Phenotypic and molecular confirmation of maize (Zea mays L.) genotypes for drought tolerance at seedling stage

Abiotic stresses such as drought is adversely affecting the crop yields as well as maize productivity. The core idea of this study was to screen and identify maize genotypes for drought tolerance through morphological and molecular characterization for Balochistan region. In-vitro experiment was carried out using four maize genotypes namely Azam, Jalal, Edhi and Pahari that were investigated for drought stress tolerance at seedling stage. Completely randomized design (CRD) was used for current study. Maize seedlings were grown in growth chamber and after two-weeks of drought stress, samples were collected at different time periods. The morphological data showed that the root architecture of Jalal was significantly different from the remaining studied genotypes. These results were further strengthened and supported by the molecular analysis. For wet lab experiment, the DNA was extracted by Plant Mini Kit method and the presence/absence of drought tolerant gene was confirmed through the Polymerase Chain Reaction (PCR). The specifically designed primers for candidate orthologues were used to obtain the targeted PCR products. Furthermore, the results of PCR were confirmed through gel electrophoresis. The results of inter simple sequence repeats (ISSR) markers showed that only ISSR-838 was attached on Jalal while remaining genotypes did not showed any amplification of the products, that mean the Jalal genotype is more diverse from the other genotypes for drought stress tolerance. The obtained results of this study will be further utilized for the development of drought resistant maize variety and also used in other future breeding programs of Balochistan.


Introduction
Plant cannot move because of their sessile nature and permanent restriction to their site of germination.Therefore, it may faces several adverse environment stresses which ultimately affect their overall yield and productivity [1,2].Apart from the poor cultural practices, both biotic and abiotic stresses are the major constrains of maize yield and production [3].More specifically, among the abiotic stresses drought is one the key environment factor, which negatively affect plant at all stages of growth and development [4].Area wise Balochistan is the largest province of Pakistan by covering about 43% of total area.Balochistan in one of the province which are naturally facing drought and going to be worse in the forthcoming years mainly due variable climate changes [5].Thus, drought is one of the biggest challenge for the agriculture sector in Balochistan [5,6].Plant respond to drought in several different ways, like physiologically, chemically or either by naturally existing defense system [7].In Balochistan, water availability is limited and maximum yield achievement of maize is not possible without focusing to develop drought tolerant varieties.Due to narrow genetic base of maize crop, it is difficult to improve or develop new varieties through phenotyping by simple field evaluation, because this tradition breeding methods only depend upon selection in the field, which is slow and laborious for evaluation and development of new genotypes.In most of the breeding programs, selection and phenotyping may be also give imprecise results because of environmental influences [8].However, the use of molecular markers is one of the most effective and advanced technique to evaluate the maize verities in response of tolerance against drought stress [9-11].The inter simple sequence repeats (ISSRs) are semi-arbitrary markers, amplified by PCR using a single primer composed of microsatellite repeated sequences [12].These novel DNA markers have higher repeatability and dominate nature with ampilicon size ranged from 100-300 base pairs.That is why; plant scientists give preference to ISSRs than other molecular markers for estimating the genetic distances among population.Previous studies showed that ISSR markers have proven to be potent amplification based on DNA fingerprinting technique.Therefore, the current study was conducted to evaluate the drought tolerance in maize genotypes through phenotypically coupled with molecular markers technique to identify the most desirable genotype at seedling stage.Furthermore, this study will ultimately facilitate the breeders and researchers to address drought tolerance in maize and other crops at initial growth stages for developing drought resistant varieties.

Plant material and growth conditions
The maize seedlings of four genotypes namely, Azam, Jalal, Edhi and Pahari were grown in small plastic pots with an equal amount of send, soil and peat moss.The experiment was laid out in a completely randomized block (CRD) with three replications.Each experimental unit was watered regularly to ensure vigorous germination.Fifteen days old maize seedlings were maintained under similar growth conditions and then subjected to drought stress by withholding water for an interval of two days.Similar drought stress treatment was given to the all genotypes such as Azam, Jalal, Edhi and Pahari.Four plants represented one treatment of drought stress for each time course.Followed by two days of withholding water supply, the plant and leaf samples were taken at different time periods from stressed as well as control plants.

Morphological parameters
After imposing artificial drought stress to all studied genotypes, the different morphological parameters including seed germination was observed.The seedling data was collected for shoot length (cm) and root length (cm) because these parameters are important indicator for drought estimation.

Molecular marker analysis (ISSR)
The total genomic (gDNA) was extracted by using DNeasy Plant Mini Kit (QIAGEN).Fresh leaves of each genotype were taken and weighing ~100 mg for gDNA extraction.After quantification of nucleic acid, gDNA of all maize genotypes and products amplification was performed by using (Thermo Fisher, USA) 10X master mix.,

Results and Discussion
To investigate the stress tolerance in maize genotypes both morphological and molecular approaches were used for this study.In modern breeding program we cannot solely rely on morphological data.Therefore, in addition to morphology data, the molecular confirmation is necessary for the identification of stress tolerance in plants.Thus, the selection of desired progenitors based on morphological traits and was supported by molecular validation would be a useful strategy for the identification of potential lines at initial seedling stage [8].Seed colour and shape For the analysis of drought tolerance, four diverse maize genotypes were investigated at seedling stage.Before germination, seed colour, size and shape was analyzed.For seed size and shape, no significant differences were observed among the genotypes.However, the seed colouration was found to be different with in the genotypes as shown in (Fig. 1).Previously, the effect of seed shape and size was determined in maize cultivars for germination and vigour.Seed traits showed considerable variability regarding size and suggested that large seed were more advantageous overs small seed fractions [10].

Seed germination (%)
When seed germination was tested among the genotypes, the results showed no differences for germination percentage among tested genotypes (Fig. 2).Data also showed that, the used seed for this study was healthy and have good ability of germination.All four genotypes showed more than 95% germination under normal condition.These findings are close in agreement with the previous study in which authors recorded 94% germination (%) in maize hybrids [13].However, in a similar study on maize, it was reported that seed germination was reduced considerably under stressed condition [14].

Shoot and root morphology
When the morphology of root and shoot was examined, it was noticed that there is significant differences among the genotypes for these traits.The highest shoot length was observed in genotype Edhi whereas lower shoot length was demonstrated by genotype Pahari as represented in (Fig. 3).Previous studies also reported meaningful variation for root morphology of maize genotypes through image analysis [15].

Shoot and root length (cm)
After germination test, the shoot length of all genotypes was measured in centimeter.When we compare the shoot length at seedling stage, shoot length was decreased as compare to control plants.However, we found no difference in shoot length among susceptible and resistance genotypes as shown in (Fig. 4).The similar results were also observed in previous study, when the drought stress intensity was increased the shoot length was decreased [16].Furthermore, similar types of results were also drought is also affected the growth, root weight and shoot length as previously noticed by different scintists [17,18].Furthermore, when we compare root lengths among the genotype, we found significant difference in the root length, Jalal and Pahari showed 20, 25 cm respectively as shown in (Fig. 5).Previous findings also suggests that, root structure is key player and will decide the drought resistance and susceptibility in any crop [19,20].On the bases of morphological data, we concluded that Jalal and Pahari were the drought resistant genotypes while the Edhi and Azam were the susceptible genotypes.To further strengthen out morphological data we confirm the drought resistance through molecular markers.

Drought tolerance gene in maize
To confirm the presence/absence of drought resistance gene in maize genotype, four drought specific ISSR markers were selected for this study and out of four only one marker (ISSR-838) was amplified and attached to the targeted region.After PCR amplification, samples were resolved on 2% agarose gel and results showed that only ISSR-838 marker was attached with Jalal genotype as depicted in (Fig. 6).Similar type of results were obtained through SSR marker screening for drought tolerance in wheat genotypes [21,22] and other researchers had also reported parallel results [15,23,24].Therefore, on the bases of morphological and molecular data it would be concluded that maize genotype Jalal genome might have drought resistant gene whereas rest of the genotypes did not show any amplification against this marker.These are not final finding, however, Jalal genotype showed some clue regarding drought stress tolerance and it would be suggested that this genotype will further confirm through RT-PCR analysis and gene sequencing.Al-Turki and Basahi [25] interpret the drought tolerance mechanism in mutant maize by using physio-biochemical traits and transcritomic analysis.The transcritomic profiling coupled with conventional indices it was suggested that two loci were highly linked with drought tolerance in maize.In case of our identified genomic ISSR-838 showed product amplification of 580 bp which is suggesting strong candidate QTL for drought tolerance enhancement by association mapping.Thus, the hybridization of Jalal genotype with high yielding local variety may have successful chances to combine desired characters in developing improved variety for Balochistan province.

Conclusion
In present study, Jalal genotype was identifed as drought tolerant cultivar whereas Azam, Edhi and Pahari were found to be susceptible genotypes on the bases of morphological and molecular data.The phenotypic data for root length among the genotypes indicated that Jalal and Pahari attended a length of 20, 25 cm respectively, but molecularly only ISSR-838 marker showed amplification of targeted product with Jalal genotype.This research might be help for the identification of drought tolerance in maize in Balochistan.Furthermore, marker assisted breeding information of this study would be utilized by researchers for drought tolerance variety development programs.

Authors' contributions
1.5 µl (10 µM), each forward and reverse primer, 3 µl of template gDNA and the total reaction volume was raised to 20 µl in each PCR tube by adding PCR grade water.The PCR programing was set in a Bio-Rad C100 Touch Thermal Cycler (Bio-Rad), as initial denaturation 95 °C for 1 min, denaturation was carried out at 95 °C for 20 s, and primers were annealed at 61 °C for 20 s, extension at 70 °C for 1 min.The PCR was performed for 35 cycles for product amplification.Final extension was done for 10 min, at 65 °C.The amplified PCR products were resolved on 2% TAE agarose gel and visualized under gel documentation system (Transilluminator Bioview UV light; Dark-box DH-30132) for results interpretation.Data analysis Data regarding shoot length, root length, and germination percentage, the experimental design was a completely randomized design with 'n' number of plants in each group.The P values for significant (P ≤ 0.05) differences were determined and shown as double asterisk.Means and standard deviations (SD) are expressed as standard error bars in the bar charts.Molecular markers results were analyzed on gel electrophoresis.

Figure 4 .Figure 5 .Figure 6 .
Figure 4. Measurement of shoot length of maize genotypes under drought stress.Values are the mean ± SD (n = 15) and (p-value < 0.05) based on t-test.Error bars represent standard error Conceived and designed the experiments: G Rasool and N Arshad, Performed the experiments: A Rehman.Analyzed the data: G Rasool & A Razak, Contributed materials/ analysis/ tools: A Jan & S Faisl, Wrote the paper: G Rasool, A Ullah & H Noor.