Transcriptome analysis of maize explant for anther culture: comparison of three different RNA extraction methods

Breeding is one method to develop better varieties. This method can be accelerated by using haploid technology. The availability of haploid inducer lines in maize makes in-vivo techniques preferred, especially for temperate maize. Developing in-vitro through anther culture in haploid breeding is still needed since haploid inducer lines are still limited in tropical maize. Like many other crops facing recalcitrancy problems, maize also experiences the same, so cracking recalcitrant traits using transcriptomic approaches is necessary. In order to enhance the success of the sequencing, obtaining a high quantity and quality of RNA product is needed since it will affect the data. This study aimed to determine the best RNA extraction methods using three different kits of maize explant for anther culture with high purity and concentrations. RNA extraction in this study was performed using Tripure, RibospinTM, Promega, and Tripure kits. The results showed that all All the kits were able to produce high concetration of RNA, where the Ribospin (Geneall) performed the best (both for the concentration and purity). Meanwhile Tripure kit were only able to produced highest concentration of RNA, but lowest purity.


Introduction
In order to develop new varieties of maize through conventional breeding, pedigree selection of F2 population is needed to produce fixed lines.This required several generations to complete and involved recurrent selection to complete pedigree methods.This process is time-consuming and needs a large of land if a large number of the targeted locus was involved.Therefore, to enhance efficiency, haploid technology can be applied [1].Using the non-traditional breeding technique to shorten the time needed is important to obtain pure lines faster so it will benefit both breeders and farmers.
Both in-vitro and in-vito technology could be used in order to obtain pure lines in one generation.Therefore, an in-vivo technique required haploid inducer lines to produce haploid plants.High capacityinducing haploid inducer rates are still unavailable in tropical maize [2].This became one of the main reasons why the adoption of haploid breeding for tropical maize is needed for temperate maize compared to tropical maize [3].[4] also reported that most of the double haploid plants derived from an in-vivo technique induced by chemical agents like colchicine being infertile, so further self-pollination will be impossible.Therefore, an in-vitro technique development in haploid breeding of maize is important to accelerate this breeding program.
Anther culture technique is one of the in-vitro haploid breeding and it involves several steps to complete including inducing a callus from immature pollen and applying a chemical agent to double the haploid chromosomes in order to produce double haploid regenerants.Moreover, the main key factors in the success of anther culture were still genotype-dependent.According to [5] most commercial varieties were recalcitrant, which became the main obstacle to adopting this technique.However, the effectiveness of this anther culture method is affected by the maize genotype.Local varieties are an important source of unique traits including culturability.Certain maize germplasm might contain useful traits such as non-recalcitrancy traits which will boost the usefulness of the haploid breeding technique by providing parental lines as well as the source of information related to the recalcitrancy mechanism.A molecular approach could be used to study and overcome the recalcitrancy problems in maize anther culture by finding responsible genes and expressions.Next-generation sequencing can be applied to obtained transcriptomic data from different growth stages and tissue or cells [6].The wide range of NGS applications is very useful and became a tool for genetic researchers to characterize and identify the association between agronomic traits with agronomic traits.Related to the NGS, RNA sequencing (RNA-seq) benefits the abilities of high-throughput sequencing to provide some comprehension into the transcriptome of targeted cells [7].RNA-Seq was also available in order to study certain aspects of biological RNA, such as translation, RNA structure, and also expressions from single-cell expression [8].
In order to enhance the success of the sequencing, obtaining a high quantity and quality of RNA product is needed since it will affect the data.[9] reports that isolating RNA for gene expression analysis was a practical difference in the commercial kits available in the market.Furthermore, [10] also reported that isolating miRNA using different methods of isolation techniques will produce different results, so in order to enhance reproducibility, robust RNA isolation methods were needed.This study aimed to determine the best RNA extraction methods using three different kits of maize explant for anther culture with high purity and concentrations.RNA extraction in this study was performed using Tripure, RibospinTM, Promega, and Tripure kits.

Materials
The materials for this analysis are 2 maize genotypes: Madura-3, a commercial hybrid variety, and Tambin, a local variety.All the genotypes tested were cultivated in a seedbed (70 x 20 cm) in a field test of Agroecotechnology, Faculty of Agriculture of the University of Trunojoyo Madura, East Java Indonesia.Nutrients were applied to the plants at three points: 7 days after planting (DAP), 25 DAP, and also 40 DAP.The applied fertilizer consists of Urea, SP36 and KCL which doses was 300 Kg ha -1 , 200 Kg ha -1 , and 100 Kg ha -1 respectively.

Preparations of the samples
Same with the previous reports [11], the uninucleate stage of microspore were used and taken from tassel at young ages in this research.Based on [12] the criteria to obtain an uninucleate microspore was when the plants had eight leaves.Moreover, to validate the microspore stages we used an aceto orcein staining to visualize the chromosome of the microspore using a microscope.After the criteria validated, we harvested the tassel and swill out in running water.The tassel was wrapped in tissue and spray with steril aquades to maintain humidity and also covered with plastic.Hereafter, the tassel was subjected to cold pre-treatment for 7 days in a cold room at 5C to reprogram microspore development from gametophytic to sporophytic.After the cold pre-treatment, spikelets that met the criteria were taken and placed in tube and incubated at -80 °C.The isolation needs 50 mg of fresh anther.

Methods
Three kits for RNA extraction were used in this study including RibospinTM plant (Geneall), Tripure TM Isolation Reagen (Roche), and SV Total RNA Isolation System (Promega).All the steps of each extraction kit were followed without any modification, and the extraction was done for three replicates for each plant.For evading the effect of the environment, anther sample was taken on the same day for both local and commercial hybrids maize.

Total RNA quantity and quality measurement
The qualities total RNA (ng.μL-1) from three kits were investigated in agarose gel electrophoresis and visualized under UV light.The quantity, concentration, and purity, were measured by nanodrop ND 1000 spectrophotometer (NanoDrop Technologies, USA) at A260:A280 and also A260:230 ratio values using a nanodrop ND 1000 spectrophotometer (NanoDrop Technologies, USA).The data was process manually using Microsoft Excel to provide the mean value as well as the standard deviation.

Results and discussion
This study used three RNA kits, namely RibospinTM plant (Geneall), SV Total RNA Isolation System (Promega), silica column-based extraction kit, and Tripure TM Isolation Reagen (Roche) a phenol and guanidine thiocyanate-based extraction.Based on table 1, two genotype samples tested in this study produced different RNA concentrations.The Tripure TM Isolation Reagen showed the highest concentration of RNA extracted than two other kits for both local varieties and Madura 3. The Ribospin kit produced low concentrations in two genotypes.[13] also reported similar findings, where compared to other kits, the Ribospin kit produced the lowest concentration of RNA in different tissues of oil palm.Meanwhile, our study found that the concentration of local varieties RNA ranges between 181.83 ±30.71 ng μl -1 to 1082.5±502. 3ng μl -1 .The highest concentration of local maize varieties was produced by the Tripure kit and the lowest by the Promega kit.A slightly different result for Madura 3, the highest concentration was generated by Tripure Kit, but the lowest concentration was produced by Ribospin Kit around 179.00 ±25.07 ng/μL.The Promega kit generated a high concentration of RNA (around 636.27±277.57ng μl -1 ) compared to Ribospin (only 179.00 ± 25.07 ng μl -1 ) in Madura 3.

Table 1. Purity and quantity of RNA of two maize genotypes using three different extraction kits
Not only RNA concentration but also RNA purity is important parameter, which indicates that the RNA can be used for further application.The purity level was measured using a spectrophotometer nanodrop with a ratio of absorbance at 260 nm and 280 nm.RNA purity was around 2.0 at A260/A280 ratio [14].The kits showed 1.80 -2.19 the ratio A260/A280.It showed that the two kits generated a good ratio of A260/A280, except for Tripure (table 1).Both in hybrid and local maize varieties, Tripure showed not only the lowest at A260/280 but also in A260/230 ratio (table 1).It indicated the presence of a high degree of contaminants that absorb at 280 nm.Trizol, phenol, Guanidine HCL, and guanidine are unwanted organic compounds that can cause a low A260/230 ratio [15].
Compared to other kits, in Madura 3 Promega performed best.It can be seen from the concentration parameter, which produced high concentration as well as good purity (2.05 for A260/280 and 1.95 for A260/230) (table 1).On the other hand, even though Ribospin produced the lowest concentrations in local maize, the purity of RNA in this kit was good (2.19 for A260/280 and 1.95 for A260/230).These findings were similar to [12]

Conclusion
All the kits were able to produce high concetration of RNA, where the Ribospin (Geneall) performed the best (both for the concentration and purity).Meanwhile Tripure kit were only able to produced highest concentration of RNA, but lowest purity.Considering concentration of RNA was not the only parameters, Ribospin kit were the best among others in extracting RNA from maize anther.

Figure 1 .
Figure 1.Visualization of RNA using gel electrophoresis where in various tissues of oil palm Ribospin produced 4quite high concentratios (>100 ng/μL) as well as the purity of RNA from 1.99-2.19.This was based on the manual of the Ribospin kit.