The feasibility of QTL analysis to study the genetic basis of pesticide residues in rice (Oryza sativa)  

Zhang Yushan1 , Naghabushana K. Nayidu2 , Margaret Y. Gruber3
1. University of Electronic Science and Technology of China Zhongshan Institute, Zhongshan 528402
2. Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, S7N 5E2 Canada
3. Agriculture and Agri-food Canada, 107 Science place, Saskatoon, SK, S7N 0X2
Author    Correspondence author
Molecular Plant Breeding, 2014, Vol. 5, No. 11   doi: 10.5376/mpb.2014.05.0011
Received: 05 Jul., 2014    Accepted: 18 Aug., 2014    Published: 29 Sep., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Yushan et al., The feasibility of QTL analysis to study the genetic basis of pesticide residues in rice (Oryza sativa), Molecular Plant Breeding, 2014, Vol.5, No. 11 47-63 (doi: 10.5376/mpb.2014.05.0011)

Abstract

Pesticide residues in Chinese rice have become one of the important issues as far as food safety is concerned. Although QTL mapping and cloning are important tools for molecular genetics, not much information has been reported yet for either QTL mapping or cloning the genes of rice pesticide degradation. In the present study, cellular pathways of pesticide metabolism, biochemical changes as a function of pesticide exposure, differences in pesticide residue levels and type among rice varieties, and detection methods of pesticide residue were reviewed. Pesticide residues in rice grain can be regarded as a genetic trait dependent on the degrading capacity of pesticide detoxification enzymes. This information led us to consider using endogenous genes of rice to degrade pesticide residues within the cell. Ways of cultivating rice varieties with low pesticide residue and transgenic and QTL methods for pesticide residue degradation will be discussed.

Keywords
Rice; QTL; Pesticide degradation; Pesticide residue; GWAS; NILs

Pesticides play an important role in controlling plant diseases, insect pests, and weeds. On the other hand, they also have negative effects on human health and on crop quality and can directly influence the crop export industry. According to a Chinese Ministry of Commerce survey, 90% of agricultural and food export enterprises in China experience an annual loss of approximately 9 billion dollars due to technical barriers against foreign trade(Xue and Tao, 2009) and part of this loss is a result of pesticide contamination in agricultural products. China is one of the largest producers and consumers of pesticides worldwide with an annual chemical sprayed area reaching 4.5 billon acres(Xue and Tao, 2009). Chemical pesticides are direct threats to food safety and environmental safety in China. According to a sanitation and quality survey from the Chinese Rice Research Institute, excessive residue proportions in rice were 6.7% and 4.8% above the standard allowable levels of pesticide residue in 2002 and 2003,respectively(Zhu, 2006). Improper or illegal pesticide use are main causes for pesticide pollution and enhanced residue levels, and more attention should be paid to training farmers in best management practices for pesticide use. However, in the present scenario of Chinese agriculture it is impractical to plant crops in such large areas without using pesticides.

Pesticides used in China are mainly divided into herbicides, insecticides and fungicides. According to their chemical structure, pesticides used in China can be roughly divided into organic phosphorus pesticides, organic chlorine pesticides and carbamate pesticides (Lu, 2011). In view of the threat to food security caused by pesticide residues, identification and cloning of pesticide residue degradation genes for these types of chemicals have become top priorities at this time. Although some components of pesticide residue degradation were found in the plant itself (Xia et al., 2009; Zhang et al., 2012), a much greater number of these genes have been cloned from microorganisms.
So far, cloning microbial genes degrading organo- phosphorus pesticide and pyrethroid pesticide as well as organochlorine pesticide have also been reported. These gene names and source of strain as well as degradation of pesticide types were shown in Table 1. However, cloning the genes of degrading pesticides residues in the plants has not been reported yet.


Table 1 Information about the cloned genes and their sources of strain as well as the types of degraded pesticide

In the last 20 years, with the completed sequencing of the rice genome and the construction of high density molecular linkage maps and various NILs populations, many quantitative trait loci (QTLs) have become available for fine mapping of any required trait in rice. Even though large numbers of QTLs, such as Ehd2, GS3, GW5, DTH8, IPA1, Ghd7, Ghd8, and Ghd7.1 (Matsubara et al., 2008; Fan et al., 2006; Weng et al., 2008; Wei et al., 2010; Jiao et al., 2010; Xue et al., 2008; Yan et al., 2010; Yan et al., 2013) have been cloned, identification and cloning of rice genes related to pesticide residue degradation have not been reported yet.
In the present review, we discuss how plants absorb pesticides and transport them, and we highlight pesticide metabolic pathways within plant cells, physiological and biochemical changes occurring in the rice-pesticide residue system, types of pesticide residues in rice grain, distribution characteristics, the differences in pesticide residue levels among rice varieties, and pesticide detection techniques. We also highlight QTL analysis to dissect the molecular basis of pesticide residue in rice, and we discuss its feasibility as a tool to use along with other ways to cultivate rice varieties with low pesticide residue.
1 Pesticide absorption and transportation in plants
When spraying pesticides, aerial tissues are the main route to pesticide absorption in plants. Pesticides adhering to plant cell surfaces are known as pesticide physical residues, and a portion of these can penetrate into the leaves, fruit, and stems via epidermal cells (because of lipotropy) and are called chemical residues. Once the pesticide is deposited on the plant surface, it is absorbed by the epidermal wax and may be accumulated or infiltrated into the cells. In the process of entering the primary cell wall, secondary cell wall and membrane, some types of pesticides can be degraded by hydrolases within the cell wall before penetration into the protoplasm. Pesticides can move a short distance through plasmadesmata between cells, whereas in vascular tissues they can move a long distance. A variety of pesticides in protoplasm can move through phloem, while the majority of systemic insecticides, such as demeton systox, dimethoate, disulfoton, and phorate, are transmitted passively within the xylem. Another major route to absorption is through the root capand root hairs of plants as they take in pesticides applied to soil and water. The pesticide is solubilized, then taken into the root, and then conducted upwards into aerial tissues via the plant transport system (Julius, 1983).
2 The metabolic pathway of pesticides in the plant cell
In the process of evolution, plants have formed a mechanism of detoxification to degrade pesticides and other exogenous chemical substances. Research showed that the main process of herbicide metabolism in plant cells consists of four stages (Van et al., 2003; Fujisawa et al., 2006; Mitsou et al., 2006): Phase I detoxification in which herbicides become soluble and toxicity is somewhat reduced by cytochrome P450 enzymatic reactions or non-enzymatic processes of oxidation, reduction, or hydrolysis; Phase II detoxification by the conjugation action of Glutathione-S-Transferase (GST) and Uridine Phosphate Glucosyl-transferase, which cause pesticides to react with sugar, amino acid and glutathione to form metabolites with high water solubility and lower toxicity within cell organelles; Phase III detoxification in which metabolites of phase II are converted into secondary conjugates that are transported to the cytoplasm or the apoplast; and Phase IV detoxification which completely removes the toxicity of secondary conjugates. A series of enzymes such as peroxidases, esterases, oxidoreductases, hydrolases, glutathione transferases, and cytochrome 450s play an important role in these different stages of pesticide metabolism (Hall et al., 2001; Hoagland and Zablotowicz, 2001; Wheelock et al., 2005).
In the rice system, cellular absorption of different pesticides will initiate a series of stress responses, and the negative effects on physiology and biochemistry will differ with each pesticide type. For example, differential impacts occurred on GST activity for different rice varieties and different kinds of pesticides(Wu et al., 2003). In addition, different rice varieties had unique superoxide dismutase (SOD) activities after spraying with several kinds of pesticides (Wu et al., 2003).
3 Parts of pesticide residues in rice grain and distribution characteristics
In the rice grain, chemical pesticide residues can be found in three different parts: the rice husk, rice bran, and endosperm tissue, but different amounts of pesticide residue were observed in these tissues. Study showed thatdistribution levels of triazophos residue in rice grain were ordered, with the levels in rice husk > endosperm tissue > rice bran(Ying et al., 2010). Moreover, triazophos in the rice husk and bran represented nearly 90% of the total pesticide residue in the grain(Ying et al., 2010).
4 Differences of pesticide residues among rice varieties
Some researchers determined the residue content of triazophos and chlorpyrifos in rice grain using 18 different kinds of indica rice cultivars as the test material (Ying et al., 2011). In doing so, they showed significant differences in individual types and levels of pesticide residues among the rice varieties and in different parts of the rice grain. It was reported that common pesticides differentially affected the physiology and biochemistry of three kinds of rice varieties (Wu et al., 2006). For example, after five kinds of pesticide treatment, the content of oxalic acid and GST activity in rice decreased differentially among rice cultivars and pesticide effects. Some researchers reported that pesticide degradation rates also varied with different rice varieties, with the fastest degradation in the Wujing 15 variety (Yu et al. 2007). Moreover, the degradation rates for pesticide residues in leaf and unexpanded leaf were faster than those of stem. These research results confirmed a genetic basis for differences in pesticide residues found in different rice varieties.
5 Detection techniques and methods to remove pesticide residues
Recently, more sensitive and effective techniques have been developed for detecting pesticide residues in rice(Chen and Li, 2012; Uddin et al., 2011; Amirahmadi et al., 2013).These include chromatography-mass spectrometry (GC/MS) methods (Shen et al., 2012), which can detect extremely low levels (traces) of rice pesticides and can help in determining many different kinds of pesticide residue simultaneously. Hence, these are powerful techniques for accurately obtaining pesticide residue phenotypes.
In view of the increasingly serious issues of pesticide residues in rice, removal methods were also studied by different researchers (Saka et al. 2008; Qian et al. 2008). Pesticide residues in rice can be reduced by using pre-harvest control methods such as varying the pesticide spraying stage, using alternative pesticide types, changing dosage, and varying pesticide spraying times (Saka et al., 2008). Post-harvest control methods include varying the rice processing steps, storage, washing, cooking, exposure to sunlight and other means (Qian et al., 2008; Zhang et al., 2007). However, these methods mainly reduce pesticide residue artificially in rice, and especially have an impact on pesticide physical residue on the rice grain surface. As for intracellular pesticide chemical residues, additional approaches (suggested below) that take advantage ofendogenous rice genesare still needed to degrade these chemicals.
6 The feasibility of using QTL analysis to clone gene of pesticide residues in rice
Ma B.H (2010) reported that the inheritance of pesticide residues was genetic after measuring residues of cucumber propamocarb. Here, she showed that the inheritance of pesticide residues was inherited quantitative and controlled by minor genes. The additive gene effect was more important than the dominant effect. This genetically controlled phenotype depends on the activity of detoxification enzymes and gene transcription in the cell. Up to date there is no report about the QTLs of pesticide residues in rice, but Ma B.H's study will provides a useful model for identification QTLs of pesticide residue in rice.
In recent years, many useful QTLs, including Ehd2, GS3, GW5, DTH8, IPA1, Ghd7, Ghd8, and Ghd7.1 1 (Matsubara et al., 2008; Fan et al., 2006; Weng et al., 2008; Wei et al., 2010; Jiao et al., 2010; Xue et al., 2008; Yan et al., 2010; Yan et al., 2013), have been identified and cloned in rice. Hence, QTL identification and map based cloning have become one of the main directions of modern genetics research. This important approach can now be applied to dissect the genetic basis of pesticide residue degradation in rice. Differences in grain pesticide residues among different rice varieties mainly reflect the variability which occurs in detoxifying enzymes and detoxification genes.Therefore, constructing segregation populations and genetic linkage maps using varieties with significantly different pesticide residue levels as parents and screening them for target trait should be a feasible method for finding these genes. Using detection method such as GC-MS etc. (which have improved in recent years) will also provide added support for obtaining pesticide residue phenotypes.
Primary QTL identification for pesticide residue traits can be achieved using conventional linkakge analysis by following steps:
A segregating population should be constructed from significantly different parents for pesticide residues and pesticide residues of the segregating populations are scored.
Genetic linkage maps can be developed using molecular markers to scan segregating populations.
Combined phenotype (pesticide residue) data and molecular marker data and also using suitable QTL identification methods such as composite interval mapping or interval mapping, primary QTL identification for pesticide residue traits can be achieved.
In recent 20 years, along with the completion of rice genome sequencing project and the development of high-throughput sequencing technology, genome-wide association study (GWAS) in rice has been used widely in exploring quantitative trait gene (Huang et al., 2010; Huang et al., 2012; Hu et al., 2010). GWAS and cloning of the gene of pesticide residues may mainly comprise the following steps: firstly, the representative rice landraces with similar growth period are selected as a natural population for association analysis. Combining with the data of pesticide residues and sequencing of the natural population, primary QTLs of pesticide residues are obtained by using genome-wide association study (GWAS). Then near isogenic lines (NILs) of rice towards major QTLs are constructed. And major QTLs can be further fine mapping using conventional linkage analysis. At last fine mapping major QTLs can be cloned by using map-based cloning method.
The availability of such pesticide degrading genes from rice could then be used to develop transgenic cultivars with enhanced levels of pesticide degrading enzymes. Additionally, varieties with reduced pesticide levels could be developed through selection using the genes as molecular markers.
Conclusion and future prospects
Many fine mapping of QTL have been cloned by map based cloning methods. So far, however, there are no reports of QTL identification about pesticide residues in rice, let alone the reports of cloning genes related to pesticide metabolism.
The first reason is that the trait of pesticide residue is a complex one which is the same as the trait of yield influenced by many factors. So the trait of pesticide residue must be decomposed in the research of pesticide residues QTL or other factors influencing the pesticide residues must be fixed and only one factor is studied. A good solution for this is to construct near isogenic lines of the target trait. Due to the similar genetic background, near isogenic lines (NILs) can eliminate the background interference as far as possible, which makes the near isogenic lines become irreplaceable material for cloning target traits genes. The second reason is that there is no more effective method to explore the genes related to pesticide metabolism. Conventional linkage analysis is QTL mapping based on segregation population constructed by two parents. This kind of study only involved the two alleles for each locus, so it is difficult to identify more excellent target gene. Genome-wide Association Study (GWAS) is association analysis based on the natural population. GWAS can realize to explore all alleles of a locus and has the potential to identify the superior alleles of target traits. The linkage analysis is more suitable for the whole gene scanning and fine mapping, whereas GWAS is suitable for primary QTL identification. Therefore, combining two methods together can be more efficient to explore efficient gene about pesticide residues metabolism in rice.
Since large-scale planting of rice without pesticides is still not practical in China, cultivating rice varieties with low pesticide residues is important for rice food safety and protection of the environment.
First of all, we naturally think of using transgenic technology to cultivate rice varieties with low pesticide residues. That is to say, cultivating rice with less pesticide residue could be achieved by cultivating transgenic rice expressing cloned microbial genes for pesticide degradation. Presently this method is still in experimental stages of cloning these microbial genes and transferring them from their source microorganisms into other "working" microorganisms that will make more efficient pesticide-degrading bacteria. Hence, cultivating transgenic rice varieties using heterologous microbial genes has not been reported yet. A secondway to reduce pesticide residues in rice is usingQTL mappingtoclonegenes for pesticide residue degrading enzymes from the rice genome. Once these genes from the rice genome are cloned, new varieties with low pesticide residue could be developed by either transgenic enhancement or molecular marker assisted breeding using these cloned genes along with conventional development of hybrids.Large-scale commercial cultivation of the rice with low pesticide residue will be highly appreciated. Since transgenic rice’s with rice genes do not have too much controversy in food safety compared with the ones with microbial genes, genesforpesticide residue degradation from the rice genome canbe directly applied in molecular plant breeding and will havestrong practical significance.
Acknowledgements:
We thank Huazhong Agricultural University Professor Yu Sibin, Professor Xing Yongzhong and Professor He Yuqing for giving valuable suggestions to the content of the paper. This work was supported by grants from the Natural Science Foundation of Guangdong Province, China (8452840301001691) and the Scientific Research Team Training Project at the University of Electronic Science and Technology of China, Zhongshan Institute (No.412YT02).
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