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Evaluation of Wheat Cultivars for Salinity Tolerance at Seedling Stage Based on Morphological and Molecular Markers

Muhammad Shahidul Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh

Shemana Mollick
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh

Nihar Ranjan Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh

Shamsun Nahar Begum
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh

Abstract/ Executive Summary:

Abiotic stresses are the major constraints to wheat cultivation in Bangladesh. The existence of genetic diversity for salt tolerance is a prerequisite for developing salt-tolerant wheat varieties. Evaluation of wheat cultivars under salt stress at the seedling stage was carried out in the present study using morphological and molecular markers. Twenty four cultivars were tested at 0, 6, 8, 10, and 12 dS/m salt stress in a hydroponic system. Based on morphological traits eight wheat cultivars namely Sourav, Pavon, Prodip, BARI Gom-25, BARI Gom-28, Gourav, Shatabdi and Aghrani were identified as salt tolerant because they showed a lower mean value of root length, shoot length, fresh weight and dry weight reduction at 12 dS/m of salt stress ultimately indicating a higher tolerance to salinity. According to Nei’s 1973, the highest value of gene diversity (0.9063) was observed in locus Xgwm577, and the lowest gene diversity value (0.8281) was observed in locus Xbarc84 with a mean value 0.4618. The PIC values ranged from moderate 0.4247 to high 0.8989. The highest PIC value was found in Xgwm577 and the lowest value was inXbarc84. Pair-wise comparison value of genetic distance (D) (Nei’s, 1973) between varieties was computed from combined data of 6 markers and ranged from 0.1667 to 1. Molecular marker based grouping indicates the Sub sub-cluster IIA contained ten cultivars; six tolerant cultivars (BARI Gom-25, BARI Gom-28, Prodip, Pavon, Shotabdi, Gourav), two moderately tolerant (Sonalika, BARI Gom-23) and two susceptible cultivars. We, therefore, identified six cultivars as saline tolerant at their seedling stage that clustered together in the same group when analysed by SSR markers linked to salinity. The findings of the present study have the potential for utilization in future wheat breeding for salinity tolerance.

Keywords: Genetic diversity, Salinity tolerance, SSR markers, Triticum aestivum L.

Location: Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur

Start Date: 00-11-2017

End Date: 00-05-2018

Program Area: Crop-Soil-Water Management

Commodity: Wheat, Soil salinity

Objectives:

To expand the cultivation and to sustain the yield of wheat in the coastal belt, the present piece of work was implemented to evaluate some agronomical, physiological, and molecular markers of wheat as screening criteria against salinity condition.

Methodology:

Plant materials: A total of 24 wheat cultivars were used in this experiment. Twenty one cultivars were collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur; two cultivars from Regional Wheat Research Center, Rajshahi and one cultivar was received from Bangladesh Institute of Nuclear Agriculture (BINA). Seed placement for germination: The morphological study was conducted from November 2017 to May 2018 at the glasshouse and the laboratory of Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. The experiment was conducted in a single factor, Completely Randomize Design (CRD), with five replications. The wheat cultivars were tested under four different salinity levels (0, 6, 8, 10 and 12 dSm^-1) in the present research. The hydroponic system was constructed according to the IRRI standard protocol (Gregorio et al., 1997) at the glasshouse to screen out the salinity tolerance seedling under salt stress. The morphological screening was done in glasshouse conditions with day/night temperatures of 20-25ºC and at least 50% RH during the day. The volume of each tray was 12 L. Water-soluble fertilizer Peters (Urea: TSP: MP=20: 20: 20) and ferrous sulphate (FeSO₄.7H₂O) were used as a nutrient solution. The concentration of nutrient solution was 1.0 g peter fertilizer and 200 mg/L ferrous sulphate were mixed carefully per liter of tap water. The pH was maintained at 5.1-5.2. Salt treatment was applied at the 2-3 leaf stage. Salinization of the nutrient solution was done by adding dry NaCl. The electrical conductivity was measured using an EC meter and adjust the solution at 12dS/m, 10dS/m, 8dS/m and 6dS/m (120 mM, 100 mM, 80 mM and 60 mM). The old solution was replaced with the new one in every weak. After 21 days of salinization, three samples of each cultivar were collected from each replication. The following data were recorded from the screening in both normal and salinized conditions following shoot length, root length, total fresh weight and total dry weight. Total dry weight = Dry weight of (Leaves+Shoot+Root). The % reduction of plant traits = [(Traits in normal – Traits in saline)/Traits in normal] × 100. DNA extraction: Fresh leaves from 21-day old seedlings were used for DNA extraction following CTAB mini-prep method (IRRI, 1997). Concentrations of DNA samples were analyzed both qualitatively and quantitatively using a nanodrop spectrophotometer. SSR marker genotyping: Six SSR markers (Huang et al., 2002; Batool et al., 2018; Ren et al., 2012; Somers et al., 2004) were used for genotyping assays. Primer name, sequences and corresponding annealing temperatures are listed (Table 1). The total of 13 μl PCR cocktail composed of 2.0 μl genomic DNA (conc. 50 ng/ml), 1.5 μl 10X PCR buffer (Tris with 15 mM MgCl₂, conc. 10X), 0.75 μl dNTPs (Contains dCTP, dGTP, dTTP and dATP all in the conc. of 10 mM)), 1.0 μl forward primer (10 pM), 1.0 μl reverse primer (10 pM), 0.5 μlTaq DNA polymerase (conc. 5 U/μl) and 8.25 μl sterile deionized water was prepared. Morphological and molecular data analysis: The collected data were managed in MS Excel® and analysed by MINITAB 14, a computer-based statistical package. Molecular weights of microsatellite products were estimated with AlphaEaseFC 4 software. The major allele frequency, the number of alleles per locus, genetic diversity and polymorphism information content (PIC) values were determined using POWER MARKER version 3.23 Liu and Muse (2005). The data were export in binary format for analysis with NTSYS-PC version 2.1 using the allele frequency data from POWER MARKER (Rohlf, 2000). The genetic similarity was calculated using 0/1 matrix in SIMQUAL subprogram (Nei and Li, 1979). The resultant similarity matrix was employed to construct a dendrogram based Unweighted Pair Group Method of Arithmetic Means (UPGMA) as implemented in NTSYS-PC (version 2.1) to infer genetic relationships and phylogeny (Rohlf, 2000).

Source of Information: J Bangladesh Agril Univ 18(2): 234–244, 2020

Article Link (Online Journal): https://doi.org/10.5455/JBAU.91898

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present study aimed to assess genetic variation for salinity tolerance in 24 wheat genotypes based on morphological and molecular marker screening. Data were collected by screening the cultivars under different salt stress. Morphological and molecular variations were observed for salt tolerance in wheat cultivars. Combining morphological findings with that of the molecular assessment, six cultivars, BARI Gom-28, Prodip, Pavon, BARI Gom-25, Shotabdi, Gourav may be considered as true salt tolerant cultivars which may contribute in a greater way in the development of salt tolerant genotypes. The findings will be helpful for both plant breeders and farmers of saline belts in general. The tolerant and moderately tolerant genotypes have been identified as resource base population, which could to be utilized suitably for further improvement programme for salt tolerance in Bangladeshi wheat.

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