HKT1;5 gene sequence characterization
Amplification of salinity tolerance gene TaHKT1;5-D was done in diverse wheat lines (Fig. 2). Amplification was achieved in all 16 lines of bread wheat. Subsequently, 12 selected lines were selectively sequenced (based on preference with salinity tolerance, sensitiveness, and/or with other interesting features such as drought/heat tolerance, etc) comprising four alleles from bread wheat. The remaining 17 sequenced accessions were wheat wild relatives and ditelosomic lines. Eight of the sequences are ~ 3 kb size from bread wheat lines, with complete CDS-ORFs. The rest of the 21 sequences are partial CDS and/or 5’ UTR of ~ 1kb size from wheat or wild relatives. Gene sequence of bread wheat varieties, wild/progenitor species, durum wheat, and ditelosomic lines was submitted at GenBank (Table 2).
Table 2
NCBI GenBank accession numbers with allele description of TaHKT1;5 gene from diverse collection of T. aestivum cultivars and wild relatives.
Wheat/wild relatives | Allele | GenBank | Size (bp) | SNPs/Allele Description |
T. aestivum WK1204 | A | KU184266 | 3073 | g.[830T > C;832T > C;2106;T > C] |
T. aestivum Dharwar Dry | B | KU212871 | 2826 | g.[830T > C;832C > T;2106;T > C] |
T. aestivum Munal#1 | B | KU184267 | 2438 | g.[830T > C;832C > T;2106;T > C] |
T. aestivum Dharwar Dry | C | KU212872 | 2826 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum OasisF86 | C | KU212870 | 3068 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum HDR-77 | C | KU212869 | 3122 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum C306 | C | KU242566 | 1066 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum Kharchia | C | KU212875 | 3074 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum Sakha8 | C | KU242567 | 965 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum YMI#6 | C | KU242568 | 1047 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum Shorowaki | C | KU242569 | 965 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum Lu-26 | C | KU242570 | 1035 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum Kharchia 65 | C | KU212873 | 2883 | g.[830T > C;832T > C;2106;C > T] |
T. aestivum Kharchia 65 | D | KU212874 | 2883 | g.[830C > T;832T > C;2106;C > T] |
A. tauschii CWI94958SH | T1 | KU253614 | 958 | g.[724G > T] |
A. tauschii CWI94956SH | T2 | KU253615 | 1009 | g.[724T > G] |
T.monococcum PI272558 | M1 | KU253607 | 994 | g. [234 A > G; 745C > G; 832C > T] |
T. monococcum PI272558 | M2 | KU253608 | 994 | g. [234 G > A; 745C > G; 832T > C] |
T. monococcum PI428150 | M1 | KU253609 | 966 | g. [234 A > G; 745C > G; 832C > T] |
T. monococcum PI326317 | M1 | KU253610 | 936 | g. [234 A > G; 745C > G; 832C > T] |
T. monococcum PI264935 | M1 | KU253611 | 672 | g. [234 A > G; 745C > G; 832C > T] |
T. monococcum PI272560 | M1 | KU253612 | 978 | g. [234 A > G; 745C > G; 832C > T] |
T. monococcum PI272560 | M3 | KU253613 | 978 | g.[ 234 A > G; 745 G > C; 832T > C] |
T.monococcum PI264935 | N1 | KY110746 | 942 | N/A |
T.monococcum PI272560 | N1 | KY110747 | 893 | N/A |
A.speltoides CWI94201SH | As2 | KY110745 | 632 | N/A |
Ditelosomic DTGH09_1566 | DS2 | KY110743 | 902 | N/A |
Ditelosomic DTGH09_1559 | DS1 | KY110744 | 879 | N/A |
T. durum DWK26 | B-Td1 | KY110748 | 1007 | N/A |
The PCR amplification derived sequencing and allele mining in bread wheat detected three SNPs, among them two SNPs at 830th and 832nd positions located at 5’UTR and the third one located at 2106th position of the first intron with reference to Byrt et al., 2007. The gene is located at chromosome 4DL between 507964629 bp to 507967781 bp (TraesCS4D02G361300) revealed with BLASTN at wheat ENSEMBL. We found four alleles were designated as A, B, C, and D among 12 geographically diverse bread wheat cultivars and allele ‘C’ is prevalent (Table 1). Variant Effect Predictor revealed that 67% upstream variants (Genomic positions: 507965458 bp, 507965460 bp) and 33% intron variants (Genomic position: 507966734 bp) without amino acids change as expected for corresponding SNPs positions at 830, 832 and 2106 on the sequence of TaHKT1;5 gene. However, a missense variant (506th Serine/Glycine) with deleterious mutation (SIFT score: 0.05) was observed in exon 3 as an inter-homoeologous variant (Genomic position: 507967636) from TaHKT1;5 gene between chromosome 4DL to 7BS (ENSEMBL: BA00457515 SNP) comparison.
Identified SNPs reflect the homozygotic nature of alleles in all the varieties with an exception in varieties Dharwar Dry (alleles ‘B’ and ‘C’) and Kharchia 65 (alleles ‘C’ and ‘D’) whereby a heterozygotic nature of the allele was observed suggesting the existence of an alternative variant of the gene TaHKT1;5-D. To check the allelic diversity in wild relatives, we used species-specific primers developed for T. monococcum and T. aestivum 4DL or A. tauschii. These primers successfully amplified TaHKT1;5-D gene in T. monococcum, A. speltoides and A. tauschii. The species-specific primers from T. monococcum amplified a native TmHKT1;5-A gene itself (M allele), also an ortholog in A. tauschii (Amplified, though not sequenced as M allele related A.tauschii (named as putative At_HKT) allele sequence directly retrieved from the public database). Likewise, T. aestivum 4DL or A. tauschii specific primers also amplified a native AtHKT1;5-D gene (Allele ‘T’) and an ortholog from T. monococcum (N allele). These results highlight the existence of two major alleles ‘M’ and ‘N’ in T. monococcum. Interestingly, we could not amplify any orthologs in T. urartu. Based on SNPs within the M allele, three sub-alleles were detected, designated as M1, M2, M3, while major allele N exhibited only a single variant (N1) (Table 1). Also, two major alleles were identified in A. tauschii. The first allele was designated as ‘T’ and consists of two sub-alleles, T1 and T2, and the second allele (of A.tauschii related to M allele of T. monococcum) was derived from a public database IWGSC-URGI (https://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST) and named as putative At_HKT.
The comparison between major alleles (M and N from T. monococcum, T and At_HKT from A. tauschii) exhibited the polymorphism at 5% level, while the comparison within the sub-category of major alleles called minor alleles showed 0 to 0.2% polymorphism. The phylogenetic tree, constructed based on maximum likelihood with 1000 bootstrap, clearly revealed some distinct clusters of major and minor alleles (Fig. 3a). In 5’ UTR region, a continuous stretch of adenine was observed at the higher number in A. tauschii gene (At_HKT allele) from 554 bp onwards, whereas in T. aestivum and T. monococcum the repetition of adenine was lower. One variant from A. tauschii (At_HKT) is a major source of the 4DL chromosome bearing HKT 1;5 gene to T. aestivum as it shows a close phylogenetic relationship with each other (Fig. 3b). The distance matrix of these alleles from corresponding species is shown in Supplementary Table 2. The alignment around exon1 between wheat and A. tauschii indicates the vicinity of evolutionary relatedness between the At_HKT allele and alleles of wheat, while the T allele (T1, T2) lies farther (Fig. 3c). Relative divergence time concerning HKT1;5 gene is recent for Triticeae members in Pooideae subfamily in Poaceae than model grass Brachypodiuum distachyon and other species belonging to the subfamily of Oryzoideae and Panicoideae as shown in Fig. 3d.
Concerning A. speltoides, amplification was achieved in some accessions accession, perhaps due to accumulated mutations in and around primer binding sites. The HKT1;5 gene partial sequence generated from A. speltoides (Allele ‘As2’) revealed an alteration of amino acids in the primary structure of protein due to the insertion of four nucleotides (CGCG) caused a frameshift mutation at exon1 suggesting a distinct allelic variant (Genbank: KY110745, Fig. 4a). Besides, a BLASTN search for additional alleles with top score sequences from IWGSC-URGI (https://urgi.versailles.inra.fr/Species/Wheat), revealed the existence of four more alleles in A. speltoides with available genomic resource. Hence, there is a need for more sequencing effort in natural accessions/populations of this species to excavate further alleles. None of the database sequences showed a frameshift mutation with CGCG insertion at exon1 as ‘As2’ allele of A. speltoides. The BLASTN of the sequences (https://urgi.versailles.inra.fr) revealed that the complete gene sequence was present only on homeolog chromosome 4D. To better understand the number of gene variants on chromosome 4D, we used two ditelosomic lines, the first one called DTGH09_1559 possessed 4D chromosome long arm (L) but lacking 4D chromosome short arm (S) (Allele ‘DS1’) and the second one namely DTGH09_1556 (Allele ‘DS2’) was the opposite of DTGH09_1559 i.e. possessing 4D chromosome short arm (S) but lacking 4D chromosome long arm (L). The alignment of sequenced homeolog from DTGH09_1559 with 4DL (-4DS) (GenBank: KY110744) revealed a 99.89% identity with T. aestivum (GenBank: KU184266), while it was 93.25% identical to T. durum (GenBank: KY110748). In contrast, the homeolog from DTGH09_1556 4DS (-4DL) (GenBank: KY110743) revealed a 98.89% identity to T. durum while it was 93.08% identical to T. aestivum. Both ditelosomic lines DTGH09_1559 and DTGH09_1556 have 93.78% identity to each other. Protein sequence alignment among bread wheat, durum wheat, and two ditelosomic lines corresponding to exon1 is shown in Fig. 4b and the nucleotide alignment with insertion and deletion is shown in Fig. 4c. This indicated that the homeolog from DTGH09_1556 4DS (-4DL) has similarities with an ortholog of durum wheat and might have amplified from the ‘A’ or ‘B’ genome rather than from the D genome of ditelosomic line DTGH09_1556 4DS (-4DL). BLASTN has further confirmed that DTGH09_1556 4DS (-4DL) gene sequence was located on chromosome 4B long arm (4BL) as it showed 100% sequence identity with the scaffold TGACv1_scaffold_320406_4BL from bread wheat. The allele DTGH09_1556 (Allele ‘DS2’) resembles an existing HKT8-B2 homeolog (DQ646341) in wheat, though there are 9 SNPs spanning the gene region of 1kb between these two forms, thus it is a novel variant of B2 homeolog. Further, our phylogenetic analysis suggests that there could be 5 or more paralogs within 4B chromosome homeolog, distinct orthologs in putative B genome donor A. speltoides and related to B-Td1 of T. durum. However, the allele ‘DS1’ from DTGH09_1559 is completely identical to HKT1;5-D at 4DL, thus it is completely belonging to the A, B, C and D alleles.
Overall, the coding sequences of TaHKT1;5-D against the survey sequences of all bread wheat revealed the presence of an entire genic region on 4DL, while remnants or portions of gene regions on 4BL, 4BS and, 4DS chromosomes. A non-autonomous mariner transposon element was found in intron 1 of the gene on all A, B, and D chromosomes with > 3120 BLASTN hits. These results indicate the mobile nature of the gene through transposition events either as a whole or part of the gene, hence multiple variants of HKT1;5 gene in bread wheat and its progenitors are highly plausible. The presence of huge variation in sequence alignment between T. durum cultivar DWK26 and T. aestivum indicated variation between the amplified gene from 4BL and 4DL respectively. The amino acid alignment evidencing that the HKT1;5 gene sequence of durum wheat was highly comparable with diltelosomic line 1556 (+ 4DS, -4DL), while T. aestivum was highly comparable with DTGH09_1559 (+ 4DS, -4DL). In A. tauschii the presence of two variants (At_HKT and T allele) revealed that these sequences sharing proximity among ditelosomic line 1559 (+ 4DL and − 4DS), bread wheat, and A. tauschii, while Ditelosomic line 1556 (+ 4DS and − 4DL) was differing with its own SNPs from others. The trend suggested that two paralogous variants existed in A. tauschii and orthologous to wheat. Besides, ditelosomic bread wheat lines showed the existence of 2 paralogous/homoeologous variants, which revealed that one was amplified from DT line 1559 (+ 4DL-4DS) while another from 1556 (+ 4DS, -4DL) of 4BL. Both the alleles from A. tauschii (At_HKT and T allele) had proximity with the sequence of DT line 1559 rather than DT1556.
Genetic diversity analysis of the HKT1;5 alleles in bread wheat
There were 2301 invariant sites, and 3 variant sites detected. Among the variant sites, 2 were singleton nucleotide variant sites (at position 830 in 5’ UTR and at 2106 in intron1) and 1 parsimony-informative site (at position 832 of 5’ UTR) with two variants. The variant sites at 5’ UTR are shown in Fig. 4d. Diversity analysis showed low polymorphism and recombination rate for this gene among the bread wheat lines concerning these four alleles in TaHKT1;5-D (Table 3). Gene flow and genetic differentiation showed a similar trend with least effective migrants (0.13) FST (0.65) among the four alleles of the defined population/genotypes. It further elucidates the least frequency of recombination in this gene owing to autogamous nature and lesser allelic variation within bread wheat lines. The conservative nature of the sequences with a lower number of mutations had resulted in low haplotype diversity of the four alleles. The observed total nucleotide difference was also significantly lower (Table 4).
Table 3
Polymorphic sites and DNA polymorphism. h-Number of haplotypes, hd-Haplotype diversity, SD-Standard deviation, θ- 4Nµ, where N is effective population size and µ is mutation rate per site per generation, π- Nucleotide diversity, η –Total number of mutations, S-Segregating sites, R-Recombination, NR- No Recombination, k-Average number of nucleotide differences, Sq-Sequence.
H | Hd | Variance | SD | π | θ from η |
4 | 0.694 | 0.02162 | 0.147 | 0.00036 | 0.00048 |
θ from S | Variance θ (NR) | SD θ (NR) | Variance θ (R) | SD θ (R) | θ from π |
0.00048 | 0.0000001 | 0.00032 | 0.0000001 | 0.00028 | 0.00036 |
θ from S | θ from η | k | θ from S/Sq | Variance θ (NR)/Sq | Variance θ (R)/Sq |
0.00048 | 0.00048 | 0.833 | 1.104 | 0.545 | 0.406 |
Table 4
Genetic diversity, differentiation and gene flow analysis from A,B,C,D alleles of TaHKT1;5-D gene. Hd-Haplotype Diversity, Kt- Average Nucleotide Differences, PiT- Nucleotide diversity, χ2- Chi Square test, Hst- Haplotype based statistics, Kst- Sequence based statistics, Z*- Rank Statistics, Snn- Near Neighbour Statistics, Gst- Differentiation of population, GammaSt- Gamma Statistics, Nst- N statistics, Fst- Fixation index, Nm- Effective number of migrants, PM- Permutation test with 1000 replicates, * 0.05 < P.
| Genetic Diversity |
Alleles | Sequences/sites | Segregating sites | Haplotypes | Hd | Kt | PiT |
A, B, C, D | 9/2304 | 3 | 4 | 0.6944 | 0.8333 | 0.00036 |
| Genetic differentiation |
Estimated | χ2 | Hst | Kst | Kst* | Z* | Snn |
Value | 9.77 | 0.15 | 0.27 | 0.12 | 3.84 | 0.43 |
PM test | 0.13 | 0.10 | 0.027* | 0.16 | 0.11 | 0.18 |
| Gene flow |
| Haplotype data | Sequence Data |
Estimated | Gst | DeltaSt | GammaSt | Nst | Fst |
Value | 0.319 | 0.00008 | 0.31624 | 0.65908 | 0.65909 |
Nm | 0.53 | 0.54 | 0.54 | 0.13 | 0.13 |
The percentage of nucleotides was 22.96% (A), 27.73% (T/U), 26.83% (C), and 22.48% (G) derived from the alleles corresponding to 2304 positions. The C to T transition mutation (pyrimidines) substitution pattern was higher (50.59%) than other types of transition mutations. Besides, the transversion mutations (purine to pyrimidine or vice versa) were lower between the ranges of 0.04 to 0.05%. Among the bread wheat cultivars, SNPs were not observed in the coding region of the gene, hence Ks/Ka (synonymous/non-synonymous mutation) analysis was not performed. Tajima D test (D: -0.93613) with negative value indicates purifying selection due to low frequency of variations along this gene among the bread wheat cultivars, which previse population size expansion. However, the Tajima D test was not significant, and thus increasing the sample size may provide a better clue.
Phylogenetic analysis of the HKT 1;5 gene
The phylogenetic tree based on all generated gene sequences and additional Poaceae member sequences (especially tribe Triticeae) from public databases revealed the presence of five orthologous groups with each group, consisting of closely related species or from closely related chromosomes in the case of polyploid species (Fig. 5). The ditelosomomic line DTGH09_1559 clustered together with the T. aestivum and A. tauschii cultivars. The alleles from T. monococcum and A. speltoides deviated from the orthologous group I and were designated as an orthologous group II. Alleles from ortholog group III existed with III sub-groups of bread wheat and durum wheat samples and have proceeded from A, B, and D putative genome donors from ortholog group II. The four alleles of A. speltoides retrieved database comes under three ortholog groups with other related species. The orthologous group IV consisted of alleles from A. sharonensis, A. speltoides, Hordeum spp, and B. distachyon. This group appeared as a distinct group from the ortholog groups I-III. Group V consists of other Poaceae members used in this study apart from Triticeae and Brachypodieae tribes.
Comparative analysis between T. aestivum and A. thaliana
Pairwise alignment between T. aestivum HKT1;5-D and A. thaliana HKT1 gene and its protein revealed the similarity at the level of 54.9% and 48.4% respectively exonic region and corresponding protein sequence of these orthologs. Since it was intriguing, the protein modeling and comparative study were done, and it indicates the structural conservation of protein sequence between A. thaliana and T. aestivum (Fig. 6a).
Nucleotide level global alignment with LAGAN revealed the presence of higher conservation in coding or exonic part of HKT1 sequences between wheat and A. thaliana. The comparison of transposable elements inside the gene showed that A. thaliana HKT1 gene has a higher number of transposon insertions or remnants of transposons inside the gene compared to bread wheat HKT1;5 gene. The conservation of the CDS region, donor, and acceptor splicing sites proved the similarity of the structural organization of this gene between A. thaliana and T. aestivum. Introns are enriched with the presence of more remnants of different transposons; however, a complete non-autonomous transposon called Mariner at intron1 in bread wheat was comparable between these two-diverse species (Fig. 6b). Physico-chemical properties of the protein comparison between A. thaliana and T. aestivum revealed that higher similarity of this orthologous gene between these two-diverse species (Supplementary Table 3). The structural organization of the corresponding putative protein sequences is also in a similar trend. The instability index (II) indicates that HKT1 protein from T. aestivum and A. thaliana are stable.
Salinity stress-induced phenotypic evaluation in selected allele designated wheat lines
To understand the effect of salinity during germination at the seedling stage, the following experimental test was done using drought/heat tolerant lines (C306, HDR-77, so far unknown for salinity tolerance or sensitiveness), WK1204 (a yellow rust-resistant line, so far unknown for salinity tolerance or sensitiveness) and a salinity sensitive line OasisF86. This test was done to understand the level of salinity tolerance in unknown varieties and these lines were further validated with subsequent experimental tests with known salinity tolerant lines such as Kharchia, Kharchia 65, etc.
A detailed description of the trait variability with LSD in the HDR-77 (Allele ‘C’), C306 (Allele ‘C’), Oasis F86 (Allele ‘C’), and WK1204 (Allele ‘A’) T. aestivum cultivars is given in Table 5. The germination test among these four cultivars showed OasisF86 (‘C’ allele) and WK1204 (‘A’ allele) to be early germinating than the other two cultivars with the least germination Mean Time (MT). Final Germination Percentage (FGP) was maximum in C306 and HDR-77, even at 200mM salt stress, suggesting that the seedlings of these two varieties are more tolerant to salt stress than others. Fresh Weight (FW) augmented upon increasing the salinity concentration, maximum at 50–100 mM level than control in all varieties, similarly FW was increased with increasing salt concentration, reached a maximum at 200mM for all, especially for HDR-77. (Fig. 7).
Table 5
Mean values and variability of traits under different treatments of salinity stress including control and mean values and variability of each genotype with underlying phenotypic features responding to treatment. Letters not sharing similarities are statistically significant to LSD in each row at P = 0.05 level. FGP = Final Germination Percentage, GE = Germination Energy, SVI = Seed Vigor Index, CL = Coleoptile Length, RL = Radicle Length, NoR = Number of radicles, ColL- Leaf Length from Coleoptile, FW = Fresh Weight, DW = Dry Weight, RWC = Relative Water Content, MT = Mean Germination Time, CVt = Coefficient Variation of Germination Time, MR = Mean Germination Rate, U = Uncertainty of the Germination Process, Z = Synchrony. Statistical significance: *P < 0.05, **p < 0.01, ***P < 0.001.
| Treatment | |
Traits | Control | 50 mM | 100 mM | 150 mM | 200 mM | LSD at P = 0.05 | F test significance |
FGP | 96.11a | 88.88ab | 88.33ab | 86.66b | 83.88b | 7.8 | 0.03957 * |
GE | 13.33a | 11.11a | 10.55a | 8.88a | 5.55a | 8.68 | 0.47182 |
SVI | 998.66a | 891.99a | 635.48b | 396.34c | 255.15d | 116.95 | < 2e-16 *** |
CL | 44.68a | 43.72ab | 38.61bc | 37.68c | 36.95c | 5.72 | 0.0205 * |
RL | 103.60a | 100.14a | 72.20b | 44.45c | 29.67d | 12.36 | < 2e-16 *** |
NoR | 3.98a | 3.98a | 3.79ab | 3.75ab | 3.59b | 0.26 | 9.31e-07 *** |
ColL | 104.49a | 99.40a | 82.81b | 70.86c | 42.47d | 8.94 | 4.39e-16 *** |
FW | 1.47a | 1.42a | 1.34a | 1.33a | 1.08b | 0.15 | 8.83e-05 *** |
DW | 0.50a | 0.49a | 0.49a | 0.43b | 0.42b | 0.04 | 0.000918 *** |
RWC | 68.93a | 66.27ab | 63.00bc | 61.91bc | 59.10c | 5.38 | 0.00613 ** |
MT | 3.24a | 3.02a | 2.68b | 2.67b | 2.65b | 0.25 | 2.51e-05 *** |
CVt | 7.71a | 7.11a | 7.11a | 6.41a | 6.39a | 1.66 | 0.4682 |
MR | 0.38a | 0.38a | 0.38a | 0.34b | 0.31b | 0.03 | 0.00052 *** |
U | 1.70a | 1.62a | 1.58a | 1.53a | 1.52a | 0.32 | 0.824 |
Z | 29.97a | 25.50ab | 22.77ab | 19.20b | 15.94b | 10.31 | 10.0772 |
| | | Genotypes | | | | |
Traits | C306 | HDR-77 | WK1204 | Oasis F86 | LSD at P = 0.05 | F test Significance |
FGP | 96.88a | 90.22ab | 84b | 84b | 6.98 | 0.00108 ** | |
GE | 18.22a | 9.77b | 8.00b | 3.55b | 7.76 | 0.00393 ** | |
SVI | 820.48a | 717.02a | 520.01b | 484.58b | 104.6 | 2.47e-08 *** | |
CL | 49.14a | 44.55a | 34.06b | 33.55b | 5.11 | 3.15e-08 *** | |
RL | 84.52a | 79.01a | 60.44b | 56.08b | 11.05 | 2.75e-06 *** | |
NoR | 4.23a | 3.89b | 3.67bc | 3.50c | 0.23 | 9.31e-07 *** | |
ColL | 94.15a | 90.73a | 77.30b | 65.85c | 7.99 | 4.99e-09 *** | |
FW | 1.44a | 1.41a | 1.36a | 1.10b | 0.14 | 2.49e-05 *** | |
DW | 0.55a | 0.50b | 0.48b | 0.34c | 0.04 | 1.73e-13 *** | |
RWC | 68.24a | 65.47ab | 62.08bc | 59.59c | 4.81 | 0.00416 ** | |
MT | 3.57a | 2.78b | 2.64bc | 2.43c | 0.22 | 6.04e-13 *** | |
CVt | 10.12a | 7.52b | 5.08c | 5.07c | 1.49 | 5.95e-09 *** | |
MR | 0.41a | 0.38ab | 0.36b | 0.28c | 0.03 | 4.21e-10 *** | |
U | 1.93a | 1.75a | 1.38b | 1.29b | 0.29 | 8.45e-05 *** | |
Z | 36.33a | 30.44a | 16.24b | 7.70b | 9.22 | 1.76e-07 *** | |
At 200 mM salinity stress, all the varieties exhibited higher dry (DW) weight at 200 mM than the control; notably, HDR-77 exhibited the highest DW than the rest of the varieties. Relative Water Content (RWC) slightly decreased by increasing salt concentration; and, at 200 mM salinity stress. RWC was higher for C306 than the rest of the varieties. RWC was comparatively higher at the control and lower at 200mM for OasisF86, it suggests that the salinity-sensitive variety Oasis F86 was able to absorb and retain the water more efficiently than the rest of the cultivar in the absence of salinity stress. Overall RWC was higher in the tolerant lines (C306 and HDR-77). Radicle Length (RL) varied or decreased corresponding to increased salinity concentration, proving the inversely proportional relationship between these two factors. A similar trend was observed with the Number of Radicles (NoR). In absence of salinity stress (control) RL was longer in C306 and HDR-77 than in WK1206 and OasisF86; while in contrast to control, the NoR were less in C306 and HDR-77 than in WK1206 and OasisF86. It suggests that at control, the salt-tolerant lines were able to produce longer radicles (RL) than sensitive lines, while sensitive lines produce more radicles (NoR) than salt-tolerant lines. This finding suggests that the sensitive line WK104 with allele ‘A’ is highly comparable for its sensitivity with the specific feature of producing more radicles (NoR) under salinity stress than lines with Allele ‘C’. (Fig. 7).
Mean Germination Time (MT) and Mean Germination Rate (MR) for both sensitive cultivars (OasisF86 and WK1206) were shorter than the other two varieties. It suggests that sensitive lines tend to germinate faster or earlier and are more susceptible to salt stress than HDR-77 and C306. This analysis revealed that the cultivar C306 is most tolerant to salinity, C306 is the second most tolerant cultivar. The allele ‘C’, was common to both salinity tolerant and susceptible cultivars such as Kharchia and OasisF86, while only one genotype WK1204 had allele ‘A’, which is saline sensitive cultivar as revealed with our analysis. Dharwar Dry is a salinity-sensitive cultivar that possessed both ‘B’ and ‘C’ alleles. Similarly, the salinity tolerant cultivar Kharchia 65 possessed both ‘C’ and ‘D’ alleles. Intriguingly, cultivars showed sensitiveness had an allele ‘A’ and some of them with allele ‘C’, while the combination of alleles ‘B’ (g.[830T > C;832C > T;2106;T > C]) or ‘D’ (g.[830C > T;832T > C;2106;C > T]) present in T. aestivum cultivar Kharchia 65), which is a known saline tolerant cultivar. (Fig. 7). Further similar experimental tests derived result among the wheat varieties Kharchia, Kharchia65, OasisF86, DharwarDry, and DWK26 is shown in Supplementary Table 4, Supplementary Table 5, Supplementary Fig. 1.
Greenhouse-based phenotypic analysis was carried out for the bread wheat cultivars and durum wheat and results were reported. Statistical results concerning wheat cultivars salinity stress are listed in Table 5. Mostly saline sensitive bread wheat cultivars OasisF86 (Allele ‘C’), WK1204 (Allele ‘A’), and durum wheat cultivar DWK26 were observed with earlier booting or heading stage in a short period (50 DAS) of the time since the salinity exposure. However, salinity stress didn’t induce early (50 DAS) booting and heading in salinity tolerant variety bread wheat variety Kharchia (Allele ‘C’), drought tolerant and heat tolerant varieties HDR-77 (Allele ‘C’) and C306 (Allele ‘C’) (Supplementary Table 6). The greenhouse-based experiment also revealed that the saline-sensitive cultivars tend to have an early maturation and short life cycle revealed through the observation and measurement of booting and heading at 50 DAS (Days After Sowing). In addition, in accordance with salt-tolerant reference line Kharchia, the drought and heat tolerant cultivars exhibited better salinity tolerance. Even the level of salinity tolerance was slightly higher for the lines C306 and HDR-77 than Kharchia (Table 6).
Table 6
Mean values and variability of traits under different treatments of salinity stress including control and mean values and variability of each genotype with underlying phenotypic features responding to treatment. Letters not sharing similarities are statistically significant to LSD in each row at P = 0.05 level. PH = Plant Height, NoT = Number of Tillers, NoET = Number of Effective Tillers, FLL = Flag Leaf Length, TS = Total Number of Spikes, SL = Spike Length. Statistical significance: .p < 1, *P < 0.05, **p < 0.01, ***P < 0.001, Ns- Not significant.
| Treatment | |
Traits | Control | 50 mM | 100 mM | 150 mM | 200 mM | LSD at P = 0.05 | F test significance |
PH | 59.44a | 59.27a | 57.72a | 56.16a | 47.88b | 7.08 | 3.20e-05 *** |
NoT | 5.05a | 4.72a | 4.66a | 4.50a | 4.38a | 1.15 | 0.52 Ns |
NoET | 4.11a | 3.38ab | 3.38ab | 3.00b | 2.72b | 0.86 | 0.000274 *** |
FLL | 55.19a | 50.86ab | 43.72ab | 42.58b | 41.94b | 12.57 | 0 0.00907 ** |
TS | 4.11a | 3.38ab | 3.38ab | 3.00b | 2.72b | 0.86 | 0.000274 *** |
SL | 25.66a | 23.22ab | 21.08ab | 19.16bc | 15.86c | 4.88 | 1.58e-06 *** |
Genotypes |
Traits | C306 | HDR-77 | Kharchia | OasisF86 | WK1204 | DWK26 | LSD at P = 0.05 | F test Significance |
PH | 62.76a | 61.56a | 60.93a | 51.70b | 50.80b | 48.83b | 8.13 | 5.11e-08 *** |
NoT | 6.60 a | 5.00 b | 4.86b | 4.53bc | 3.73bc | 3.26c | 1.33 | 9.8e-10 *** |
NoET | 3.80a | 3.80a | 3.40ab | 3.33ab | 2.86ab | 2.73b | 0.99 | 0.004342 ** |
FLL | 63.80a | 62.90a | 59.90a | 35.06b | 33.21b | 26.30b | 14.44 | 3.08e-15 *** |
TS | 3.80a | 3.80a | 3.40ab | 3.33ab | 2.86ab | 2.73b | 0.99 | 0.004342 ** |
SL | 25.43a | 22.63ab | 22.53ab | 21.36ab | 19.66bc | 14.36c | 5.6 | 2.74e-06 *** |
Statistical results from the greenhouse experiment revealed that bread wheat variety C306 (Allele ‘C’) is more saline tolerant (PH: 62.76a, LSD at P = 0.05, F test significance 5.11e-08, P < 0.001) (Table 5) than other bread wheat varieties. Oasis F86 (Allele ‘C’) is saline sensitive cultivar comparable with WK1204 (Allele ‘A’) and durum for salt sensitiveness (Fig. 8). The overall experiment revealed the salinity sensitiveness or tolerance among the varieties through the possible comparison of phenotypes with alleles.
Our greenhouse experiment demonstrates that the varieties C306 and HDR-77 even showed slightly better salt tolerance over known salinity tolerant reference line Kharchia. However, wheat varieties like Munal#1 evinced slightly better tolerance with germination-related salinity stress than varieties WK1204, OasisF86 and Dharwar Dry. Germination-related studies showed that the variety WK1204 is slightly better salinity tolerant than the variety OasisF86, while the greenhouse experiment is contrary, as OasisF86 exhibits slightly better phenotypic features with salinity stress than the variety WK1204. Perhaps, a known saline-sensitive variety OasisF86 is very sensitive during germination with salinity stress. Nonetheless, the varieties C306 and HDR-77 had better tolerance in greenhouse study and germination-related salt stress study (Table 5, Table 6, Supplementary Table 7).