Estimation of Heterografting Associated DNA Methylation Changes in Tree Crops by MSAP Analysis.

The genetic incompatibility of the seedlings which are used as rootstocks (stock-scion interactions) and the mechanical stress induced by grafting are two major factors responsible for the high intraclonal variations observed in tree crops which are propagated through bud grafting. Since stress-induced DNA methylation changes associated with heterografting is a major contributor of such variations in grafted tree crops, a proper assessment of this epigenetic phenomenon is inevitable to devise strategies for the development of more uniform planting materials with minimal intraclonal variations in the future. In order to evaluate and establish the effects of heterografting on the epigenome of plants, availability of ideal plant materials and a standard procedure for testing is very essential. Development of genetically uniform own-rooted seedlings through induction of cleavage polyembryony by a novel technique of half ovulo embryo culture is the first step. Grafting of buds from these genetically and epigenetically uniform plants to genetically divergent rootstock and identification of DNA methylation polymorphism among them forms the second part of the methodology for detecting epigenetic changes associated with grafting in tree crops. Methylation-sensitive amplification polymorphism technique (MSAP), a modified version of AFLP using a pair of methylation-sensitive and insensitive isoschizomers (such as HpaII and MspI), is an ideal methodology to assess DNA methylation polymorphisms on a genomic scale in such plants. Comparative analysis of two sets of restriction digestion products (EcoRI/HpaII and EcoRI/MspI) allows the identification of DNA methylation polymorphisms induced by grafting and will aid in the detection of differentially methylated regions (DMRs) among grafted plants. This chapter describes a detailed protocol for inducing multiple embryos of single zygotic origin and regeneration of seedlings in rubber tree (Hevea brasiliensis), grafting of buds from these genetically uniform own-rooted seedlings to divergent rootstocks, identification of epigenetic changes induced by grafting or stock-scion interactions through MSAP analysis, and locating the differentially methylated genomic region. The methodology described here could be applied to any tree species commercially propagated through grafting for detecting epigenetic changes putatively associated with intraclonal variability.

Development of Novel Markers for Yield in Hevea brasiliensis Muell. Arg. Based on Candidate Genes from Biosynthetic Pathways Associated with Latex Production.

Scarcity of functional genetic markers associated with candidate genes (CGs) is a serious constraint for marker-assisted selection in the natural rubber producing tree, Hevea brasiliensis. In order to develop markers associated with rubber yield, five CGs involved in latex biosynthesis were characterized from 16 popular Hevea varieties. Novel SNPs and indels were identified and developed into markers using simple genotyping techniques like allele-specific PCR, CAPS, etc. A progeny population was genotyped using these markers to validate them, to understand their segregation pattern and to map them to a genetic linkage map. Parent-specific maps were constructed using pseudo-test cross strategy with the help of additional markers. The sequence structure information generated will be useful for future studies on gene mapping, functional relevance of coding SNPs and evolution of rubber biosynthesis genes in Hevea. Concurrently, the markers developed may serve as powerful tools for yield-based selection and for genetic diversity and pedigree studies in Hevea. Above all, the marker assays designed for genotyping could be economically carried out in any laboratory having basic molecular biology infrastructure and expertise.

Distinguishing CPT gene family members and vetting the sequence structure of a putative rubber synthesizing variant in Hevea brasiliensis.

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. cis-PTs catalyze the cis-1,4-polymerization of isoprene units to generate isoprenoids with carbon skeletons varying from C10 (neryl pyrophosphate) to C > 10,000 (natural rubber). Though the previously reported CPTs in Hevea are designated based on sequence variations, their classification was done mostly by phylogenetic analysis using a mixture of partial as well as full length sequences often excluding the UTRs. In this context an attempt was made to reclassify the CPTs strictly based on their sequence similarity and distinguish the members putatively associated with rubber biosynthesis from the others. Extensive computational analysis was carried out on CPT sequences obtained from public resources and whole genome assemblies of Hevea. Based on the results from BLAST analysis, multiple sequence alignments of protein, nucleotide and untranslated regions, open reading frame analysis, gene prediction analysis and sequence length variations, we conclude that there exists mainly three CPTs namely RubCPT1, RubCPT2 and RubCPT3 putatively associated with rubber biosynthesis in Hevea brasiliensis. The rest were categorised as variants of dehydrodolichyl diphosphate synthase (DHDDS) involved in the synthesis of dolichols having short chain isoprenoids. Analysis of the sequence structure of the most highly expressed RubCPT1 in latex revealed the allele richness and diversity of this important variant prevailing in the popular rubber clones. Haplotypes consisting of SNPs with high degree of heterozygosity were also identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor towards the generation of allelic diversity rather than point mutations. Alternatively, gene expression analysis indicated the possibility of association between specific haplotypes and RubCPT1 expression in Hevea clones which may have downstream impact up to the level of rubber production. The conclusions from this study may pave way for the identification and better understanding of CPTs directly involved with natural rubber biosynthesis in Hevea and the SNP data generated may aid in the development of molecular markers putatively associated with yield in rubber.

Impact of an intragenic retrotransposon on the structural integrity and evolution of a major isoprenoid biosynthesis pathway gene in Hevea brasiliensis.

Isoprenoids belong to a large family of structurally and functionally different natural compounds found universally from prokaryotes to higher animals and plants. In Hevea brasiliensis, the commercially important cis-polyisoprene (rubber) is synthesised as part of its defence mechanism in addition to other common isoprenoids like phytosterols, growth hormones etc. Farnesyl diphosphate synthase (FDPS) is a key enzyme in this process which catalyses the conversion of isoprene units into polyisoprene. Although prior sequence information is available, the structural variants of the FDPS gene presently existing in Hevea population are largely unknown. Since gene structure has a major role in gene regulation, extensive sequence analysis of this gene from different genotypes was carried out to identify the prevailing structural variants. We identified several SNPs and large indels which were associated with a partial transposable element (TE). Modification of key regulatory motifs and splice sites induced by the retroelement was also identified in the first intron. Screening of popular rubber clones, wild germplasm accessions and Hevea species revealed that the retroelement is responsible for the generation of new alleles with varying degrees of sequence homology. Segregation analysis of a progeny population confirmed that the alleles are not paralogs and are inherited in a Mendelian mode. Our findings suggest that the first intron of the FDPS gene has been subjected to various chromosomal rearrangements due to the interaction of a retrotransposon, resulting in novel alleles which may substantially contribute towards the evolution of this major gene in rubber. Moreover, the results indicate the possible existence of a retrotransposon-mediated epigenetic gene regulatory mechanism in Hevea.