Genomic analysis of chloroplast matK and rbcL gene from Flacourtia inermis Roxb for plant DNA barcoding

Identification of medicinally significant plant species is helpful for their utilization as medicine. Flacourtia inermis Roxb are important medicinal plant. Aim: To Genotypic characterization of Flacourtia inermis through DNA sequence of matK and rbcL gene. Flacourtia inermis Roxb DNA was isolated using NucleoSpin® Plant II Kit (Macherey-Nagel gene extraction) Kit. Isolated DNA was amplified by using PCR (matK and rbcL gene primers). Amplified matK and rbcL gene was sequenced by ABI 3500 DNA analyzer and the sequence quality was checked using sequence scanner software v1. The results exposed basic variability of the sequences. The matK and rcbL gene phylogenic tree was constructed using MEGA 6 software. Flacourtia inermis Roxb DNA was successfully isolated using NucleoSpin® Plant II Kit (MachereyNagel gene extraction) Kit and confirmed by agarose gel electrophoresis. Isolated matK and rbcL genomic DNA was successfully amplified by using PCR. From the matK sequence, BLASTn analysis result Flacourtia inermis species showed 99.20% relationship with Flacourtia jangomas and rbcL BLASTn analysis result of Flacourtia inermis species showed100% relationship with Flacourtia jangomas at NCBI database. From this study, the result concluded the matK and rcbL genomes can be used for DNA identification of Flacourtia inermis. The information obtained from the study would be helpful for further research involving genotypic identification of Flacourtia sp.


Introduction
Flacourtia inermis plant belonging to family Flacourtiaceae, is a autotrophic plant found in South India, and also found in Malaysia, Indonesia, the Philippines etc. [1] The most important task for a botanist is the identification of correct plant species. [2] DNA bar-coding is the standard method for species identification and differentiation. DNA bar-coding technique growing rapidly, and functional tool for species diversity examination and monitoring of the genomic evolution. [3] Flacourtia inermis Roxb is a medium size tree that grow up to 15m height. Genomic evaluation, a short genome sequence is used as a molecular marker for identifying the diversity that present among the plants. Plant genomic analysis sequence locus of nuclear ribosomal cistron internal transcribed spacer (ITS) regions is most commonly used. Several genes of mitochondria, chloroplast and nucleus genes have been used for evaluation sequence differences at genomic level. [4] Phylogenetic evaluation is emerging as a capable tool for exploring correlations between the phylogenetic diversity and useful attributes of medicinal species. [5] Among all the genes matK and rbcL gene sequence are used for the study of plant genomic evaluation. [6,7] The development of DNA barcoding method to recognize these herbs is critically needed for the safety of patients. [8] Maine objective of work genotypic characterization of Flacourtia inermis through DNA sequence of matK and rbcL gene.

DNA isolation using NucleoSpin ® Plant II Kit (Macherey-Nagel)
About 100 mg of the tissue is homogenized using liquid nitrogen and the powdered tissue is transferred to a microcentrifuge tube. Four hundred microlitres of buffer PL1 is added and vortexed for 1 minute. Ten microlitres of RNase A solution is added and inverted to mix. The homogenate is incubated at 65 o C for 10 minutes. The lysate is transferred to a Nucleospin filter and centrifuged at 11000 x g for 2 minutes. The flow through liquid is collected and the filter is discarded. Four hundred and fifty microlitres of buffer PC is added and mixed well. The solution is transferred to a Nucleospin Plant II column, centrifuged for 1 minute and the flow through liquid is discarded. Four hundred microlitre buffer PW1 is added to the column, centrifuged at 11000 x g for 1 minute and flow though liquid is discarded. Then 700 µl PW2 is added, centrifuged at 11000 x g and flow through liquid is discarded. Finally 200 µl of PW2 is added and centrifuged at 11000 x g for 2 minutes to dry the silica membrane. The column is transferred to a new 1.7 ml tube and 50 µl of buffer PE is added and incubated at 65 o C for 5 minutes. The column is then centrifuged at 11000 x g for 1 minute to elute the DNA. The eluted DNA was stored at 4 o C.

Agarose Gel Electrophoresis for DNA Quality check
The quality of the DNA isolated was checked using agarose gel electrophoresis. 1µl of 6X gel-loading buffer (0.25% bromophenol blue, 30% sucrose in TE buffer pH-8.0) was added to 5µl of DNA. The samples were loaded to 0.8% agarose gel prepared in 0.5X TBE (Tris-Borate-EDTA) buffer containing 0.5 µg/ml ethidium bromide. Electrophoresis was performed with 0.5X TBE as electrophoresis buffer at 75 V until bromophenol dye front migrated to the bottom of the gel. The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad).

Agarose Gel electrophoresis of PCR products
The PCR products were checked in 1.2% agarose gels prepared in 0.5X TBE buffer containing 0.5 µg/ml ethidium bromide. 1 µl of 6X loading dye was mixed with 5 µl of PCR products was loaded and electrophoresis was performed at 75V power supply with 0.5X TBE as electrophoresis buffer for about 1-2 hours, until the bromophenol blue front had migrated to almost the bottom of the gel. The molecular standard used was 2-log DNA ladder (NEB). The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad).

ExoSAP-IT Treatment
ExoSAP-IT (GE Healthcare) consists of two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase (SAP), in a specially formulated buffer for the removal of unwanted primers and dNTPs from a PCR product mixture with no interference in downstream applications. Five micro litres of PCR product is mixed with 2 µl of ExoSAP-IT and incubated at 37oC for 15 minutes followed by enzyme inactivation at 80oC for 15 minutes.

Sequencing using BigDye Terminator v3.1
Sequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems , USA) following manufacture protocol.

Post Sequencing PCR Clean up
Make master mix-I of 10µl milli Q and 2 µl 125mM EDTA per reaction. Add 12µl of master mix I to each reaction containing 10µl of reaction contents and mix properly. Make master mix II of 2 µl of 3M sodium acetate pH 4.6 and 50 µl of ethanol per reaction. Add 52 µl of master mix II to each reaction. Contents are mixed by inverting. Incubate at room temperature for 30 minutes. Spin at 14,000 rpm for 30 minutes and decant the supernatant and add 100 µl of 70% ethanol. Spin at 14,000 rpm for 20 minutes and decant the supernatant and repeat 70% ethanol wash. Finally decant the supernatant and air dry the pellet. The cleaned up air dried product was sequenced in ABI 3500 DNA Analyzer (Applied Biosystems).

Sequence Analysis
The sequence quality was checked using sequence scanner software v1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.1.

Phylogenetic analysis
The basic sequence analysis including nucleotide frequencies, transition/ transversion ratio and variability in different regions of sequences were computed by Molecular Evolutionary Genetics Analysis by using MEGA. [9,10].

DNA isolation and Identification
Flacourtia inermis DNA was isolated using NucleoSpin ® Plant II Kit. The Isolated DNA was checked using agarose gel electrophoresis. The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad). (Figure 1).

matK and rbcL gene
From the Isolated genomic DNA, matK and rbcL gene amplified by using primers 390f forward, 1326r reverse and rbcLa forward, rbcL724 reverse. Amplified matK and rbcL gene were confirmed by agarose gel electrophoresis. The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad). (Figure 2). matK gene 1KB rbcL Gene 800bp

Sequences
The amplified matK and rbcL gene sequence quality was checked using sequence scanner software v1 (Applied Biosystems) and sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.1

Conclusion
In this work, genomes of Flacourtia inermis species were sequenced and evaluated. Two variant regions were screened and were formed to be potential DNA barcodes for the identification of Flacourtia inermis. The results exposed basic variability of the sequences. The matK and rcbL gene phylogenic tree was constructed using MEGA 6 software. Flacourtia inermis species show 99.20% relationship with Flacourtia jangomas. The phylogenic trees indicate that the matK and rcbL genomes can be used for the identification of Flacourtia inermis. The information obtained in this work would be a useful source for further research involving the identification and phylogenetic analysis of Flacourtia sp (Flacourtiaceae).