm the effects of QTLs on the basis of expression stability, we first prioritizedand. Unfortunately, an RHL forwas unavailable from the RIL population.

On the basis of genotypes of markers RM250 and RM207 flanking, RHL945 carrying the heterozygous interval of RM263–RM207 on chromosome 2 were screened from the RIL population. RHL945 carried two large segments in heterozygous interval on chromosomes 3 and 8, and one small segment in heterozygous interval on chromosome 5. No QTLs were detected in these intervals on the basis of mapping in the RIL population. In addition, five cross-over regions distributed on chromosomes 2, 4, 9, 10 and 12 were observed. Of them, a single cross-over region on chromosome 10 coincided with the interval in(Fig. 3).

RHL945 showed Cd concentrations of 0.77 and 1.10 mg/kg in 2014 and 2015, respectively. RHL945 self-crossed to develop an F2population. Cd concentrations in the F2population varied greatly, ranging from 0.01 to 1.50 mg/kg with an average of 0.51 mg/kg. We observed a skewed distribution towards low Cd concentrations (Fig. 4). QTL analysis showed thatwas detected in the interval of RM5404– RM3774, displaying a phenotypic variance of 15.52% and an LOD value of 4.38. The allele from Katy increased Cd concentration in the rice grains (Fig. 4).

Discussion

Cd is harmful to both animals and humans. Understanding the genetic control of Cd in food crops is therefore imperative. Seven QTLs were identified in this study. Five QTLs from Katy alleles increased grain Cd concentrations and two QTLs from Katy alleles decreased Cd concentrations. These results implied that favourable QTLs that regulate Cd were scattered among the parents.

is co-localize with the QTLfor Cd translocation from root to shoot (Tezuka et al, 2010), and has been subsequently cloned (Miyadate et al, 2011). The locus has been repeatedly identified in several independent studies (Uenoet al, 2009b; Ishikawa et al, 2010), confirming it as a major and stably expressed QTL. In previous studies,co-localizes with one QTL for shoot Cd concentration (Ueno et al, 2009a). However, QTL analysis of the shoot Cd concentrations was not performed. Thus, it is difficult to determine whether the locus is pleiotropic or consists of multiple Cd-related alleles.is consistent with the locus for Cd concentration in brown rice, which is identified using CSSLs containing a Kasalath segment under a Koshihikari genetic background (Ishikawa et al, 2005).

Cd concentration in rice grains is affected by environmental factors, including temperature, water management and soil pH. Recent evidence reveals that iron and fertilizer (N and P) also influence Cd uptake in rice (Gao et al, 2016; Yang et al, 2016a, b). Thus, minor QTLs are barely detectable across continual years. In this study, two QTLs (and) were repeatedly detected in both years, while the others were identified only in a single year. In addition,was further confirmed by the F2population derived from RHL945. Cd concentrations in the F2population varied significantly and distributed in an irregular segregation pattern, suggesting that other genetic effects besidesare present. In general, the effects of minor QTLs can be significantly magnified when the major QTLs are masked (Nagaoka et al, 2017). However,displayed a modestly higher phenotypic contribution and higher LOD value in the F2population than in the RIL population. This could be attributed to the partly heterozygous background of RHL945, in whichwas mapped in the cross-over region on chromosome 10. We cannot yet exclude the possibility that other QTLs were segregating in the population. Hence, the genetic background of RHL945 should be further considered before further dissection and fine-mapping of. At present, a series of RHLs overlapping with the target region will be selected from advanced progeny, paving the way for fine- mapping and cloning of.

Acknowledgements

The work was jointly supported by Hunan Natural Science Foundation (Grant No. 2016JJ6061), Hunan Academy of Agricultural Sciences Scientific and Technological Innovation Project (Grant No. 2017NQ05), China National Key Research and Development Project (Grant No. 2016YFD0100101-12) and the earmarked fund for China Agriculture Research System (Grant No. CARS-01-14).

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25 March 2018;

27 July 2018

Li Xiaoxiang (xiaoxiang66196@126.com)

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