Flow cytometric estimation of nuclear DNA content for the subfamily Zingiberoideae optimized in a modified nuclear isolation buffer. A phylogenetic tree was constructed based on interspecies variation in nuclear DNA content.
SYNOPSIS
Background
While the number of species cultivated for human survival is so small, the high level of intra-species, inter-species and inter-generic diversity in agricultural plants and their wild relatives leads to the development of plant genetic resources (PGR). Plant genetic diversity is susceptible to "genetic erosion", the loss of individual alleles/genes and combinations of alleles/genes, such as those found in adapted landraces.
Zingiberaceae
The unique wealth of biological resources in NE makes it a potential hub for economic growth of the country to a competent level, if used and tapped efficiently. Based on molecular data, a reorganization of the genera of the Zingiberaceae into four subfamilies (Kress et al. 2002) is possible: the Siphonochiloideae (only the genus Siphonochilus), the Tamijioideae (the single genus Tamijia), the Alpinioideae (most of the former Alpinieae) and the Zingiberoideae (including the former tribes Hedychieae, Zingibereae and Globbeae) were done (Fig. 1.1). The subfamily Zingiberoideae consists of medicinally important genera of Boesenbergia, Caulokaempferia, Cautleya, Curcuma, Curcumorpha, Haniffia, Hedychium, Kaempferia, Scaphochlamys and Stahlianthus.
Chromosome studies
Hedychium species are often cultivated for their fragrance and as a useful raw material for paper production. Taxonomic classification of Kaempferia is difficult due to the morphological similarity of vegetative parts between species of the same genus and other genera in Zingiberaceae, such as Boesenbergia, Cornukaempferia, Curcuma and Scaphochlamys, while taxonomic identification based on reproductive parts is limited due to the relatively short flowering period.
Genetic diversity
Many of the sedum plants are found throughout northeastern India in the wild. Therefore, the study of genetic diversity using molecular markers would be useful for any kind of future analysis using members of the Zingiberaceae family.
Genome size
- Zingiberoideae
Current study aims to provide comprehensive information on the nuclear DNA content estimation of some of the members of Zingiberoideae plants. Chromosome number observations and karyo-morphometric analysis of selected species of the subfamily Zingiberoideae.
Ethnobotanical studies
Cytological studies
There is no literature available on chromosome number studies of the subfamily Zingiberoideae, mainly from Northeast India. The present study attempts to investigate the number of chromosomes in the germplasm of the subfamily Zingiberoideae.
Molecular techniques assisted diversity and phylogenetic studies
The use of the various reference standards for flow cytometric estimation of nuclear DNA content estimation has been given (Fig. 2.11). It is preferable to use colorless plant organs rather than those stained by anthrocyan (a fluorescence inhibitor) (Greilhuber et al. 2007). The suitability of seeds as a material for flow cytometric estimation has been investigated (Sliwinska et al. 2005).
Nuclear DNA content across Zingiberoideae
Despite several studies, there is a lack of information on the action of the color inhibitors and no universal method to completely avoid their effects on DNA content estimation (Greilhuber et al 2007). They reasoned that the variation in nuclear DNA content is not caused by aneuploidy, but by heterochromatin variability. The chapter also determines the chromosome number determination of some of the members of the subfamily.
Despite the economic advantage of the family, several orders of polyploidization (2x-15x) and hybridization have created phylogenetic and taxonomic confusion (Skornickova et al 2007; Zaveska et al 2012) opening options for further work. Ethnobotany is the study of the different uses of plants in the daily life of the ethnic communities (Srivastava et al 2010).
Methods
A botanical specimen consists of the entire plant, complete with roots, stem, leaves, flowers and, if possible, fruits. Most plants wilt very quickly after being cut or dug from the ground. Washing the root tips with distilled water followed by soaking in filter paper was done to remove the traces of water.
The root tips were pre-treated in a saturated solution of para dichlorobenzene (PDB) at room temperature for 3 hours. The material was reheated over a lamp to increase the intensity of the staining.
Result and discussion .1Collection of germplasm
Most plant species grow naturally in different regions and their properties are important in traditional herbal medicine. Rhizomes were used in most cases, followed by fruits, leaves, shoots and flowers. Chromosome numbers of 9 species of the subfamily Zingiberoideae were investigated by the conventional rapid squash technique.
It may be noted that species of Kaempferia, from morphological considerations, fall under apparently two types, such as those showing morphological specialties characterized by the two lateral petaloid staminodes free from the deeply bilobed labellum, and others with fused labellum and staminodes ( Omanakumari & Mathew 1984). The former morphologically less advanced species are Asian, and the latter advanced ones from Africa (Mahanty 1970). The common feature noted in the subfamily Zingiberoideae was the wide variety of chromosomes with very small sizes in most of the species.
Conclusion
From the cytological studies of the 9 investigated Zingiberoideae germplasm, it can be concluded that speciation and evolution has been possible as a result of increased variability through changes in base numbers, as well as numerical and structural changes in the number of chromosomes. The diverse cellular phenomenon, for example, autopolyploids, allopolyploids, protoautoploidy, amphiploidy, ascending and descending dysploidy may have resulted in the variability of basic numbers in the family. The wide range of chromosome numbers observed in many genera in the present study marks an important contribution that aneuploidy and polyploidy have played in the evolution of various taxa at the generic and species level.
It also turned out that different types of abnormalities have played a key role in the evolutionary diversification of the family. This chapter discusses the genetic diversity status of the turmeric varieties of different states of Northeast India.
CHAPTER 4A
PCR amplification of the genomic DNA was performed using 20 RAPD and 20 ISSR (Operon Tech, USA) primers to study the intra-variety and inter-variety genetic relationship (Table 4A.2). The annealing temperature for 1 min ranged from 38 - 50 oC for ISSR primer, depending on the melting temperature of the primer (Table 4A.2). AMOVA (P < 0.001) of RAPD data showed that 25% of the total genetic variation could account for the differences between the turmeric varieties from 4 different states of NE India.
Nei's unbiased measures of genetic identity and genetic distance between cultivars were also calculated (Table 4A.11). Accurate information on genetic variability among turmeric cultivars in NE India is important to establish basic germplasm collections and assist in breeding.
CHAPTER 4B
Detailed analysis of RAPD, ISSR and AFLP marker data is mentioned in section 4a.2.4. Subgroup II of group II consisted of the single species (H. . flavescens) with a similarity coefficient of 0.551 and 0.614 with H. The highest cumulative contribution of the first three principal components to the total variation was found by RAPD-ISSR markers compared to their individual use.
The correlation coefficient of the similarity matrix was higher in the RAPD-ISSR dendrogram (r = 0.48), followed by ISSR-AFLP (r = 0.06) and the RAPD-AFLP (r = -0.26) dendrogram, respectively. The strong aroma of the flowers may be an evolutionarily derived trait of the species.
CHAPTER 4C
Conclusion
Therefore, the combination of the RAPD-ISSR marker reflected a reliable method for calculating genetic relationships as it reflects coding and non-coding regions of the genome and could be well used in aiding identification and classification of the Zingiberaceae using more species in the genus. Further research into single nucleotide polymorphism (SNP), together with gene content, morphological data and chromosome studies from more species, should reduce any confusion in species identification and help gain a better understanding of evolutionary phylogeny, especially when combined with a survey to genes. specific analysis.
CHAPTER 4D
The detailed data analysis of the RAPD and ISSR markers has been discussed in Section 4A.2.4. The correlation coefficient of the similarity matrix derived from RAPD (0.88), ISSR (0.85) and RAPD+ISSR (0.85) data was calculated to detail the correlation between both types of molecular markers. So the phylogenetic interpretation of the Zingiberaceae family has been done only by RAPD markers.
Subgroup I of cluster II consisted of species belonging exclusively to the genus Curcuma, subgroup II consisted of the species of Kaempferia and Zingiber. The results of the cluster analysis were strongly supported by principal coordinate analysis (Fig. 4D.1).
CHAPTER 5A
The youngest fully developed leaf sheath was used to prepare the suspension of intact kernels. Subsequently, the use of 100 micron and 30 micron filter mesh resulted in a significant decrease in cell number in bright field microscopic analysis (Figure 5A.2 C, F &J). Decreased cell cluster intensity was also seen in the scatter plot of the assay (Fig 5A.2 G. & K).
A and D are the bright field microscopy image of the nuclear suspension prepared in hypotonic PI of Z. B and E are the fluorescence microscopy image of the nuclear suspension prepared in hypotonic PI of Z.
CHAPTER 5B
The variation of nuclear DNA content with tissue types (mitotically active cells, non-dividing cells along with flowers) of Zingiberaceae has not been documented. Rhizome showed a different order of nuclear DNA content than that of other parts of the plants. The vegetative parts (leaf, root, rhizome) resulted in significant variation in nuclear DNA content (P = 0.49 > 0.05).
It can be said that the content of nuclear DNA in all the studied tissue types of Zingiberaceous species was not uniform. This chapter provides information on the variation in nuclear DNA content of cultivated varieties of turmeric.
CHAPTER 5C
A variation in nuclear DNA content of 15.1% has already been documented in Indian turmeric samples (Skornickova et al 2007). Eleven groups of turmeric cultivars with significant differences in nuclear DNA content were identified against O. The highest nuclear DNA content of Arunachal turmeric cultivars (6) was found to be 2.83 pg (9x) and the lowest nuclear DNA content (9) was the same was found.
Fold changes in nuclear DNA content were found to be minimal for varieties occurring in Manipur. This is the first study on the nuclear DNA content of northeast Indian saffron varieties.
CHAPTER 5D
