Mol Genet Genomics DOI 10.1007/s00438-015-1066-y
ORIGINAL PAPER
Association mapping of soybean seed germination under salt stress Guizhen Kan1 · Wei Zhang1 · Wenming Yang1 · Deyuan Ma1 · Dan Zhang2 · Derong Hao3 · Zhenbin Hu1 · Deyue Yu1
Received: 5 November 2014 / Accepted: 12 May 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Soil salinity is a serious threat to agriculture sustainability worldwide. Seed germination is a critical phase that ensures the successful establishment and productivity of soybeans in saline soils. However, little information is available regarding soybean salt tolerance at the germination stage. The objective of this study was to identify the genetic mechanisms of soybean seed germination under salt stress. One natural population consisting of 191 soybean landraces was used in this study. Soybean seeds produced in four environments were used to evaluate the salt tolerance at their germination stage. Using 1142 singlenucleotide polymorphisms (SNPs), the molecular markers associated with salt tolerance were detected by genomewide association analysis. Eight SNP-trait associations and 13 suggestive SNP-trait associations were identified using a mixed linear model and the TASSEL 4.0 software. Eight SNPs or suggestive SNPs were co-associated with two salt tolerance indices, namely (1) the ratio of the germination Communicated by L. Xiong. Electronic supplementary material The online version of this article (doi:10.1007/s00438-015-1066-y) contains supplementary material, which is available to authorized users. * Deyue Yu [email protected] 1
National Center for Soybean Improvement/National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
2
Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
3
Jiangsu Yanjiang Institute of Agricultural Sciences, Nantong 226541, China
index under salt conditions to the germination index under no-salt conditions (ST-GI) and (2) the ratio of the germination rate under salt conditions to the germination rate under no-salt conditions (ST-GR). One SNP (BARC-02134704042) was significantly associated with these two traits (ST-GI and ST-GR). In addition, nine possible candidate genes were located in or near the genetic region where the above markers were mapped. Of these, five genes, Glyma08g12400.1, Glyma08g09730.1, Glyma18g47140.1, Glyma09g00460.1, and Glyma09g00490.3, were verified in response to salt stress at the germination stage. The SNPs detected could facilitate a better understanding of the genetic basis of soybean salt tolerance at the germination stage, and the marker BARC-021347-04042 could contribute to future breeding for soybean salt tolerance by markerassisted selection. Keywords Soybean · Salt tolerance · Germination · Genome-wide association mapping · Single-nucleotide polymorphisms
Introduction Soil salinity is a major abiotic stress that threatens the agricultural ecological environment and agriculture sustainability worldwide (Hamwieh and Xu 2008; Hamwieh et al. 2011; Wang et al. 2011). Approximately 20 % of agricultural land is currently saline (Flowers and Yeo 1995). Because of ineffective irrigation and fertilization (Flowers and Yeo 1995), soil salinity will become progressively more severe. Specifically, more than 50 % of all arable land is predicted to become saline by 2050 (Ashraf 1994). Soybean is classified as a moderately salt-tolerant crop (Ashraf 1994; Phang et al. 2008); however, soybean yields
13
are reduced when the soil salinity exceeds 5 dS/m (Ashraf 1994; Phang et al. 2008). Salt stress has been reported to severely affect the agronomic traits of soybeans by inhibiting seed germination and plant growth, promoting leaf chlorosis and bleaching, reducing the number of internodes, the biomass, and the final yield, and by changing the seed quality. In addition, salt stress can result in plant death (Pathan et al. 2007; Phang et al. 2008). In fact, salt tolerance is a complex trait that is affected by numerous genetic and non-genetic factors (Ashraf and Foolad 2013; DeRose-Wilson and Gaut 2011; Long et al. 2013; Pathan et al. 2007; Wang et al. 2011). The development of salttolerant cultivars is the most effective method for improving salinized land. However, the insufficient amount of precise salt tolerance measurements for breeding lines has hampered the breeding of salt-tolerant soybeans in fields with uneven distributions of salt. Therefore, understanding genetic architecture for soybean salt tolerance is crucial in developing salt-tolerant soybean cultivars (Pathan et al. 2007). The plant growth cycle includes germination, vegetative, and reproductive stages. Of these stages, seed germination plays an essential role (Bewley 1997). The germination of seeds in salt-affected soils is a primary factor in the agricultural production of soybean because it enables plant survival and reproduction. Thus, the salt tolerance of soybean seeds is critical for enhancing soybean yields. Soybean germplasms have different degrees of salt tolerance at different development stages (Pathan et al. 2007; Phang et al. 2008). In recent years, some markers linked closely to salt tolerance and several results from quantitative trait loci (QTL) analyses for salt (including alkaline salt) tolerance in soybean have been reported (Chen et al. 2008; Guo et al. 2002; Ha et al. 2013; Hamwieh and Xu 2008; Hamwieh et al. 2011; Lee et al. 2004; Pathan et al. 2007; Tuyen et al. 2010, 2013). These QTLs were mapped on linkage group (LG) D2 (Tuyen et al. 2010, 2013), LG G (Chen et al. 2008) and LG N (Lee et al. 2004; Hamwieh and Xu 2008; Hamwieh et al. 2011; Ha et al. 2013; Guan et al. 2014), and the QTL located on the LG N was confirmed in the same genetic region by the various researchers. The causal gene underlying this salt tolerance locus had been cloned by both whole-genome sequencing (Qi et al. 2014) and map-based cloning (Guan et al. 2014). These studies mainly focused on salt tolerance at the soybean seedling stage. A few studies regarding salt tolerance during germination have been conducted for tomato, rice, and barley (Long et al. 2013; Wang et al. 2011). However, limited information regarding salt tolerance during germination has been reported for soybean (Hosseini et al. 2002; Na et al. 2009; Qiu et al. 2011; Shao et al. 1994; Xu et al. 2011; Zhang et al. 2014b). The above QTLs were identified using bi-parental QTL mapping. Compared with bi-parental QTL analysis,
13
Mol Genet Genomics
association mapping based on linkage disequilibrium (LD) studies in natural populations with high natural variation provides higher resolution, which facilitates fine mapping and gene discovery (Famoso et al. 2011). Therefore, association mapping has been widely used in plants such as soybean (Hao et al. 2012a, b; Hu et al. 2013; Zhang et al. 2014a). However, although association analyses of salt tolerance have been reported for Arabidopsis and barley (DeRose-Wilson and Gaut 2011; Long et al. 2013), few reports are available regarding soybean (Zhang et al. 2014b). The salts that affect soils include 12 types of salt composed of three types of cations (i.e., Na+, Ca2+, and Mg2+) and four types of anions (CO32−, HCO32−, Cl−, and SO42−). Among these cations and anions, the proportions of Cl− and Na+ account for approximately 60 and 88 % of total salt ions, respectively (Wang et al. 1993). This study aimed to identify the genetic mechanisms underlying salt (NaCl) tolerance at the seed germination stage in diverse soybean accessions using association analysis. The objectives of the study were to (i) analyze the genetic variations for traits contributing to salt tolerance at the seed germination stage, (ii) analyze the salt tolerance indices in a diverse soybean collection, and (iii) identify the SNPs and possible candidate genes underlying salt tolerance at the seed germination stage in soybean. The major loci and possible candidate genes detected in this study would be useful to genetically improve soybean for salt tolerance.
Materials and methods Plant materials An association mapping panel consisting of 191 soybean accessions from different geographic origins was selected and successfully applied in a whole-genome association analysis for several soybean agronomical traits, including yield-related traits and phosphorus efficiency (Hao et al. 2012b; Zhang et al. 2014a). The field trials were conducted in the following four environments: the Jiangpu Experimental Station of Nanjing Agricultural University (32.12°N 118.37°E) in Nanjing, China, in 2012 and 2013; the Experimental Farm of the Jiangsu Yanjiang Institute of Agricultural Sciences (31.58°N 120.53°E) in Nantong, China, in 2012; and the Experimental Farm of the Agricultural College of Yangzhou University (32.23°N 119.25°E) in Yangzhou, China, in 2012. All accessions were planted in three-row plots in 200-cm-long rows with a row spacing of 50 cm. A randomized complete-block design was used for all experiments and was replicated three times. Seeds were collected at maturity to evaluate the phenotypes related to germination under conditions with and without salt stress.
Mol Genet Genomics
Salt tolerance evaluation Forty healthy seeds were selected, weighed, and placed in sterilized Petri dishes (9 cm diameter) containing two sheets of filter paper. Next, 15 mL of the NaCl solutions (0 or 150 mM) were added to the dishes. The seeds were incubated in the dark in a temperature-controlled germination chamber at 25 ± 1 °C. After 24 h, the imbibed seeds were weighed and the number of germinated seeds was counted. Seeds were considered to have germinated when the radicle and plumule lengths were equivalent to the seed lengths or half of the seed length, respectively. Subsequently, the seeds were rinsed and placed into new dishes with filter paper before adding 5 mL of NaCl solution (0 or 150 mM) to the dishes. Over the next 6 days, the germinated seeds were counted before rinsing the seeds and adding 5 mL of NaCl solution (0 or 150 mM) to the dishes every 24 h. This seed germination experiment was repeated three times for the salt treatment and for the control. The evaluated germination traits included the imbibition rate [IR % = (W2 – W1)/W1 × 100, where W2 represents the seed weight after imbibition for 24 h and W1 represents the dry seed weight before imbibition] (Wang et al. 2011), the germination index (GI = ΣGt/Dt, where Dt represents N days of seed germination and Gt represents the number of germinated seeds on day N), and the germination rate [GR % = (number of germinated seeds/total seed number used in the test) × 100]. The salt tolerance (ST) was defined as the ratio of the germination-related traits (IR, GI, and GR) under salt conditions to the same traits under saltfree conditions (Long et al. 2013). Phenotypic data analysis Statistical analysis of all phenotypic data was performed with the software SAS version 9.0 (SAS Institute, Inc., Cary, NC, USA). Descriptive statistics and correlation analyses were performed using the mean values of all phenotypic data from the traits of 191 soybean accessions across four environments. An analysis of variance (ANOVA) was performed for all traits using the PROC GLM, and the Pearson’s correlations between the traits were calculated using the mean values in the PROC CORR. SNP genotyping A total of 2435 random SNPs evenly covering the genome were selected from the Soybean SNP database (http://bfgl. anri.barc.usda.gov/soybean/) (Choi et al. 2007). Based on their quality scores, the best 1536 SNPs were chosen to design the GoldenGate assay (Shen et al. 2005). SNP genotyping was performed via the GoldenGate assay on the Illumina Beadlab system at the National Engineering
Center for Biochip (Shanghai, China). The SNP genotyping procedure was described by Hao et al. (2012a, b) and Hu et al. (2013). The SNP genotyping and genome-wide association mapping of the 191 accessions were conducted with 1536 SNP markers. Among the 1536 SNPs, 222 SNPs with minor allele frequency (MAFs)
ORIGINAL PAPER
Association mapping of soybean seed germination under salt stress Guizhen Kan1 · Wei Zhang1 · Wenming Yang1 · Deyuan Ma1 · Dan Zhang2 · Derong Hao3 · Zhenbin Hu1 · Deyue Yu1
Received: 5 November 2014 / Accepted: 12 May 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Soil salinity is a serious threat to agriculture sustainability worldwide. Seed germination is a critical phase that ensures the successful establishment and productivity of soybeans in saline soils. However, little information is available regarding soybean salt tolerance at the germination stage. The objective of this study was to identify the genetic mechanisms of soybean seed germination under salt stress. One natural population consisting of 191 soybean landraces was used in this study. Soybean seeds produced in four environments were used to evaluate the salt tolerance at their germination stage. Using 1142 singlenucleotide polymorphisms (SNPs), the molecular markers associated with salt tolerance were detected by genomewide association analysis. Eight SNP-trait associations and 13 suggestive SNP-trait associations were identified using a mixed linear model and the TASSEL 4.0 software. Eight SNPs or suggestive SNPs were co-associated with two salt tolerance indices, namely (1) the ratio of the germination Communicated by L. Xiong. Electronic supplementary material The online version of this article (doi:10.1007/s00438-015-1066-y) contains supplementary material, which is available to authorized users. * Deyue Yu [email protected] 1
National Center for Soybean Improvement/National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
2
Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
3
Jiangsu Yanjiang Institute of Agricultural Sciences, Nantong 226541, China
index under salt conditions to the germination index under no-salt conditions (ST-GI) and (2) the ratio of the germination rate under salt conditions to the germination rate under no-salt conditions (ST-GR). One SNP (BARC-02134704042) was significantly associated with these two traits (ST-GI and ST-GR). In addition, nine possible candidate genes were located in or near the genetic region where the above markers were mapped. Of these, five genes, Glyma08g12400.1, Glyma08g09730.1, Glyma18g47140.1, Glyma09g00460.1, and Glyma09g00490.3, were verified in response to salt stress at the germination stage. The SNPs detected could facilitate a better understanding of the genetic basis of soybean salt tolerance at the germination stage, and the marker BARC-021347-04042 could contribute to future breeding for soybean salt tolerance by markerassisted selection. Keywords Soybean · Salt tolerance · Germination · Genome-wide association mapping · Single-nucleotide polymorphisms
Introduction Soil salinity is a major abiotic stress that threatens the agricultural ecological environment and agriculture sustainability worldwide (Hamwieh and Xu 2008; Hamwieh et al. 2011; Wang et al. 2011). Approximately 20 % of agricultural land is currently saline (Flowers and Yeo 1995). Because of ineffective irrigation and fertilization (Flowers and Yeo 1995), soil salinity will become progressively more severe. Specifically, more than 50 % of all arable land is predicted to become saline by 2050 (Ashraf 1994). Soybean is classified as a moderately salt-tolerant crop (Ashraf 1994; Phang et al. 2008); however, soybean yields
13
are reduced when the soil salinity exceeds 5 dS/m (Ashraf 1994; Phang et al. 2008). Salt stress has been reported to severely affect the agronomic traits of soybeans by inhibiting seed germination and plant growth, promoting leaf chlorosis and bleaching, reducing the number of internodes, the biomass, and the final yield, and by changing the seed quality. In addition, salt stress can result in plant death (Pathan et al. 2007; Phang et al. 2008). In fact, salt tolerance is a complex trait that is affected by numerous genetic and non-genetic factors (Ashraf and Foolad 2013; DeRose-Wilson and Gaut 2011; Long et al. 2013; Pathan et al. 2007; Wang et al. 2011). The development of salttolerant cultivars is the most effective method for improving salinized land. However, the insufficient amount of precise salt tolerance measurements for breeding lines has hampered the breeding of salt-tolerant soybeans in fields with uneven distributions of salt. Therefore, understanding genetic architecture for soybean salt tolerance is crucial in developing salt-tolerant soybean cultivars (Pathan et al. 2007). The plant growth cycle includes germination, vegetative, and reproductive stages. Of these stages, seed germination plays an essential role (Bewley 1997). The germination of seeds in salt-affected soils is a primary factor in the agricultural production of soybean because it enables plant survival and reproduction. Thus, the salt tolerance of soybean seeds is critical for enhancing soybean yields. Soybean germplasms have different degrees of salt tolerance at different development stages (Pathan et al. 2007; Phang et al. 2008). In recent years, some markers linked closely to salt tolerance and several results from quantitative trait loci (QTL) analyses for salt (including alkaline salt) tolerance in soybean have been reported (Chen et al. 2008; Guo et al. 2002; Ha et al. 2013; Hamwieh and Xu 2008; Hamwieh et al. 2011; Lee et al. 2004; Pathan et al. 2007; Tuyen et al. 2010, 2013). These QTLs were mapped on linkage group (LG) D2 (Tuyen et al. 2010, 2013), LG G (Chen et al. 2008) and LG N (Lee et al. 2004; Hamwieh and Xu 2008; Hamwieh et al. 2011; Ha et al. 2013; Guan et al. 2014), and the QTL located on the LG N was confirmed in the same genetic region by the various researchers. The causal gene underlying this salt tolerance locus had been cloned by both whole-genome sequencing (Qi et al. 2014) and map-based cloning (Guan et al. 2014). These studies mainly focused on salt tolerance at the soybean seedling stage. A few studies regarding salt tolerance during germination have been conducted for tomato, rice, and barley (Long et al. 2013; Wang et al. 2011). However, limited information regarding salt tolerance during germination has been reported for soybean (Hosseini et al. 2002; Na et al. 2009; Qiu et al. 2011; Shao et al. 1994; Xu et al. 2011; Zhang et al. 2014b). The above QTLs were identified using bi-parental QTL mapping. Compared with bi-parental QTL analysis,
13
Mol Genet Genomics
association mapping based on linkage disequilibrium (LD) studies in natural populations with high natural variation provides higher resolution, which facilitates fine mapping and gene discovery (Famoso et al. 2011). Therefore, association mapping has been widely used in plants such as soybean (Hao et al. 2012a, b; Hu et al. 2013; Zhang et al. 2014a). However, although association analyses of salt tolerance have been reported for Arabidopsis and barley (DeRose-Wilson and Gaut 2011; Long et al. 2013), few reports are available regarding soybean (Zhang et al. 2014b). The salts that affect soils include 12 types of salt composed of three types of cations (i.e., Na+, Ca2+, and Mg2+) and four types of anions (CO32−, HCO32−, Cl−, and SO42−). Among these cations and anions, the proportions of Cl− and Na+ account for approximately 60 and 88 % of total salt ions, respectively (Wang et al. 1993). This study aimed to identify the genetic mechanisms underlying salt (NaCl) tolerance at the seed germination stage in diverse soybean accessions using association analysis. The objectives of the study were to (i) analyze the genetic variations for traits contributing to salt tolerance at the seed germination stage, (ii) analyze the salt tolerance indices in a diverse soybean collection, and (iii) identify the SNPs and possible candidate genes underlying salt tolerance at the seed germination stage in soybean. The major loci and possible candidate genes detected in this study would be useful to genetically improve soybean for salt tolerance.
Materials and methods Plant materials An association mapping panel consisting of 191 soybean accessions from different geographic origins was selected and successfully applied in a whole-genome association analysis for several soybean agronomical traits, including yield-related traits and phosphorus efficiency (Hao et al. 2012b; Zhang et al. 2014a). The field trials were conducted in the following four environments: the Jiangpu Experimental Station of Nanjing Agricultural University (32.12°N 118.37°E) in Nanjing, China, in 2012 and 2013; the Experimental Farm of the Jiangsu Yanjiang Institute of Agricultural Sciences (31.58°N 120.53°E) in Nantong, China, in 2012; and the Experimental Farm of the Agricultural College of Yangzhou University (32.23°N 119.25°E) in Yangzhou, China, in 2012. All accessions were planted in three-row plots in 200-cm-long rows with a row spacing of 50 cm. A randomized complete-block design was used for all experiments and was replicated three times. Seeds were collected at maturity to evaluate the phenotypes related to germination under conditions with and without salt stress.
Mol Genet Genomics
Salt tolerance evaluation Forty healthy seeds were selected, weighed, and placed in sterilized Petri dishes (9 cm diameter) containing two sheets of filter paper. Next, 15 mL of the NaCl solutions (0 or 150 mM) were added to the dishes. The seeds were incubated in the dark in a temperature-controlled germination chamber at 25 ± 1 °C. After 24 h, the imbibed seeds were weighed and the number of germinated seeds was counted. Seeds were considered to have germinated when the radicle and plumule lengths were equivalent to the seed lengths or half of the seed length, respectively. Subsequently, the seeds were rinsed and placed into new dishes with filter paper before adding 5 mL of NaCl solution (0 or 150 mM) to the dishes. Over the next 6 days, the germinated seeds were counted before rinsing the seeds and adding 5 mL of NaCl solution (0 or 150 mM) to the dishes every 24 h. This seed germination experiment was repeated three times for the salt treatment and for the control. The evaluated germination traits included the imbibition rate [IR % = (W2 – W1)/W1 × 100, where W2 represents the seed weight after imbibition for 24 h and W1 represents the dry seed weight before imbibition] (Wang et al. 2011), the germination index (GI = ΣGt/Dt, where Dt represents N days of seed germination and Gt represents the number of germinated seeds on day N), and the germination rate [GR % = (number of germinated seeds/total seed number used in the test) × 100]. The salt tolerance (ST) was defined as the ratio of the germination-related traits (IR, GI, and GR) under salt conditions to the same traits under saltfree conditions (Long et al. 2013). Phenotypic data analysis Statistical analysis of all phenotypic data was performed with the software SAS version 9.0 (SAS Institute, Inc., Cary, NC, USA). Descriptive statistics and correlation analyses were performed using the mean values of all phenotypic data from the traits of 191 soybean accessions across four environments. An analysis of variance (ANOVA) was performed for all traits using the PROC GLM, and the Pearson’s correlations between the traits were calculated using the mean values in the PROC CORR. SNP genotyping A total of 2435 random SNPs evenly covering the genome were selected from the Soybean SNP database (http://bfgl. anri.barc.usda.gov/soybean/) (Choi et al. 2007). Based on their quality scores, the best 1536 SNPs were chosen to design the GoldenGate assay (Shen et al. 2005). SNP genotyping was performed via the GoldenGate assay on the Illumina Beadlab system at the National Engineering
Center for Biochip (Shanghai, China). The SNP genotyping procedure was described by Hao et al. (2012a, b) and Hu et al. (2013). The SNP genotyping and genome-wide association mapping of the 191 accessions were conducted with 1536 SNP markers. Among the 1536 SNPs, 222 SNPs with minor allele frequency (MAFs)
