Assessment of Marker Based Genotype Diversity of Saltol QTL in Relation to Seedling Stage Salinity Tolerance in Selected Rice Genotypes

Salinity stress is one of the major soil problems in relation to rice production in Sri Lanka. Development of salinity tolerant rice through rice breeding is a key solution for salinity problem. Saltol is the major Quantitative Trait Loci (QTL) responsible for salt tolerance at the seedling stage of the rice plants which is mapped on chromosome one of the rice genome. To select most salt tolerant rice genotypes for future breeding programmes and test the usefulness of the Microsatellite (SSR) markers associated with Saltol QTL, 18 rice varieties were screened using electrical conductivity 12 dSm-1 nutrient solution for salt tolerance at the seedling stage using a visual score of 1-9 scale. Six polymorphic SSR markers linked to the Saltol QTL were used to determine the impact of these markers associated with salt tolerance in rice. Results of the phenotypic screening of 18 rice genotypes showed different responses for salt stress at the seedling stage. Based on the phenotypic screening rice genotypes were categorized into four groups, and Pokkali accessions were the most tolerant genotypes for salinity stress which can be useful as donor parents for future rice breeding programs. Microsatellite marker RM10843 showed highest allelic number while RM140 and RM10748 showed lowest allelic number. Polymorphism information content (PIC) value varied from 0.1780 for RM10748 to 0.7659 for marker alleles. Based on the six SSR markers, 18 rice genotypes were clustered into two groups and salinity tolerant Pokkali accessions were grouped in a separate cluster. Salinity tolerant and susceptible genotypes were clearly discriminated by RM10852 marker in 3% agarose gel and positively associated with the result of the phenotypic screening. Therefore, the marker will be useful in Marker Assisted Selection (MAS) in rice breeding programmes for salinity tolerance.


ABSTRACT
Salinity stress is one of the major soil problems in relation to rice production in Sri Lanka. Development of salinity tolerant rice through rice breeding is a key solution for salinity problem. Saltol is the major Quantitative Trait Loci (QTL) responsible for salt tolerance at the seedling stage of the rice plants which is mapped on chromosome one of the rice genome. To select most salt tolerant rice genotypes for future breeding programmes and test the usefulness of the Microsatellite (SSR) markers associated with Saltol QTL, 18 rice varieties were screened using electrical conductivity 12 dSm -1 nutrient solution for salt tolerance at the seedling stage using a visual score of 1-9 scale. Six polymorphic SSR markers linked to the Saltol QTL were used to determine the impact of these markers associated with salt tolerance in rice. Results of the phenotypic screening of 18 rice genotypes showed different responses for salt stress at the seedling stage. Based on the phenotypic screening rice genotypes were categorized into four groups, and Pokkali accessions were the most tolerant genotypes for salinity stress which can be useful as donor parents for future rice breeding programs. Microsatellite marker RM10843 showed highest allelic number while RM140 and RM10748 showed lowest allelic number. Polymorphism information content (PIC) value varied from 0.1780 for RM10748 to 0.7659 for marker alleles. Based on the six SSR markers, 18 rice genotypes were clustered into two groups and salinity tolerant Pokkali accessions were grouped in a separate cluster. Salinity tolerant and susceptible genotypes were clearly discriminated by RM10852 marker in 3% agarose gel and positively associated with the result of the phenotypic screening. Therefore, the marker will be useful in Marker Assisted Selection (MAS) in rice breeding programmes for salinity tolerance.

INTRODUCTION
Rice is one of the most important cereal crops which serves as the staple food for one-third of the global population. It is the world's third-largest cereal crop after wheat and maize (Mohommad-Nejad et al., 2010). However, many abiotic stresses such as salinity, iron toxicity, acidity and drought greatly affect the rice production in these regions. At present, salinity is the second most widespread abiotic problem next to drought (Islam et al., 2010). Around 21.5 million hectares of the land area is seriously affected by salinity in the south and southeast Asian region alone, where majority of the rice-growing areas of the world are situated (Li et al., 2014). Salinity is a major constraint to expanding rice cultivation in Sri Lanka. Nearly 70% of the coastal rice lands are reported to be affected by salinity (De Costa et al., 2012). Sirisena and Herath (2009) stated that salinity is gradually spreading in rice lands of Sri Lanka both in coastal and inland regions. Out of 12,000 ha of cultivated rice lands in Jaffna, about 3,000 ha are unproductive due to salinity (Rajadurai, 2003).
Rice plant is highly sensitive for salt stress, especially at early seedling and late reproduction stages (Zeng and Shannon, 2000), where salinity greatly affects rice yield. Vinod et al. (2013) reported a 12% yield reduction for every unit of dSm -1 of electrical conductivity (EC) above the threshold level of tolerance of 3.0 dSm -1 .
Breeding a salinity tolerant rice variety is one of the best approaches to overcome the effect of adverse saline soil condition. The rapid advancement of DNA based molecular marker technologies and their applications in plant breeding programmes were reported in the last two decades. Unravelling of rice genome has given an infinite opportunity to produce SSR markers throughout the rice genome (Thomson et al., 2010). Therefore, a higher number of SSR markers are developed which are tightly linked to the important genes and QTLs regulating different important mechanisms in rice plant. Saltol is one of the major QTLs for salt tolerance at the seedling stage (Bonilla et al., 2002). Recently the Saltol QTL has been mapped by several studies (Bonilla et al., 2002;Thomson et al., 2010;Soda et al., 2013). One of the studies reported that Saltol QTL is located between the SSR marker RM1287 and RM6711 (10.8Mb -16.4Mb) in the short arm of the rice chromosome one (Thomson et al., 2010). Platten et al. (2006) reported SKC1 (later named as gene OsHKT1;5) is a causal gene underlying the Saltol QTL which is supported to control sodium/potassium (Na + /K + ) ratio. SKC1 was found to encode a sodium transporter protein that helps control Na + /K + homeostasis through the unloading of sodium from the xylem, which has been functioning mainly in roots to reduce the amount of Na + ions that are transported to the leaves (Ren et al., 2005). There are many functional SSR markers which are tightly linked to the important loci of the Saltol QTL. Therefore, those markers will be very useful in genetic studies of the Saltol region and will provide significant assistance for future salinity tolerance rice breeding programmes. The objectives of the present study were as follows: The assessment of salinity tolerant ability of the popular local rice varieties and group those varieties based on their salinity tolerance level at the seedling stage, select the useful donor parents for future salinity tolerant rice breeding programmes, identification of positively associated SSR marker/markers for salinity tolerance with the results of phenotype screening for salinity tolerance, and identification of useful SSR markers for marker assisted selection to assist future salinity tolerant rice breeding programmes.

MATERIALS AND METHODS
The laboratory work was conducted at the Rice Research and Development Institute (RRDI) at Bathalagoda from May 2016 to November 2017. Sixteen rice varieties and two Pokkali accessions were used for the study. Seeds of each variety were obtained from RRDI, Bathalagoda and Plant Genetic Resource Center, Gannoruwa, Sri Lanka.

Morphological screening
The salinity tolerant ability at the seedling stage of the 16 rice varieties namely Bg369, Bg359, IR42, IR46, Bg250, Bg300, Bg360, Bg358, Bg370, Bg357, Bg310, Bg94/1, IR36, Bg380, At303, At362 and two Pokkali accessions were tested by using the method developed by Gregorio et al. (1997b) with some modifications. Varieties were selected based on different criteria such as reported salinity tolerant ability under field conditions, popularity of the variety and usability of each variety for future breeding programmes as donor and recipient parents. Pokkali IRRI accession, which is the most salinity tolerant IRRI accession and, Pokkali local accession 5557, which is one of the most salinity tolerant local accessions (Senanayake et al., 2017a) were used as known salinity tolerant genotypes.
The morphological screening was done in the green house of the RRDI, Bathalagoda. Two replicates (two trays) were used for each variety in Complete Randomized Design (CRD) for the experiment. Each variety was separately planted in the each replicate with twenty-five seedlings. Rectangular Styrofoam sheets of 50 cm (Length, L) x 30 cm (Width, W) x 1.25 cm (Depth, D) were used for anchoring seedlings on the nutrition solution. Holes of 1 cm diameter were drilled, which were spaced 2.5 cm within the rows and 4 cm between the rows. A nylon net with sieve size less than 1 mm was fixed on one side of each styrofoam board. Plastic trays of 55 cm (L) x 35 cm (W) x 8 cm (D) were used to fill the nutrient solution and the prepared styrofoam board floated on these trays without letting them move.

Seed germination and maintenance during the first twelve days
Sterilized seeds were placed in petri dishes on moistened filter paper and incubated for 48 hours to facilitate germination as in Gregorio et al. (1997b). Two pre-germinated seeds were inserted per each hole on the styrofoam seedling float and radicle was carefully inserted through nylon mesh.
Pre-germinated seeds were maintained under the water for three days to recover the damage of roots. After three days, the water was replaced by 2 dSm -1 Albert solution at pH 6.5. The seedlings were maintained for nine days in these conditions. One well grown seedling was retained while removing the poorly grown seedling in each hole. Twenty-five healthy seedlings were remained in each replicate. Freshly prepared Albert solution at the 2 dSm -1 was added to maintain the constant level of the nutrient solution to avoid plants getting dried in first 12 days.

Initiating salt stress condition and evaluation of the seedlings
Pre-washed table salt was used to prepare a solution with electrical conductivity (EC) 10 dSm -1 . Albert nutrient solution was added to the salt solution to increase the EC up to12 dSm -1 and pH was adjusted to 6.5 using 1M sodium hydroxide solution. This solution was used as the stress solution to evaluate the response of the rice seedlings. The initial nutrient solution (2 dSm -1 ) was replaced by using 12 dSm -1 stress solutions to start the salt stress on rice seedlings, when seedlings were 12 days old. The rice seedlings were exposed for the salt stress condition for 14 days. The control experiment (unstressed seedlings) consisted of 10 plants of each variety which was maintained with Albert solution at 2 dSm -1 EC and pH 6.5 simultaneously. The EC and pH of the stress solution were checked every 3 days and kept constant throughout the stress period by adding freshly prepared 12 dSm -1 solution as same to the initial stress solution.
The seedlings were visually inspected for salt stress symptoms at 7, 10 and 14 days after imposing the stress solution. Salt stress symptoms in each plot were inspected separately and tolerance score was assigned based on modified standard evaluating score system (Table 1) developed by Gregorio et al. (1997b). Finally average tolerance score was given for each variety and categorized as in Table 1. To check the differences of response among varieties for salinity tolerance at seedling stage, ANOVA was performed using General Linear Model (GLM) procedure followed by the Duncan Multiple Range Test (DMRT). The evaluated results were used for the molecular markerbased genotype diversity analysis.  (Table 2). However, Senanayaka et al. (2017b) reported that the variety Bg369 was categorized under the salinity tolerant category at seedling stage. Therefore, the popular local variety Bg369 will be useful for cultivation in moderately saline regions. However, the salinity tolerance response of these varieties both at the vegetative and reproductive stages should be evaluated to arrive at a more precise conclusion.
Most part of the leaves of the Bg357, Bg310, Bg94/1, IR36, Bg380, At303 and At362 varieties were dried and seedling growth was severely retarded with the salinity stress at the end of 12 days. The tolerance score of these varieties was significantly different from moderately tolerant varieties. Average evaluating score of 7 was given for these varieties based on the symptoms and categorized under the salinity susceptible (S) category. The varieties Bg250, Bg300, Bg360, Bg358 and Bg370 almost withered and dried during seedling stage itself and no vegetative growth could be observed. The tolerance score of these varieties was significantly different from susceptible varieties. Average evaluating score of 9 was given for those varieties and were categorized under highly susceptible (HS) group. None of the varieties in the control experiment show any salt stress symptom and hence their salinity tolerance score was 1. Salinity affects all growth stages of the rice plant in varying degrees from germination to maturation. The rice plant is especially very sensitive to salinity during the early seedling and late reproductive stages (Lafitte et al., 2004). Therefore, the result of the morphological screening will be very useful for selecting the suitable varieties for the saline soils in Sri Lanka. Therefore, a prospective study to test the varietal response for salinity stress at the vegetative and reproductive stages would be helpful for further validation of the results. The final response of the 18 rice varieties for the salt stress after 14 days is shown in table 2. Bg370 j IR555178/9-3R//IR65515-47-2-1/IR6551-47-2-1-91 9 HS

Molecular marker analysis
The six markers were spanning for a 3.0 Mbp length in the Saltol region of the chromosome one (Thomson et al., 2010). The SSR markers were based on the allele diversity which ranged from one hexa-allelic marker RM10843, one penta-allelic marker RM3412, one quard-allelic marker RM10852 and three bi-allelic markers RM8094, RM140 and RM10748. The RM10843 marker will be useful to perform a molecular diversity analysis between different Pokkali accessions in Sri Lanka, because Pokkali alleles were slightly distinguishable from other alleles of the different genotypes. Nineteen separate alleles were identified for six SSR markers with 3.5 average alleles per marker. Highest number of genotype number was six and it was given by RM 10843 markers. Mohommad-nejad et al. (2010) and Chowdhury et al. (2016) reported that RM8094 marker showed highest PIC value and highest number of marker alleles. The present study did not agree with those studies where RM8094 showed only two alleles for eighteen genotypes with low PIC value. Chowdhury et al. (2016) also reported that RM 10843 and RM3412 showed only three marker alleles among 54 rice genotypes in 6% poly acrylamide gel electrophoresis. In the present study same markers have given a high number of alleles even in 3% agarose gel electrophoresis.
The polymorphic information content (PIC value) in the present study varied from 0.1780 to 0.7659 with an average 0.4650. Based on the PIC value three markers, i.e. RM3412, RM10843 and RM 10852, were considered as highly informative because the PIC value was greater than 0.5. The highest PIC value was reported by RM10843 SSR marker as 0.7659. RM8094 SSR maker showed a PIC value of 0.3711 and it was considered as an informative marker because PIC ranges were 0.25-0.5. The markers RM140 and RM 10748 were considered as less informative because they showed a PIC value less than 0.25 (Table 3).
The marker alleles of RM10852 (Figure 1) was positively associated with the salinity tolerance genotypes. The marker alleles of the Pokkali IRRI accession and local accession were unique and discriminated salt tolerant and susceptible genotypes. The RM10852 markers fairly discriminated the salinity tolerance genotypes even in 3% Agarose gel. It is easy and less laborious compared with the polyacrylamide gel electrophoresis. Hence, this marker would be an effective tool for marker-assisted back-crossing. Huyen et al. (2013) reported RM10852 marker could discriminate the salinity tolerance and susceptible genotypes in 6% polyacrylamide gel electrophoresis. Therefore, further validation with polyacrylamide gel electrophoresis will be helpful to re-confirm the results of the present study. Furthermore, Pokkali marker allele RM10852 will be useful to select the back-crossed population, if the same Pokkali accessions is used as donor parents in marker assisted back-crossed programmes. The Pokkali allele of the RM10843 marker showed some separation from other alleles except for a few exceptions. However, polyacrylamide gel electrophoresis is needed to confirm whether it is clearly discriminate two salinity tolerance genotypes form others (Figure 2). RM 8094 is one of the most tightly linked markers in Saltol QTL with highest LOD peak value and the maximum alleles were found for the RM8094 marker (Thomson et al., 2010;Islam et al., 2011). The previous studies conducted by Mohammad-Nejad et al. (2010), Thompson et al. (2010) and Babu et al. (2014) used RM8094 and RM3412 to test the discriminating ability of salinity tolerance and susceptible genotype. In those studies, maximum alleles were found for the RM8094 marker. Rice genotypes having Pokkali allele for RM8094 marker were strongly associated with tolerant or highly tolerant phenotypes during the morphological screening. However, in this study, this positive association was not identified for the RM8094 marker.

Cluster analysis
According to the similarity matrix introduced by Nei et al. (1983), lowest genetic distance (0.333) was reported between Pokkali IRRI and Pokkali local accession which reflected the genetic closedness between the Pokkali local and Pokkali IRRI varieties. The UPGMA analysis defined all 18 rice genotypes into two clusters (Figure 3). Cluster one consisted of the salinity tolerance genotypes which consisted of only the Pokkali IRRI accession and Pokkali local accession. These two varieties were clearly divergent from the moderately tolerant, susceptible and highly susceptible varieties during the phenotypic screening too. Therefore, phenotypic screening results were further confirmed by salinity tolerance accessions by the cluster analysis. The results also showed that the salinity tolerance of the Pokkali accessions share a common allele for the markers.
Therefore, screening of all local Pokkali accessions and genotyping with more tightly linked SSR markers to Saltol region will be useful to further explain the relationship between Pokkali allele of the marker and salinity tolerance response during the seedling stage.
The second cluster was divided into two subclusters ( Figure 3). Moderately salinity tolerant and susceptible varieties were distributed in these two clusters and it was difficult to explain the common relationship of the varieties in cluster 02 and sub-cluster of the cluster 02. The moderately tolerant varieties Bg359 and Bg369 were clustered in sub cluster 02. Therefore, the salinity tolerance relationship among these varieties at the seedling stage is difficult to explain by these markers, because the salinity tolerance mechanism of seedling stage regulation is done by many other QTLs in addition to Saltol QTLs. Gregorio (1997a) reported that the Saltol QTL governed the Na + /K + uptake by 64.3 to 80.2%, and Thomson et al. (2010) reported the Saltol is responsible for 60% total phenotypic variations of the seedling stage salinity tolerance in rice. Many QTLs which are responsible for seedling stage salinity tolerance in rice were mapped in the chromosome 2, 3, 4, 6, 9 and 12 of the rice genome (Thomson et al., 2010) (Figure 3).

CONCLUSIONS
Pokkali IRRI accession and Pokkali local accession (RRDI Batalagoda accession 5557) were tolerant to salinity stress at the seedling stage. Hence, those varieties will be useful as donor parents for future salinity tolerance improvement programmes. The popular local rice varieties Bg359 and Bg369 were moderately tolerant for salinity stress at the seedling stage. Therefore, these varieties will be useful for the soils which are moderately affected by the salinity.
The microsatellite marker (SSR) RM10852 was positively associated with salinity tolerance rice genotypes in the 3% agarose gel electrophoresis. Therefore, this marker will be useful for Marker Assisted Selection (MAS) in rice breeding programmes and detecting the salinity tolerant Pokkali allele in the back-crossed population derived from the same Pokkali accession as donor parent. However, further validation will be needed to test the discriminating ability of salinity tolerant and susceptible genotypes by RM 10843 marker with polyacrylamide gel electrophoresis.