Identification of Candidate Gene SalT and Designing Markers Involving in Salt Tolerance of Vietnamese Rice Landraces

. Rice ( Oryza sativa L.) is a principle crop as the main economic importance in Vietnam, providing daily food for over 90 million people in this country. However, a large rice growing areas and rice production are being seriously affected by salinity intrusion, the threats of devastation from climate change. The need to develop salinity tolerance rice varieties to cope with adverse climate change is very imperative. In this study, based on the genome sequence databases of 36 Vietnamese rice landraces, we have identified nine Vietnamese rice landraces carrying nine SalT candidate genes with the sequence similarity to O. sativa SalT (the published GenBank: Z25811.1) which have shown salinity tolerance are included: Te Nuong, Khau mac buoc, Chan thom, Khau giang, Tan ngan, Nang thom cho dao, OM5629, Hom rau and Thom Lai landraces). Amongst them, four rice landraces, Nang thom cho dao, OM5629, Hom rau and Thom lai have revealed two fragments of deletion with six and seven nucleotides which were the most identical to the SalT reference gene. Two primer pairs have been successfully designed to identify the SalT candidate genes in Vietnamese rice landraces. This study provides useful information of salinity tolerance of some Vietnamese rice landraces for breeding programs.


Introduction
Salinity tolerance of rice derives from genes that limit the rate of salt uptake from the soil and the transport of salt across the plant, adjust the ionic and osmotic balance of cells in roots and shoots, and regulate leaf development and onset of senescence [1]. Salinity is a key abiotic constraint devastating crop production in the world. One-fifth of irrigate arable lands in the world has been reported to be adversly affected by high soil salinity. Saline soils occur naturally in both coastal areas, where ground water is contaminated by sea water rise, and in areas subjected to irrigation and/or draining. Salt stress is one of the major serious factors limiting the productivity of rice crop in many worldwide areas. According to report of FAO [2] over 800 million ha of worldwide arable lands are severely salt affected and approximately 20% of irrigated areas. In Asia, 21.5 million hectare of land areas are being influenced by salinity and estimated to cause the loss of up to 50% fertile land by the 21 st midcentury [3].
Rice (Oryza sativa L.) is the most important food in Vietnam, providing daily food for over 90 million people in this country. Presently, Vietnam is a second biggest rice exporter in the world, accounting for 50% of the world rice trade [4]. Most of Vietnam's rice is produced in the areas of Mekong and Red River Deltas where most acute rice growing areas in close vincity of the sea are vulnerable to salinity. Moreover, this country has been ranked as one of the countries most affected by climate change, as a consequence, rice production is particularly vulnerable [5]. Salt intrusion due to sea level rise has caused adverse effects on one million cultivated hectares. equally with 3.0% of total areas of this country, causing the economic loss by salt intrusion in 2005 was up to 45 million USD [6]. To date, approximately 600.000 ha of rice growing areas in this country are being severely influenced by drought and saline intrusion in the early year of 2016, has caused seriously economic losses up to 15 trillion VND in rice only (≈ 670 million USD) and reduced rice harvest by up to 50% [7]. Therefore, the need to develop salinity tolerance rice varieties to cope with adverse climate change is very imperative.
To enhance salt tolerance of crops, included rice crop, it undergoes a variety of changes from physiological adaptations to gene expression. There are numberous such as reclamation, irrigation, and drainage are used to minimize soil salinity. However, it is not always shown economical or practical. Based on the known mechanisms of rice plant to response salt stress, the three major approaches to enhance rice salinity tolerance in rice are being utilized: (i) conventional breeding, (ii) molecular breeding and (iii) genetic engineering. Among them, the most feasible methods to develop the rice varieties with salinity tolerance is widely applied via molecular breeding. Salinity tolerance in rice is a complex traits, controlled by the typical at least 5 to 8 significant genes/QTLs which related in both the whole plant level and cellular level, involving interactions of stress with molecular, biochemicals and physiological processes at different stages of plant growth and development [8][9] .In order to initially launch the breeding program, it requires new and accurate genetic sources of this tolerance and more efficient techniques to evaluate the salt tolerance germplasm. Advanced powerful molecular tools, included the complete genome sequence of rice was initially generated in 2005 are available [10], which may be facilitated to find the new tolerant genetic resouces. SalT gene is known as one of most reported, co-localizing with the QTL Saltol on the chromosome 1 of rice. SalT protein was first isolated and chracterized from the roots of salttreated rice plant by Claes et al [11]. Some previous studies on induction of the SalT protein in response to salinity were also reported [12][13]. Numberous QTLs/genes, candidate genes involving in salt tolerance in rice have been so far identified such as SalT, RSS1, SCK1, saltol etc [1,[10][11][12][13][14]. However, there is no report on identifying SalT gene in Vietnamese rice landraces. Hence, based on the genome sequence databases of 36 Vietnamese rice landraces, the objectives of this study were to evaluate the similarities (homologous segments of DNA and proteins), then designing marker to accurately identify candidate gene SalT to further utilize for molecular breeding program to develop salt tolerance rice varieties.

Plant Materials
Total 36 Vietnamese rice landraces (Table 1) were kindly provided by Vietnam Plant Resouces Center in 2016, and their genomes were previous re-sequenced [15]. The databases of genome sequence of the rice landraces were used for study.
The method to design the specific primers were conducted as following: Based on the difference of the rice genome sequence, the Primer 3.0 software was used to design the primer sequence (http://primer3plus.com/web_3.0.0/primer3web_input.htm). All the software products are opening sources and available to use as the above links.

Method to test resistance of candidate gene:
Leaf samples of each rice landrace were collected and extracted DNA following the CTAB methods with some modification [16]. PCR reactions were perfomed by Veriti 96-well Thermal cycler. Total volume was 15 µl, included: 5µl DNA; 0.15µM primer; 0.2 mM dNTPs; 1X Buffer PCR; 2.5mM MgCl 2 and 0.25 Taq polymerase.
PCR products were performed on electrophoresis on 6% gel polyacrylamide for further analysis.The gels were stained in 0.5 mg/ml ethidium bromide and were documented using Alpha Imager 1220 (Alpha Innotech, CA, USA).

Identification of homologous sequence to SalT and nucleotide analysis in CDS (Coding DNA Sequence) and amino acid analysis of candidate gen SalT
The databases of genome sequences of 36 rice landraces were utilized to find homologous sequences of SalT gene (salinity tolerance) based on the published GenBank code: Z25811.1 [17]. We have found nine sequence segments which shown equivalent length to the SalT reference gene from genome sequences of nine rice landraces: Te Nuong, Khau mac buoc, Chan thom, Khau giang, Tan ngan, Nang thom cho dao, OM5629, Hom rau and Thom Lai. The nucleotide sequences of nine SalT homologous genes (SalT candidate genes) were analyzed and shown in Further analysis of nucleotide sequences in CDS (coding DNA sequence) and amino acid contents of nine candidate genes were shown in Table 3, and Table 4, respectively. These results showed that all candidate genes had similar length in CDS with 438 nucleotides and resemble polypeptide length with 145 amino acids in comparison with that of reference gene. However, the contents of nucleotide in CDS and contents of amino acids of nine candidate genes were shown difference between two groups of landraces. The first group containing four landraces: Nang thom cho dao, OM5629, Hom rau, and Thom Lai, had candidate genes that were akin to the reference gene with both percentage of nucleotide in CDS and amino acid content. While, the second group which contained the rest of landraces had different contents of nucleotide and amino acid (percentage of four amino acids: His, Leu, Pro, and Gln) when compared to that of SalT reference gene. These results imply that the difference in length of nine candidate genes in comparison with reference gene (Table 2) occur in the intron region, which is not effected to the length of CSD and polypeptide.
In last decades, some studies reported that the molecular response of rice to NaCl stress caused by SalT and has been weakly expressed in untreated plants, but showed high level protein in sheaths after plants are osmotically changed [11,14]. Similarly, Benitez et al. [18] reported that SalT gene apparently is not directly involved in salinity tolerance by inducing at a higher level in the sensitive varieties. Other report of Teraoka et al. [19] stated that novel mannose-binding (MRL) rice lectin composed of some insolectins and relation to a stress-inducible SalT gene, specifically, the cDNA and the genomic clones encoding the MRL 4.85 were highly homologous to the SalT gene induced by salt and drought stresses in rice plant in both nucleotide sequences of DNA and the deduced amino acid sequence [20].

Designing primers to identify SalT candidate genes
By software analyses, the results of prediction and comparing gene sequences of SalT candidate gene from 36 rice genome sequences showed that the frequency of single nucleotide polymorphisms (SNPs) and segment insertion, deletion (InDels) between landraces were rather high. To identify the distinctive features in nucleotide sequences of candidate genes, these nucleotide sequences were aligned and compared with sequence of the reference gene. The result in Fig. 1 showed clearly the difference between two groups of candidate genes. The first group International Letters of Natural Sciences Vol. 63 containing four candidate genes from landraces: Nang thom cho dao, OM5629, Hom rau, and Thom Lai had the similar nucleotide sequence to the reference gene which showed two nucleotide segments due to deletion by six nucleotides and seven nucleotides. While, the candidate genes from five other rice landraces in the second group showed insertion at these two segments, and also had different nucleotide sequence at several positions when compare with the reference gene (Fig.1).   Fig. 1. Nucleotide sequence alignment of SalT reference gene and candidate genes from nine rice landraces, (A: 6 nucleotide deletion segment; B: 7 nucleotide deletion segment).
Based on the difference in nucleotide sequence between candidate genes and SalT reference gene, two primers namely SalT1_add7 with the sequence: SalT1 add7F: AAACTTGACGATTCCAAAAT/SalT1add7R: GTAAATACAAAAGCACATCTT and SalT1_add6 with the sequence: SalT1 add6F: TACGTGTTGCACCGATCCGT/SalT1 add6R: TGCTGAACATGTACAAATGAcc were designed by using Primer 3.0 software. It is able to amplify the candidate gene with the calculated size in the rice landraces carrying the candidate genes involved in salt tolerance, which demontrated identical to the reference gene and different sequence to compare with the published sequence segments ( Fig. 2A). Similarly, by analysis the PCR product of reactions with primer pair SalT1_add6, the band size 113bp was found in 20 rice landraces including: Tam xoan Bac Ninh, Tam xoan Hai Hau, Nang thom cho Dao, Thom Lai, Nep man, Chiem do, Mot bui do, Chiem nho Bac Ninh 2, Nep lun, OM6377, Xuong ga, OM5629, Tep Thai Binh, Toc lun, Hom rau, Nep ong tao, OM3536, Lua goc do, Chiem da and IS1.2 landraces. While, the band size 116bp was found in the rest of landraces (Fig.2B). Therefore, these primers should be used to identify the SalT candidate genes in the parental materials, progenies to develop salinity tolerance rice lines via breeding program. Recently, the development of salt responsive candidate gene based on the genome sequence databases of plants has been paid much attention. The previous report demonstrated that SalT gene is possible to be regulated by ABA-dependant and ABA-independent pathways [14]. In another study reported the evidence of correlation of SalT expression with osmoprotectants, such as trehalose and proline, potential role in pathogen agglutination [21][22]. The SalT gene is highly expressed in the youngest not fully expanded leaves, but as plants get older, the higher expression levels are found in the oldest leaves of mature plant . Hence, the role of SalT protein is not limited to environmental stress responses and that it may be included in a broader response/sensor mechanism to the imposed stress [23].
In general, based on appropriate data mining and candidate genes identification, it can help reduce the laborious nature and high cost of both fine-mapping and cloning a large number of genes. It even becomes possible to efficiently process quite large QTL intervals and feasible for using to accelerate the QTL cloning. In crop plant resources, often untapped, the landraces as resources of genetic variation are potential sources of novel salt stress mechanisms and/or QTLs and genes, with their close genetic identity to current crops following candidate genes to be introduced into commercial lines by conventional breeding approaches [24][25]. Molla et al. [26] validated the salt responsive cgSSRs and analyzed their possible to distinguish salt tolerant and susceptible rice genotype. They reported that the repeat length variations in the designed cgSSR loci may play a role in the manifestation of differential behavior of rice genotypes to salt stress. Further functional analysis of the candidate genes within the target region should be then required to determine which gene or genes are responsible for the phenotype. The known candidate genes for salinity tolerance should be introgressed into elite lines and tested in field conditions.

Conclusions
In this study, we have identified nine Vietnamese rice landraces carrying nine SalT candidate gene with the sequence similarity of O.sativa SalT (the published GenBank: Z25811.1) which have shown salinity tolerance included: Te Nuong, Khau mac buoc, Chan thom, Khau giang, Tan ngan, Nang thom cho dao, OM5629, Hom rau and Thom Lai landraces. Amongst them, four rice International Letters of Natural Sciences Vol. 63 landraces including Nang thom cho dao, OM5629, Hom rau and Thom lai revealed two fragments of deletion with six and seven nucleotides and showed to be similar with the reference SalT gene. It has been successfully designed two primer pairs to identify the SalT candidate genes in Vietnamese rice landraces for future breeding program.