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.

Hevea brasiliensis coniferaldehyde-5-hydroxylase (HbCAld5H) regulates xylogenesis, structure and lignin chemistry of xylem cell wall in Nicotiana tabacum.

KEY MESSAGE: The HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl-guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants. Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.

Heterografting induced DNA methylation polymorphisms in Hevea brasiliensis.

MAIN CONCLUSION: Heterografting induced intraclonal epigenetic variations were detected among rubber plants. Interaction between genetically divergent root stock and scion tissues might have triggered these epigenetic changes which may eventually lead to intraclonal variability in rubber. DNA methylation in response to stress may be associated with the alteration in gene transcription leading to morphological changes in plants. Rubber tree is commercially propagated by bud grafting where the scion of a high yielding variety is grafted on to a genetically divergent root stock. Still, significant levels of intraclonal variations exist among them. Epigenetic changes associated with heterografting may be partly responsible for this conundrum. In the present study, an attempt was made to identify the impact of divergent root stock on the epigenome of scion in grafted rubber plants. Heterografts were developed by grafting eye buds from a single polyembryony derived seedling on to genetically divergent root stocks of unknown parentage. The plants were uniformly maintained and their DNA was subjected to MSAP analysis. Polymorphic DNA methylation bands corresponding to CG as well as the plant-specific CHG types of methylation were observed. Cloning of selected polymorphic regions and bisulfite sequencing confirmed the presence of methylation in the promoter and coding region of important genes including an LRR receptor kinase gene. Since divergent root stock is the major factor differentiating the grafted plants, the changes in DNA methylation patterns might have been triggered by the interaction between the two genetically different tissues of stock and scion. The study assumes importance in Hevea, because accumulation and maintenance of epigenetic changes in functional genes and promoters during subsequent cycles of vegetative propagation may contribute towards intraclonal variability eventually leading to altered phenotypes.

Analysis of Genetic Diversity and Resistance to Foliar Pathogens Based on Genotyping-by-Sequencing of a Para Rubber Diversity Panel and Progeny of an Interspecific Cross.

Para rubber trees (Hevea brasiliensis) are the largest major source of natural rubber in the world. Its major pathogens are Phytophthora spp., Corynespora cassiicola, and Colletotrichum spp. A rubber diversity panel of 116 clones using over 12,000 single nucleotide polymorphisms (SNPs) from DArTSeq genotyping revealed clear phylogenetic differences in clones that originated from different geographical regions of the world. An integrated linkage map constructed with an F1 progeny of 86 from an interspecific cross between H. brasiliensis and H. benthamiana using 23,978 markers [10,323 SNPs and 13,655 SilicoDArTs] spanned 3947.83 cM with 0.83 cM average marker-interval. The genome scaffolds that were anchored to the linkage map, covering 1.44 Gb of H. brasiliensis reference genome, revealed a high level of collinearity between the genetic map and reference genome. Association analysis identified 12 SNPs significantly associated with the resistance against Phytophthora, Corynespora, and Colletotrichum in six linkage groups: 2, 6, 12, 14, 17, and 18. Kompetitive Allele-Specific PCR marker assays were developed for those 12 SNPs, screened with 178 individuals, and detected clear separation between two genotypes. Within the proximity to those SNPs, 41 potentially key genes that have previously been reported to associate with plant disease resistance were predicted with high confidence.

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.

Genetic and epigenetic uniformity of polyembryony derived multiple seedlings of Hevea brasiliensis.

Hevea brasiliensis Muell. Arg (Para rubber tree) is a tropical tree species of Amazonian origin widely cultivated in several parts of the world for natural rubber, a highly priced commodity inevitable for the world rubber industry. Large, tree to tree variation in growth and latex yield among individual plants of high yielding Hevea clones is a common phenomenon observed in mature rubber plantations. The genetic heterogeneity of the seedlings which are used as rootstocks for propagation through budgrafting is considered as a major factor responsible for this variation. In order to minimize this variation, attempts were made to develop highly uniform rootstock material via an in vitro technique by inducing zygotic polyembryony in Hevea. Immature open pollinated fruits of a high yielding clone RRII 105 were cultured by half ovulo embryo culture technique. Multiple embryos were induced from the 8-10-week-old zygote with a novel combination of gibberellic acid (GA3), kinetin, and zeatin. Plantlets were successfully generated from the multiple embryos and raised in the field post hardening. Screening using genetic and epigenetic molecular markers revealed that the multiple seedlings developed are highly uniform and are of single zygotic origin. Development of plants having genetic and epigenetic uniformity suggests that this technique is ideal for raising uniform rootstock material in Hevea which may significantly reduce intraclonal variations. Moreover, these plants could serve as ideal material for physiological and molecular investigations towards the understanding of stock-scion interaction process 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.