Objective This study aimed to determine the best protein extraction method of Metroxylon sagu for the two-dimensional gel electrophoresis and the comparative analysis. Results To perform good proteome research, the most critical step is to establish a method that gives the best quality of extracted total proteins. To develop an optimized protein extraction protocol for two-dimensional polyacrylamide gel electrophoresis (2-DE) analysis of Metroxylon sagu, five protein extraction protocols were compared; polyethene glycol (PEG) fractionation method, SDS/phenol method, TCA/acetone method, combination SDS/phenol and TCA/acetone and imidazole method. The PEG fractionation method was found to give the most reproducible gels with the highest number of spots and highest protein concentration followed by SDS/Phenol method. The lowest number of spots were observed in Imidazole method. The PEG fractionation method provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethene glycol (PEG) fractionation provides clearer detection of low-abundance protein. Hence the result from this study propose PEG fractionation as the effective protein extraction method for 2-DE proteomic studies of Metroxylon sagu.
Climatic changes affect various organisms, including plant species, becoming unfavorable for the environment and socioeconomic value, prompting an increase in activities related to plant resources conservation. The successful conservation of plant genetic resources depends on the proper identification and characterization of plant material. One of the notable developments in genetic conservation is the use of molecular markers for assessing the conservation and use of plant genetic resources. This review is devoted to the use of molecular marker techniques for genetic assessment of plant genetic variations. Development in these techniques provides smooth, reliable, and effortless ways for assessing known and unknown taxa, between and within species. These techniques provide a revelation to researchers on taxonomical and evolutionary questions which were not possible earlier. The polymerase chain reaction-based molecular markers give rise to various novel techniques due to the simplicity and high reproducibility of the methods. Examples including a combination of earlier techniques, such as restriction fragment length polymorphism, random amplification of polymorphic DNA, simple sequence repeat, intersimple sequence repeats, single nucleotide polymorphism, and amplified fragment length polymorphism, have been used for plant genetic variations and polymorphism studies. Progress in the advanced high-throughput sequencing techniques or next-generation sequencing technologies has been rapidly utilized to study genetic diversity broadly and to identify suitable genes and alleles rapidly. These techniques offer a practical resolution to the challenges in crop genomics. This review explains the recent advances in the molecular marker techniques, along with the advantages, uses, and limitations. Each technique differs in resolving the genetic variations and polymorphism in plant species.
Background Sago palm (Metroxylon sagu) is an important staple crop in the rural area of Sarawak, Malaysia. The palms grow well in the rough, swampy peat, from low flooded areas to uplands and acidic to neutral soils. Among the starch-producing crops, this palm is the most productive and promising, where it can store a significant amount of carbohydrate in the trunk. Due to the lack of molecular study of sago palm, the work described here aimed to develop molecular markers for identifying M. sagu and accessing the genetic variations of sago palm in different locations of Sarawak. Results Amplified fragment length polymorphism was employed to determine the genetic variations of sago palm between fifteen populations collected from three different locations: Mukah, Pusa and Samarahan. Based on 35,016 polymorphic fragments generated from 32 primer combinations, polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.269, 0.005157 and 51.52039 per primer combination, respectively, were obtained, signifying the effectiveness and accuracy of the AFLP marker. Mukah showed higher diversity (I = 0.428, H = 0.232), while Pusa showed the lowest (I = 0.367, H = 0.240). The average value of the Shannon information index was 0.404. AMOVA showed 99% of the variation was found within the population. The Mantel test was performed with 9999 permutations. A significantly positive correlation (r = 0.220, p = 0.010) was observed between the genetic divergence of the population (Nei genetic distance) with the geographical distance. Conclusions Our study considered AFLP is as an effective and reliable method to assess the genetic variations and the identification of M. sagu. Graphical Abstract
Background: Sago palm (Metroxylon sagu) is a versatile crop and has been hailed as the next viable commodity in Sarawak, Malaysia. Sago palm can thrive in the harsh swampy peat, from low flooded areas to uplands and in acidic to neutral soils. Sago palm has been neglected and unfortunately, very little is known about the sago palm proteome. This study aimed to determine the best protein extraction method of Metroxylon sagu for the two-dimensional gel electrophoresis (2-DE) and its comparative analysis. Results: To perform good proteome research, the most critical step is to establish a method that gives the best quality of extracted total proteins. Five different protein extraction protocols: polyethylene glycol (PEG) fractionation method, phenol extraction method, TCA-acetone method, the combination of phenol and TCA-acetone extraction method and imidazole method were compared to develop an optimized protein extraction method for two-dimensional gel electrophoresis analysis of Metroxylon sagu. The PEG fractionation method was found to give the most reproducible gels with the highest number of spots and highest protein concentration followed by phenol extraction method. The lowest number of spots was observed in the imidazole method. The PEG fractionation method provides improved resolution and reproducibility of 2-DE and reduces the time required to analyze samples. Partitioning Rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low abundance protein. Hence, the results from this study propose PEG fractionation as the effective protein extraction method for 2-DE proteomic studies of Metroxylon sagu. Conclusion: In this study, the PEG fractionation method is considered as the best extraction method for 2-DE proteomic studies of Metroxylon sagu in terms of yield, gel quality, spot numbers, and quantities of proteins.
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