Transcriptome assembly and sequencing
Transcriptome analysis of the twenty-four Salt tolerant (J194) and sensitive (J7) samples using Illumina paired-ends sequencing technology was used to explore the DEGs related to NaCl stress in jute (utilizing two biological replicates in each case; tissue (leaf and roots) and time (0, 6 and 12 hours) (Additional Table S3). The comparison of J194 and J7 due to salt exposure is also presented in Figure (1a-b). The 1518813542 raw reads were generated, 759406771 clean reads were obtained, and the sequence was mapped to the jute genome (Additional Table S3). The GC contents range between 44.24–45.59%, and the Q30 scores are greater than 90% (Additional Table S3 and S4). The clean reads were used to assemble the 27720 transcripts (Additional Table S3) to be used as a reference sequence for downstream analysis and 26463 unigenes (Additional Table S4) 7272 unigenes. Comparing the clean reads with the reference genome when assigned into exon, intron, and intergenic regions indicated that the exon-region sequence accounts for more than 80% of the genome-mapped sequences as expected (Additional Figure S1). These results present a higher degree of annotation accuracy. Additionally, the box plot comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPK) indicates that sequence results were reliable; the sample yielded equivalent reads and coverage depths between duplicates (Fig. 1b).
To comprehend the DEGs annotated unigenes functions, 26463 unigenes were annotated in all eight databases. It was observed that most of the unigenes (26413) were aligned with that of the NR database, followed by eggNOG_Annotation (22539) and Pfam_Annotation (19493). However, annotation in the KEGG database (8992 unigenes) presented the least number of unigenes (Fig. 1c). DEGs annotations across the treated samples (Additional Table S5) indicated that (J7R_0H Vs. J7R_6H) had the highest number of annotated unigenes across all the databases, followed by (J7L_0H Vs. J7L_6H), whereas (J194L_0H Vs. J194L_6H) had the least number across all the databases (Additional Table S5). It was observed that the most common enrichment analysis was related to plant hormone signal transduction pathways (Additional Table S6) as vital for jute response to NaCl exposure.
Identification of differentially expressed genes in response to salt treatment
Analysis of DEGs in J7 and J194 on different tissues (roots and leaves) under control and salt stress conditions. Indicated that 26463 unigenes were differentially expressed (Additional Table S7). Cluster analysis revealed that differential gene expression profiles were similar within the same tissue types and time exposure rather than between different genotypes and time duration (Fig. 2a). The dispersion of these differentially expressed genes is presented in (Additional Figure S2).
Additional Figure S2 (i) indicated the same germplasm combination for up-regulated and down-regulated DEGs in J7 and J194 leaf tissues at all durations of NaCl exposure. Up-regulated DEGs (Figure c) indicated no DEGs were recorded across all the germplasms in all durations. However, J7_0H Vs. J7_6H (1075) and J194_0H Vs. J194_6H (6) recorded the highest and lowest DEGs, respectively. Moreover, (J7_0H Vs. J7_6H) Vs. (J7_0H Vs. J7_12H) (1052) had a high combination, but none were observed (J194-0H Vs. J194-6H) Vs. J7-0H Vs. J7_12H) (0). However, 16 and 2 DEGs were recorded in (((J7_0H Vs J7_6H) Vs (J7_0H Vs J7_12H) Vs (J194_0H Vs J194_12H))) and (((J7_0H Vs J7_6H) Vs (J194_0H Vs J194_12H) Vs (J194_0H Vs J194_6H))) combinations, respectively. Figure (a) indicated the down-regulated DEGs for the same germplasm combination across all durations of exposure to NaCl indicated that 11 DEGs were recorded across all the germplasms and the time of the exposure to salt treatment. J7_0H Vs. J7_6H (840) and J194_0H Vs. J194_6H (6) had the highest and lowest number of DEGs. Still, (J7_0H Vs. J7_6H) Vs. (J7_0H Vs. J7_12H) (1093) recorded the highest DEGs, whereas none was observed in (J7_0H Vs. J7_12H) Vs. (J194_0H Vs. J194_6H) (0). 43 and 4 DEGs were recorded in (((J7_0H Vs J7_6H) Vs (J7_0H Vs J7_12H) Vs (J194_0H Vs J194_12H))) and, (((J7_0H Vs J7_6H) Vs (J7_0H Vs J7_12H) Vs (J194_0H Vs J194_6H))) combinations, respectively. Moreover, figure b and d indicated the DEGs under different germplasms combined for down and up-regulated leaf tissue, indicating that about 33 and 21 DEGs were recorded in (J194_6H Vs. J7_6H) Vs. (J194_12H Vs. J7_12H) and (J194_6H Vs. J7_6H) Vs. (J194_12H Vs. J7_12H) respectively.
Additional Figure S2 (ii) indicated different germplasm combination for up and down-regulated DEGs in J7 and J194 root tissues at all durations of exposure to NaCl. For Up-regulated DEGs (Figure c), indicated that 67 DEGs were recorded in all the germplasms across the duration of the exposure to NaCl. Still, J7_0H Vs. J7_6H (1380) and J194_0H Vs. J194_6H (50) recorded the highest and lowest number of DEGs, respectively. Interestingly, (J7_0H Vs. J7_6H) Vs. (J7_0H Vs. J7_12H) Vs. (J194_0H Vs. J194_6H) (168) and (J194_0H Vs. J194_12H) Vs. (J7_0H Vs. J7_12H) Vs. (J194_0H Vs. J194_6H) (4) possess the highest and lowest number of DEGs. Hitherto, 291 and 9 DEGs were recorded for (J7_0H Vs. J7_6H) Vs. (J7_0H Vs. J7_12H) and (J194_0H Vs. J194_6H) Vs. (J194_0H Vs. J194_12H) combinations, respectively. J7 and J194 down-regulated DEGs roots are presented in Figure (a), in which 258 DEGs were recorded across all germplasms at a different time of NaCl exposure J7_0H Vs. J7_6H(1044) and J194_0H Vs. J194_12H (38) had the highest and lowest numbers. Additionally, (J7_0H Vs. J7_6H) Vs. (J7_0H Vs. J7_12H) (550) and (J7_0H Vs. J7_12H) Vs. (J194_0H Vs. J194_6H) (6) recorded the highest and lowest DEGs combination, respectively. Moreover, 218 and 8 DEGs were recorded in (J7_0H Vs. J7_6H) Vs. (J7_0H Vs. J7_12H) Vs. (J194_0H Vs. J194_6H) and (J194_0H Vs. J194_12H) Vs. (J7_0H Vs. J7_12H) Vs. (J194_0H Vs. J194_6H) combinations, respectively. Also, (Figures b and d) indicated the DEGs under different germplasms combination for down and up-regulated roots tissue, respectively; the result revealed that about 16 and 12 DEGs were observed in (J194_6H Vs. J7_6H) Vs. (J194_12H Vs. J7_12H) and (J194_6H Vs. J7_6H) Vs. (J194_12H Vs. J7_12H) respectively.
Comparison of Up/down-regulated DEGs for J7 and J194 leaves and roots at 0, 6, and 12 hours of exposure to NaCl are presented in (Fig. 2b). The results indicated that J7R_0H Vs. J7R_6H (2413) had the highest number of up-regulated DEGs, followed by J7L_0H Vs. J7L_6H (2152), whereas J194L_0H Vs. J194L_12H (38) had the lowest number. However, J7L_0H Vs. J7L_6H (2256) had the highest number of down-regulated DEGs, followed by J7L_0H Vs. J7L_6H (2000) whereas J194L_0H Vs. J7L_0H (74) recorded the least number. However, J194R_0H Vs. J194R_6H recorded up and down-regulated DEGs of 703, 689 respectively.
Cluster analysis revealed that differential gene expressions were more similar within the same tissue types and time exposure than between different genotypes and time duration (Fig. 2a). We observed that genes within the same tissues under the exact period of exposure to NaCl were clustered together. Additionally, replicating the treated and control samples was highly consistent, signifying the RNA-seq results’ reproducibility.
The results showed that plant hormone signal transduction (ko04075) was the most enriched pathway in root tissues at six-hour exposures to NaCl stress. Plant hormones such as auxins, jasmonic acid, and abscisic acid play significant roles in the plant response to stresses by regulating hormone signal transduction pathways [19, 20]. Among the 62 enriched DEGs observed in both J194 and J7 root tissues at six hours of exposure to NaCl Stress (Additional Table S8), 17 had unknown functions, while the remaining 45 were generally related to abiotic stress responses.
Plant hormonal signal transduction pathways of the up-and down-regulated DEGs in the two germplasms’ root tissues (J194 and J7) at six-hour of exposure to NaCl stress are presented (Fig. 2c and Additional Figure S3). The result indicated that three DEGs were involved for AUXIN1, including Corchorus_capsularis_newGene_739, Cc.01G0031930, and Cc.04G0019670. These genes were down-regulated in J194 and J7, respectively. For IAA, five DEGs were identified; however, only Cc.07G0002340 (down-regulated) and Cc.07G0003430 (up-regulated) were recorded in J194. Additionally, two DEGs (Cc.01G0010780 and Cc.07G0003430) and three DEGs (Cc.04G0043620, Cc.04G0045370 and Cc.07G0002340) were up-and down-regulated in J7, respectively (Fig. 2c). For ARF, three DEGs were involved, but only one (Cc.06G0025590) was down-regulated in both J194 and J7. another one (Cc.06G0027580) was down-regulated in J7 only. For IAA, one DEG (Cc.07G0005110) was up-regulated in both J194 and J7, While for the GH3 signaling pathway, two up-regulated DEGs were found (Cc.01G0028710 and Cc.02G0006030) in both J194 and J7. SMALL AUXIN UP RNAs (SAUR) involves eight DEGs, among which four (Cc.03G0019440, Cc.03G0029630, Cc.04G0007220 and Cc.05G0005100) were up-regulated in both J194 and J7. However, the remaining four (Cc.02G0011530, Cc.02G0022670, Cc.04G0014260, and Cc.06G0027410) were up-regulated only in J7.
For PYL, one DEG (Cc.03G0016680) was only up-regulated in J7 whereas six DEGs were involved in the PP2C pathway, out of which three (Cc.03G0000600, Cc.03G0030800, and Cc.07G0001880) were up-regulated in both J194 and J7, Cc.03G0016550, and Cc.07G0028160 were up-regulated in J194 while Cc.06G0030850) was only up-regulated in J7. SnRK2 signaling pathway had only three DEGs, including Cc.02G0003620 and Cc.04G0017920 that were up-regulated, and Cc.02G0021190, which was down-regulated in J7. For ABA-responsive element binding factor (ABF), three DEGs were involved; one (Cc.04G0004780) was up-regulated in both J194 and J7, whereas Cc.01G0035870 and Cc.06G0010680 were respectively up-and down-regulated specifically in J7. Two DEGs were involved in JASMONATE ZIM DOMAIN (JAZ) pathway; however, only one (Cc.06G0030170) was up-regulated in both J194 and J7. In the MYC2 pathway, only one gene (Cc.04G0013900) was down-regulated in both J194 and J7.
Quantitative reverse transcription-PCR to validate the RNA-seq result is presented in (Additional Figure S4). The result indicated that all the study genes confirmed theirs expression in both the qRT –PCR and RNA-seq, except that sample (J7R_12H Vs. CR) for ABF/bzip genes were down-regulated in the qRT –PCR rather than up-regulated.
Correlation analysis between germination rate related traits with seedling stage parameters and qRT-PCR with RNA-seq results are presented in supplementary (Additional Figure S5 and S6) respectively. The results indicated a significant positive correlation between germination related traits (RGS and RGVS) and seedling stage parameters exposed to NaCl stress, except DRW, RL, RFW, and DSW. Moreover, a significant positive correlation was also observed between qRT-PCR and RNA-seq results (Additional Figure S6).
The relative expression levels of seven randomly selected candidate genes in J194 and J7 at one and two weeks duration of exposure to NaCl stress are presented in (Additional Figure S7a-b) validating the candidate genes. The gene relative expression levels of the J194 and J7 at one week is shown in (Additional Figure S7a and c). The relative expression of the genes in leaf and root tissues of J194 and J7, were higher in J194 compared to J7, though the expression of Cc.06G0024090 (MYB) was higher in the control condition than that of the treated samples of both J194 and J7. The gene relative expression levels of the J194 and J7 at two weeks is presented in (Additional Figure S7b and d). The result indicated that the expression levels were slightly higher than that of one week. Still, the genes expressed high in treated J194 root and leaf tissue samples compared to that of J7; however, Cc.06G0024090 (MYB) of the control leaf sample had the highest expression compare to the treated samples. It was observed that the genes expressed more elevated in the leaf than in the root.
Identification of Corchorus capsularis PP2C gene family
The Corchorus capsularis PP2C gene family and their homologs in other species are presented in (Table 1); the table indicated 89.8–100% relative similarities and most of the homologous organisms were Theobroma cacao (15 genes), Herrania umbratica (8 genes), and Duriozibethinus (5 genes). Moreover, the identified PP2C Arabidopsis and Corchorus capsularis conserved domains’ after removing redundancies sequences, and non-PP2C using SMART and PFAM (Additional Table S10). About 78 Arabidopsis PP2C were identified, which used as a queries sequences to search against the Corchorus capsularis genome using NCBI local blast as stated in the materials and methods, after confirming the presence of PP2C catalytic domains using SMART and PFAM, about 38 genes were identified (Additional Table S10).
To study Corchorus capsularis and Arabidopsis and PP2C protein families’ relationship, we generated an alignment for full-length protein sequences of 76 and 38, respectively (Fig. 3). To be consistent with the previous report of Arabidopsis PP2C [21], we followed the same name for twelve common subfamilies (A, B, C, D, E, F (1and 2), G, H, I, J, K, and L) [21]. Moreover, three novel groups M (AT2G40860 and Cc.05G0014550), N (AT4G27800 and Cc.03G0020320), and O (AT5G19280 and Cc.04G0042080) were identified with 100% bootstrap support. Group F, as previously reported, and was divided into two sub-groups (F1 and F2) with 99 and 100% bootstrap support, indicating that these genes in the same sub-family shared a similar evolutionary origin. We also observed that in-group F2 (AT5G53140 and Cc.03G0016550) as well as (AT4G31750 and Cc.07G0024730) shared 100% bootstrap, revealing they do share an evolutionary relationship. However, F1, B, and K groups of Arabidopsis PP2C proteins did not share similarities’ with Corchorus capsularis PP2C. In-group A, Cc.06G0030850 had 57% bootstrap support with AT1G17550, ATG72770, AT4G26080, and AT5G57050. Moreover, in-group G, Cc.07G0010300 had 100% bootstrap similarities with AT2G25620, Cc.01G0009520 had 100% bootstrap support with AT3G51570 and AT2G33700. Cc.06G0018350 in-group I had 100% bootstrap support with AT2G25070 and AT4G31860. Group M and N, Cc.05G0014550 and Cc.03G0020320 had 100% bootstrap support with AT2G40860 and AT4G27800. Group H, Cc.040004590 and Cc.05G0029950 had 100% bootstrap support respectively. Additionally, Cc.07G0002210 and Cc.04G0045880 had 100% similarities respectively. For group E, Cc.05G0029870 had 100% bootstrap support with AT5G27930 and AT3G05640, however 66% with Cc.04G0047530. Additionally, in this group, Cc.06G001817 had 100% bootstrap with AT1G16220 and AT1G79630; lastly, AT5G36250 and AT3G02750 had 100% and 99% bootstraps support respectively with Cc.02G0024270. Furthermore, in the J group, Cc.04G0009430 had 100% bootstrap with AT3G63320 and AT3G63340. Regarding group C, Cc.05G0002960 and Cc.07G0028160 had 99 and 100% bootstrap support with (ATG07630, AT2G28890, AT3G09400, and AT5G02400) and (ATG07630 and AT2G28890), respectively. Moreover, AT2G46920, AT2G35350 and AT3G16560 had 100% bootstrap support with Cc.0440013340, Cc.03G0022250 and Cc.04G0046980 respectively. In-group D, Cc.05G0010230 and Cc.04G0048240 had 100% bootstrap support with AT5G06750 and AT3G17090 respectively. Moreover, AT4G33920 had 100, 52% bootstrap similarities with Cc.02G0005900 and Cc.01G0028490 respectively, furthermore Cc.03G0003110 had 100 and 97% similarities with AT3G55050 and AT3G12620 respectively. Similarly, Cc.01G0032360 and Cc.02G0000770 had 73% bootstrap with AT3G51370 and AT5G66080. Group O Cc.04G0042080 had 100% bootstrap support with AT5G19280.
Table 1
Corchorus capsularis PP2C gene family and theirs homologs in other species
|
Similar Jute gene bank ID
|
|
Homologous genes
|
Gene bank ID
|
Gene bank ID
|
RS(%)
|
Gene length(bp)
|
Protein(aa)
|
Gene bank ID
|
Cc.01G0009520
|
OMO83396.1
|
100
|
1074 bp
|
357 aa
|
XP_021284306.1(Herrania umbratica)
|
Cc.01G0028490
|
OMO73934.1
|
100
|
112028 bp
|
381 aa
|
XP_021276140.1(Herrania umbratica)
|
Cc.01G0032360
|
OMO74554.1
|
100
|
48004 bp
|
397 aa
|
XP_007041776.2[Theobroma cacao]
|
Cc.02G0000770
|
OMO55759.1
|
100
|
96995 bp
|
397 aa
|
XP_007046602.1[Theobroma cacao]
|
Cc.02G0005900
|
OMO56534.1
|
100
|
46132 bp
|
380 aa
|
XP_022751230.1[Durio zibethinus]
|
Cc.02G0024270
|
OMO52614.1
|
96.02
|
26475 bp
|
493 aa
|
XP_021282751.1[Herrania umbratica]
|
Cc.03G0003110
|
OMP08102.1
|
97.31
|
38078 bp
|
331 aa
|
XP_007009638.1[Theobroma cacao]
|
Cc.03G0016650
|
OMO99628.1
|
100
|
34627 bp
|
354 aa
|
XP_021295755.1[Herrania umbratica]
|
Cc.03G0020320
|
OMO87933.1
|
100
|
85758 bp
|
392 aa
|
XP_022734118.1[Durio zibethinus]
|
Cc.03G0022250
|
OMO77235.1
|
100
|
50299 bp
|
789 aa
|
XP_007039547.2[Theobroma cacao]
|
Cc.04G0004590
|
OMO78977.1
|
100
|
71295 bp
|
428 aa
|
XP_017981229.1[Theobroma cacao]
|
Cc.04G0009430
|
OMO77631.1
|
99
|
86720 bp
|
1088 aa
|
EOX94652.1 [Theobroma cacao]
|
Cc.04G0013340
|
OMO82327.1
|
89.8
|
36529 bp
|
896 aa
|
EOX95490.1[Theobroma cacao]
|
Cc.04G0042080
|
OMO61226.1
|
100
|
169640 bp
|
590 aa
|
XP_022762030 [Durio zibethinus]
|
Cc.04G0045880
|
OMO98359.1
|
98.59
|
67103 bp
|
426 aa
|
XP_021290662.1[Herrania umbratica]
|
Cc.04G0046980
|
OMO53660.1
|
100
|
151752 bp
|
531 aa
|
XP_022760559.1[Durio zibethinus]
|
Cc.04G0047530
|
OMP11874.1
|
95.09
|
14476 bp
|
410 aa
|
XP_021290531.1 [Herrania umbratica]
|
Cc.04G0048240
|
OMO53492.1
|
99.41
|
22592 bp
|
1173 aa
|
XP_021291718.1[Herrania umbratica]
|
Cc.05G0002960
|
OMO66259.1
|
100
|
49282 bp
|
597 aa
|
XP_007027029.2[Theobroma cacao]
|
Cc.05G0010230
|
OMO63000.1
|
100
|
122420 bp
|
388 aa
|
XP_017977120.1 [Theobroma cacao]
|
Cc.05G0014550
|
OMO53906.1
|
97.42
|
58784 bp
|
659 aa
|
XP_022742528.1[Durio zibethinus]
|
Cc.05G0029870
|
OMP11453.1
|
99.73
|
101706 bp
|
369 aa
|
XP_017977558.1[Theobroma cacao]
|
Cc.06G0018170
|
OMO83325.1
|
100
|
15947 bp
|
497 aa
|
EOY26142.1[Theobroma cacao]
|
Cc.06G0018350
|
OMO74037.1
|
98.9
|
106105 bp
|
362 aa
|
XP_021298273 [Herrania umbratica]
|
Cc.06G0030850
|
OMO70679.1
|
100
|
96546 bp
|
539 aa
|
XP_007024551.2 [Theobroma cacao]
|
Cc.07G0002210
|
OMO82641.1
|
100
|
103588 bp
|
1085 aa
|
XP_017983109.1[Theobroma cacao]
|
Cc.07G0010300
|
OMO80598.1
|
93.68
|
58017 bp
|
364 aa
|
XP_007013785.1[Theobroma cacao]
|
Cc.07G0028790
|
OMO67773.1
|
98.76
|
53567 bp
|
733 aa
|
XP_007016425.2[Theobroma cacao]
|
The distance tree was inferred using the neighbor-joining methods based upon an alignment of full-length amino-acid sequences of the PP2C conserved domains. They were grouped according to the PP2C Arabidopsis genes previously reported.
For identification of motifs and gene structure, 11 Corchorus capsularis PP2C motifs were identified; their sequences and motifs distributions are presented in (Fig. 4a). The results indicated that motifs 3, 2,5,11, 4, and 1 were wide spreads among the Corchorus capsularis PP2C groups. However, other motifs such as 9 and 6 were specific to two groups (D and C), whereas 10 and 7 were exclusively specific to group D. Moreover, the 8 motif was specific to group C and E.
The phylogenetic tree was constructed from alignments of 38 amino acid sequences of PP2C conserved domains in Corchorus capsularis PP2C; it was grouped according to PP2C Arabidopsis genes previously reported. The Sequences and the length of the conserved motifs in the amino acid sequences of Corchorus capsularis PP2C genes are also indicated at the bottom.
The arrangement of exon-intron will play an important role in the process of Corchorus capsularis PP2C diversification. As such, exon-intron organization was analyzed, using online tools GSDS as stated in the methodology. Figure 4b illustrated the distribution of the Corchorus capsularis PP2C exon-intron; the result revealed that most of the Corchorus capsularis PP2C members in the same subfamily shared similar exon number with different exon and intron length, the number of exon-intron ranges between (3–21) and (2–20), respectively. Cc.04G0046980 had the least number of exon (3), and intron (2) subfamily group C. Whereas Cc.04G0048240 in subfamily member of group D had the highest exon and intron number of 20 and 19 respectively. Group G and A had four exons, except for Cc.06G0018350 of group G, which had ten. Cc.03G0016550 and Cc.07G0024730 of group F2 had a similar number (8) of the exon. Cc.05G0029870 and Cc.04G0047530 of group E had the same number (5) of the exon. Cc.05G0002960, Cc.07G0028160 and Cc.03G0022250 of group C had the same number (4) of exon. Moreover, group D member (Cc.02G0005900, Cc.05G0010230, Cc.03G0003110, Cc.01G0032360, and Cc.02G0060770) had the same number (4) of exon.
The phylogenetic tree was constructed from alignments of 38 amino acid sequences of PP2C conserved domains in Corchorus capsularis PP2C; they were grouped according to previously reported Arabidopsis PP2C. The exon/intron structure of each Corchorus capsularis PP2C gene was proportionally displayed according to the scale at the bottom. Yellow boxes represent exons, gray lines represent introns and blue boxes represent untranslated regions.
The result further indicated that Corchorus capsularis PP2C genes were localized on 7 chromosome groups, and were unevenly distributed (Additional Figure S8). Three genes each were mapped on chromosome 1 and 2, however about 4 genes were mapped on chromosome 7. Moreover, chromosome 4 and 5 had the highest number of map genes (Cc.04G0004590, Cc.04G0009430, Cc.04G0013340, Cc.04G0042080, Cc.04G0046980, Cc.04G0048240, Cc.04G0045880, and Cc.04G0047530) and (Cc.05G0010230, Cc.05G0029870, Cc.05G0002960, Cc.05G0014550, and Cc.05G0029950), respectively.
Expression pattern of PP2C (reference marker) genes in J194 and J7
Response to NaCl stress was assessed by monitoring the expression pattern of the key stress marker genes in J194 and J7 under control and stress conditions, as mentioned in the materials and methods. As such, we conducted qRT-PCR base expression analysis from the well-established stress marker genes such as; RAB18, RD29B, KIN1, and RD29A using J194 and J7 root and leaf tissue samples exposed to NaCl at 0, 6, and 12 hours as previously stated. Besides, J194 and J7 root and leaf tissue samples at one and two weeks’ of exposure to NaCl were conducted.
The relative gene expression level of Cc.04G002237(RAB18), Cc.03G0029910(RD29B), Cc.01G0008270(KIN1), and Cc.03G0029910(RD29A) at 0, 6, and 12 hours of exposure to NaCl are presented in (Figs. 5a-d). The result indicated salt-tolerant germplasm (J194) had the highest relative gene expression level across the periods. The result further revealed that J194 root tissues at six-hour exposure to NaCl recorded the highest gene expression levels in all the studied genes.
Moreover, these genes’ relative expression levels at one and two-week exposure to NaCl are presented (Figs. 6a-d). The results still indicated the J194 had the highest relative gene expression level in all studied genes across the periods of exposure to NaCl. However, the relative expression level of Cc.04G002237 (RAB18) and Cc.03G0029910 (RD29B) were higher in J194 roots tissues across the period of NaCl exposure whereas, Cc.01G0008270 (KIN1) and Cc.03G0029910 (RD29A) had the highest expression level in J194 leaf tissues at all duration of exposure to NaCl.
The qRT-PCR analysis was conducted to generate the relative expression profile level of key established stress marker genes such as RAB18, RD29B, and KIN, and RD29A (a to d respectively) in J194 and J7 leaf (L)and root (R) tissues at CT(0), 6, and 12hours as indicated in X-axis and the relative expression level is indicated in Y-axis. Data from the mean of the replicated samples are presented as columns and an error bar denotes the standard deviation. *p-value < 0.05 and **p-value- < 0.01 indicate statistically significant level.
The qPCR analysis was conducted to generate the relative expression profile level of key established stress marker genes such as RAB18, RD29B, and KIN, and RD29A (a to d, respectively) in J194 and J7 leaf (L)and root (R) tissues at 0(control), one, and two weeks duration of the exposure to NaCl. The X-axis represents the samples at control (C) and Treated (T) and the relative expression level is indicated in Y-axis. Data from the mean of the replicated samples are presented as columns and an error bar denotes the standard deviation. *p-value < 0.05 and **p-value- < 0.01 indicate statistically significant level.