Effects of droughts on tomato landraces at the seedling stage.
Several parameters such as root fresh weight, root dry weight, root growth rate, shoot fresh weight, shoot dry weight, shoot growth rate was measured on the 46 tomato landraces. Seedlings were grown under three levels of water stress (control, 70% FC, 40% FC), however, all the parameters were shown high significant differences at 40% FC as compared to the control treatment, which also was significant at 70%FC (Table S1). All morphological traits had a lower mean performance under drought stress (70% FC and 40% FC) than under normal conditions as expected. On average, all parameters had a reduction due to drought stress at 70% FC and 40% FC as compared with the control treatment (Table S3).
Drought stress is significantly affected by the proline concentrations in the leaves tissue for 46 tomato landraces (Table S1). A highly significant increment in proline content was detected at 40% FC as compared with the control treatment (100% FC) by 304.2%.
Morphological characterization among tomato landraces
In this study, Tomato Analyzer (TA) was used to assess the fruit shape variation of landraces by measuring the morphological characterizations rapidly, accurately, and quantifying traits that are impossible to quantify manually. The analysis of variance for all the morphological characters indicates to presence wide range of variability among tomato landraces including, the area, perimeter, maximum width, maximum height, curved fruit shape index, average red, average green, fruit shape index external I, fruit shape index external II, curved fruit shape index, fruit shape index internal (Table S4).
Genetic variation among tomato landraces revealed by SSRs.
Our investigation tested 14 SSRs primers, of these, 6 yielding polymorphic amplification products were used and the remaining eight SSRs primers either yielded no amplification product or no polymorphic. The banding patterns of SSRs are shown in Supplementary figures (1- 6). Of 46 tomato landraces, the genetic relationship among thirty-six tomato landraces was analyzed using six SSRs primers pair. Two hundred forty-seven amplified bands were produced of 12 loci, of them (11) loci were polymorphic and (1) loci were monomorphic shown in (Table 3) and these index on the high allele variation. The molecular weights ranged from 128 to 1170bp. The number of alleles per locus varied from 1 for (CT114 and Asr2) markers to 3 for (LEat018 and LEat020) SSRs markers. The percentage of polymorphic was 91.67% with a range between (zero to 0.49) (Table 4). The highest values of the effective number of alleles (Ne*) (Kimura and Crow 1964), the gene diversity (h*) (Nei 1973), and the Shannon Index (I*) (Lewontin 1972), were recorded to LEct004 primer (350bp loci) with values of 1.99, 0.4996, and 0.6927, respectively. While the lowest value (zero) of the effective number of alleles (Ne*), the gene diversity (h*) and the Shannon Index (I*) was shown by Asr2 primer (536bp loci) (Table 3-4).
Table 3
SSRs names, total number of alleles, loci, monomorphic and polymorphic loci, percentage of polymorphism and Polymorphism Information Content (PIC):
SSRs Name | Total bands/primer | No. of loci | Monomorphic Loci | Polymorphic Loci | Polymorphism % |
LEat018 | 46 | 3 | 0 | 3 | 100 |
LEct004 | 40 | 2 | 0 | 2 | 100 |
Lea014 | 46 | 2 | 0 | 2 | 100 |
LEta020 | 46 | 3 | 0 | 3 | 100 |
CT114 | 33 | 1 | 0 | 1 | 100 |
Asr2 | 36 | 1 | 1 | 0 | 0 |
Total | 247 | 12 | 1 | 11 | |
Table 4
Allele Frequency, Genotype Frequency, Heterozygosity for primers:
SSRs Marker | Locus | Sample size | Ne* | H* | Average h* | PIC | * |
LEat018 | 139 | 36 | 1.52 | 0.34 | 0.34 | 0.34 | 0.52 |
| 132 | 36 | 1.26 | 0.21 | | 0.20 | 0.36 |
| 128 | 36 | 1.95 | 0.49 | | 0.48 | 0.68 |
LEct004 | 364 | 34 | 1.60 | 0.38 | 0.43 | 0.37 | 0.56 |
| 350 | 34 | 1.99 | 0.49 | | 0.49 | 0.69 |
Lea014 | 175 | 36 | 1.38 | 0.27 | 0.27 | 0.28 | 0.45 |
| 190 | 36 | 1.38 | 0.27 | | 0.28 | 0.45 |
LEta020 | 223 | 36 | 1.75 | 0.43 | 0.36 | 0.42 | 0.62 |
| 217 | 36 | 1.65 | 0.39 | | 0.39 | 0.58 |
| 206 | 36 | 1.34 | 0.25 | | 0.25 | 0.42 |
CT114 | 1170 | 36 | 1.69 | 0.41 | | 0.41 | 0.60 |
Asr2 | 536 | 36 | 1.00 | 0.00 | | 0.00 | 0.00 |
Mean | | 36 | 1.55 | 0.33 | | 0.33 | 0.49 |
Ne* = Effective number of alleles |
h*= gene diversity |
I*= Shannon's Information index |
The number of polymorphic loci is: 11 |
The percentage of polymorphic loci is: 91.67 |
UPGMA Dendrogram and Similarity.
Genetic variation among tomato landraces was assessed based on bands obtained from SSRs profiling using Nie genetic distance and Unweighted Pair Group with Arithmetic Averages (UPGMA). The coefficient of genetic similarity ranged from 0.30 between JOR956 and JOR966 landrace to 1 between JOR950 and JOR951, JOR964 and JOR965, JOR955 and JOR979, JOR955 and JOR980, JOR979 and JOR980, JOR955 and JOR988, JOR979 and JOR988, JOR980 and JOR988. The most similar tomato landraces reported above are from Kharja, Rhaba, Ain Al-Biada whereas the most different are from Qasfa and Rhaba showed in Table 5.
Table 5
Scoring of SSRs Markers for 41 tomato landraces. 1 and 0 are used for present and absent alleles, respectively.
SSR Marker | Fragment size | c1 | c2 | c3 | 1 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 30 | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | 41 |
LEat018 | 139 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 |
| 132 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
| 128 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 |
LEct004 | 364 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | . | . |
| 350 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | . | . |
LEta014 | 175 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| 190 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
LEta020 | 223 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 1 |
| 217 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| 206 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 1 |
CT114 | 1170 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Asr2 | 536 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Table 6
BLAST corresponding Solyc (Solanum lycopersicum) gene sequences and annotation for SSR marker sequences.
SSR Name | Solyc gene | Annotation result |
LEat018 | Solyc11g005330 | ACTIN–RELATED PROTEIN; contain Interpro domain IPR004000 (Actin family domain) |
LEct004 | Solyc02g089940 | HOMEOBOX PROTEIN TRANSCRIPTION FACTORS; contains Interpro domain(s) IPR009057(Homeodomain-like), IPR006563 (POX domain), IPR008422 (Homeobox KN domain), IPR016039 (Thiolase-like), IPR001356 (Homeobox domain). |
LEta014 | Solyc03g031970 | AUXIN RESPONSE FACTOR 8; contains Interpro domain(s) IPR003340 (B3 DNA binding domain), IPR010525 (Auxin response factor), IPR003311 (AUX/IAA protein), IPR015300 (DNA-binding pseudobarrel domain). |
LEta020 | Solyc01g097450 | THIOREDOXIN FAMILY TRP26; contains Interpro domain(s) IPR008979 (Galactose-binding domain-like), IPR010400 (PITH domain). |
CT114 | Solyc10g011690 | Protein suppressor of PHYA-105 1 (SPA1); contains Interpro domain(s) IPR000719 (Protein kinase domain), IPR017986 (WD40repeat-containing domain), IPR002290 (Serine/threonine/dual-specificity protein kinase, catalytic domain), IPR001680 (WD40 repeat), IPR015943 (WD40/YVTN repeat-like-containing domain), IPR011009 (Protein kinase-like domain). |
Asr2 | Solyc04g071580 | ABA/WDS induced protein (ABA_WDS); contain Interpro domain IPR003496 (ABA/WDS induced protein). |
Moreover, similarity values for all tomato landraces using UPGMA dendrogram were shown in Figure 1. At a genetic similarity value of 0.62, the dendrogram is divided into two groups except 25 (JOR978) landraces from Rhaba in a separate group. The first group consists of three sub groups:1 (JOR111), 5 (JOR953), 30 (JOR984) landraces with 80% similarity, 6 (JOR954), 7 (JOR955), 26 (JOR979), 27 (JOR980), 34 (JOR988), 22 (JOR972), 19 (JOR968), 28 (JOR981), 18 (JOR967), 23 (JOR973), 20 (JOR970), 33 (JOR987), 35 (JOR989), 36 (JOR990) landraces with 76% similarity, 17 (JOR966), 21 (JOR971) landraces with 88% similarity, these tomato landraces reported in the first group from kharja, Al-al, Rhaba, Afra, Abel, Ain Al-Baida. The second group consists of three sub-groups: 2 (JOR950), 3 (JOR951), 8 (JOR956), 12 (JOR961) landraces with 76.5% similarity, 4 (JOR952), 11 (JOR959), 10 (JOR958), 15 (JOR964), 16 (JOR965), 9 (JOR957), 13 (JOR962), 32 (JOR986) landraces with 85% similarity, 14 (JOR963), 29 (JOR982), 31(JOR985) landraces with 77% similarity, these tomato landraces reported in the second group from kharja, Qasfa, Shtafina, Al-al, Sakib, Hebras, Ain-Jannah, Abel, Afra, and Rhaba. The number of alleles and the PIC value for each SSRs marker are presented in Table 4.
Polymorphic information content (PIC) values were ranged from 0.00 to 0499 (mean 0.33) which confirm that the SSRs markers are highly informative. The SSRs marker had the highest PIC value (LEct004) followed by LEat018 (0.488), while the (Asr2) marker had the lowest PIC value. In this study, we found no relationship between the number of nucleotides per repeat and PIC shown in Table 4. For example, LEat018 with the lower PIC (0.34) has 29 repeats compared to LEta020 which has PIC (0.36) with 11 repeats.
Functional Annotations of the Gene-Associated SSRs in Tomato
Solanum lycopersicum genomic sequence was used as a template for searching the sequence of SSRs primers to predict the potential functions of our six genes. Then, more function annotations for these genes were predicted by various databases, such as Phytozome, NCBI, InterPro, and KEGG databases. In context, these genes were related to a wide range of functions, which indicated that these gene-associated SSRs were potentially associated with very important biological functions, such as the LEat018 SSR marker that associated with ACTIN–RELATED PROTEIN gene (Solyc11g005330) (Table 7). Also, the LEct004 SSR marker was associated with the HOMEOBOX PROTEIN TRANSCRIPTION FACTORS gene (Solyc02g089940). Moreover, AUXIN RESPONSE FACTOR 8 gene (Solyc03g031970) was associated with the LEta014 SSR marker. While the LEta020 SSR marker was associated with the THIOREDOXIN FAMILY TRP26 gene (Solyc01g097450). Furthermore, the CT114 marker was associated with the Protein suppressor of the PHYA-105 1 (SPA1) gene (Solyc10g011690). In addition, the ABA/WDS induced protein (ABA_WDS) gene (Solyc04g071580) was associated with the Asr2 SSR marker (Table 7).
Putative tissue expression pattern of our target genes based on Solanum lycopersicum transcript expression database.
We analyzed gene expression profile maps of our target genes based on Solanum lycopersicum transcript expression database for further understanding the functions of our genes at different nineteen tissues from Solanum lycopersicum (Figure 2). Expression profiles were built using the tomato Electronic Fluorescent Pictograph Browsers (tomato eFP browsers (http://bar.utoronto.ca/eplant_tomato/)). The arrow points to the expression scale (the more intense the red color, the more gene expression). It is clear from the Tomato Electronic Fluorescent Pictograph Expression Profiles Browsers that the gene Solyc11g005330 which is related to the ACTIN–RELATED PROTEIN was highly expressed in all tomato tissues especially in Mature Green Fruit, Breaker Fruit, 3cm Fruit, Root and Breaker Fruit + 10 (Figure 2 and Table 7). While the highest expression levels for Solyc02g089940 gene concerning HOMEOBOX PROTEIN TRANSCRIPTION FACTORS was observed at Fully Opened Flower, Leaves, Pimpinellifolium Leaf, Unopened Flower Bud, and Root (Figure 2 and Table 7). Also, the highest expression levels for Solyc03g031970 gene which is encoding to AUXIN RESPONSE FACTOR 8 was recorded at 1cm Fruit, 3cm Fruit, 2cm Fruit, Unopened Flower Bud, Mature Green Fruit, and Leaves. Unlike, Solyc01g097450 gene which is related to THIOREDOXIN FAMILY TRP26 did not show any clear expression level at any tomato tissue. On the other side, the highest expression levels for Solyc10g011690 gene concerning with Protein suppressor of PHYA-105 1 (SPA1) was observed at Root, 3cm Fruit, Pimpinellifolium Immature Green Fruit, Pimpinellifolium Breaker Fruit, Mature Green Fruit, and Breaker Fruit. In addition, Solyc04g071580 gene which is related to ABA/WDS induced protein (ABA_WDS) was highly expression in Breaker Fruit + 10, Breaker Fruit, Mature Green Fruit, Pimpinellifolium Immature Green Fruit, 3cm Fruit and Root.
Putative subcellular localizations of our target genes based on Solanum lycopersicum transcript expression database.
Cell Electronic Fluorescent Pictograph Browsers (Cell eFP browsers (http://bar.utoronto.ca/eplant_tomato/ )) tools were used to predict the putative subcellular localizations of our target genes based on tomato protein localization at different cell organelles (such as cell plate, cytoskeleton, cytosol, endoplasmic reticulum, extracellular, Golgi, mitochondrion, nucleus, peroxisome, plasma membrane, plastid, unclear, unknown and vacuole). It is clear from the Tomato Cell Electronic Fluorescent Pictograph subcellular localizations profiles that Solyc11g005330 and Solyc01g097450 genes were highly expressed and presented in the cytosol (Figure 3). While, the Solyc02g089940, Solyc03g031970, Solyc10g011690, and Solyc04g071580 genes were highly expressed and presented in the nucleus (Figure 3).
Putative root cell types- and tissues-specific of our target genes based on Solanum lycopersicum transcript expression database.
Root Electronic Fluorescent Pictograph Browsers (Root FP browsers (http://bar.utoronto.ca/eplant_tomato/ ) tools were used to predict the putative function of our candidate genes in root cell types- and tissues-specific (such as. Columella, Lateral root cap, Quiescent center, Epidermis, Exodermis, Cortex, Endodermis, Pericycle, Phloem, Procambium, Xylem, and Vascular initials) under the effect of various promoters (Such as AtWER, SIPEP, AtPEP, SICO2, SISCR, SISHR, AtS32, AtS18, SIWOX5, SIRPL11C, and 35S). Interestingly, we observed the highest expression levels of Solyc11g005330 gene in all root cell types under 35Spro, followed by Phloem under AtS32pro, Endodermis and Cortex under SIPEPpro (Figure 4). Also, Solyc02g089940 gene was highly expressed in all root cell types under 35Spro, then Endodermis and Cortex under SIPEPpro, and Cortex under AtPEPpro. Moreover, the highest expression levels of Solyc03g031970 gene were reported at all root cell types under 35Spro, followed by Epidermis and Procambium under AtS18. In addition, highly expressed Solyc01g097450 gene was observed in all root cell types under 35Spro, followed by Endodermis and Cortex under SIPEPpro, Epidermis and Lateral root cap under AtWER. Furthermore, the highest expression levels of Solyc10g011690 gene were observed at all root cell types under 35Spro, Columella, and Cortex under SICO2pro, then Exodermis and Cortex under SIPEP. While, the Solyc04g071580 gene was highly expressed in Exodermis and Cortex under SIPEP, the Cortex under AtPEPpro (Figure 4).