Pratik Satya

Next generation sequencing technologies for next generation plant breeding

As a term, “next generation plant breeding” is increasingly becoming popular in crop breeding programmes, conferences, scientific fora and social media (Schnable, 2013). Being a frontier area of crop science and business, it is gaining considerable interest among scientific community and policymakers and funds flow from entrepreneurs and research funding agencies. Plant breeding is a continuous attempt to alter genetic architecture of crop plants for efficient utilization as food, fodder, fiber, fuel or other end uses. Although the scientific concepts in plant breeding originated about 100 years ago, domestication and selection of desirable plants from prehistoric periods have contributed tremendously to ensure human food security (Gepts, 2004). During the past few decades, well supported crop improvement programmes for major crops started reaping benefits from cutting edge technologies of biological sciences, particularly in the form of molecular markers and transgenic crop development, which in combination with conventional phenotype based selection, defines the current generation plant breeding practices. Different types of molecular markers have been developed and extensively used during the last three decades for identifying linkage between genes and markers, discovering quantitative trait loci (QTLs), pyramiding desired genes and performing marker assisted foreground and background selections for introgression of desired traits (Varshney and Tuberosa, 2007). However, these markers are based mostly on electrophoretic separation of DNA fragments, which limits detection of genetic polymorphism. In large plant breeding populations, genotyping may take up several months depending on marker system, adding more cost to genotyping. The next generation plant breeding would thus demand more efficient technologies to develop low cost, high-throughput genotyping for screening large populations within a smaller time frame.

Start codon targeted (SCoT) polymorphism reveals genetic diversity in wild and domesticated populations of ramie (Boehmeria nivea L. Gaudich.), a premium textile fiber producing species

Twenty-four start codon targeted (SCoT) markers were used to assess genetic diversity and population structure of indigenous, introduced and domesticated ramie (Boehmeria nivea L. Gaudich.). A total of 155 genotypes from five populations were investigated for SCoT polymorphism, which produced 136 amplicons with 87.5% polymorphism. Polymorphism information content and resolving power of the SCoT markers were 0.69 and 3.22, respectively. The Indian ramie populations exhibited high SCoT polymorphism (> 50%), high genetic differentiation (GST = 0.27) and moderate gene flow (Nm = 1.34). Analysis of molecular variance identified significant differences for genetic polymorphism among the populations explaining 13.1% of the total variation. The domesticated population exhibited higher genetic polymorphism and heterozygosity compared to natural populations. Cluster analysis supported population genetic analysis and suggested close association between introduced and domesticated genotypes. The present study shows effectiveness of employing SCoT markers in a cross pollinated heterozygous species like Boehmeria, and would be useful for further studies in population genetics, conservation genetics and cultivar improvement.

Genome synteny and evolution of AABB allotetraploids in Hibiscus section Furcaria revealed by interspecific hybridization, ISSR and SSR markers

Two tetraploid species of Hibiscus section Furcaria, H. acetosella and H. radiatus, have an AABB genomic constitution. The diploid species, H. cannabinus (AA) and H. surattensis (BB), were hybridized to develop interspecific alloploid (AB) hybrids. The synthetic interspecific hybrid exhibited intermediate morphological characters, with expression of domestication-related traits, but exhibited higher genomic association with the B genome donor. Evolution of allopolyploids in section Furcaria was found to be associated with mutations in repetitive sequences, leading to higher variation in the tetraploid genome. Allopolyploidization was observed to be associated with both loss of repetitive sequences and appearance of new alleles. Genetic diversity analysis using ISSR and cross-species SSR markers revealed a closer association of diploid genomes and high variability of tetraploid genomes. The evolution of AABB tetraploids in this section possibly took place by hybridization of the A and B genome in geographically isolated regions.

The draft genome of Corchorus olitorius cv. JRO-524 (Navin)

Here, we present the draft genome (377.3 Mbp) of Corchorus olitorious cv. JRO-524 (Navin), which is a leading dark jute variety developed from a cross between African (cv. Sudan Green) and indigenous (cv. JRO-632) types. We predicted from the draft genome a total of 57,087 protein-coding genes with annotated functions. We identified a large number of 1765 disease resistance-like and defense response genes in the jute genome. The annotated genes showed the highest sequence similarities with that of Theobroma cacao followed by Gossypium raimondii. Seven chromosome-scale genetically anchored pseudomolecules were constructed with a total size of 8.53 Mbp and used for synteny analyses with the cocoa and cotton genomes. Like other plant species, gypsy and copia retrotransposons were the most abundant classes of repeat elements in jute. The raw data of our study are available in SRA database of NCBI with accession number SRX1506532. The genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession LLWS00000000, and the version described in this paper will be the first version (LLWS01000000).

Jute Genomics : Emerging Resources and Tools for Molecular Breeding

Perhaps no other natural fibre crop is as versatile as jute ( Corchorus capsularis L. and C. olitorius L.; Malvaceae s. l.) in the industrial and engineering uses of textiles. Yet, genomics research was rather delayed in this typical lignocellulosic bast fibre crop, which has been characterized by slow responses to classical genetic selection due to several limitations, the most important being narrow genetic base, strong sexual incompatibility and dearth of compatible genetic and/or genomic resources. Recent depreciation of the genome sizes of Corchorus spp. concomitant with a drastic fall in the cost of next-generation sequencing has now made it possible to explore and characterize jute genomes at affordable prices. This has resulted in the development and validation of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers from transcriptomic unigenes, genome-wide discovery of single nucleotide polymorphisms (SNPs) using reduced-representation restriction-site-associated DNA (RAD) sequencing de novo, construction of a dense RAD-SNP-based genetic linkage map, detection of QTL and candidate gene analysis for bast fibre yield and its components, and generation and characterization of reference bast transcriptomes using next-generation RNA-seq. A structured association mapping panel has been developed for C. olitorius , and a large number of RAD-SNP markers are being used for genome-wide association mapping of complex bast fibre quality traits. Current results have further addressed some of the basic issues of jute genome biology, ranging from chromosomal evolution to comparative genomics to cellulose and lignin biosynthesis in bast fibres. Here, we have discussed the present status of jute genomics, with a historical perspective on DNA markers development and utilization. The potentials of genomic selection to accelerate the rate of genetic gain in selection for bast fibre quality traits and mutagenesis-based reverse genetic approach for developing low-lignin jute are also discussed.

Comparative analysis of diversification and population structure of kenaf (Hibiscus cannabinus L.) and roselle (H. sabdariffa L.) using SSR and RGA (resistance gene analogue) markers

Multiple DNA marker systems and complementary analytical approaches are often useful in population genetic analysis and speciation of plants. We investigated population structure of kenaf (Hibiscus cannabinus) and roselle (H. sabdariffa) for gaining insight in evolution and geographic separation of these crop species using SSR and RGA (resistance gene analogues) markers through Bayesian clustering and principal coordinate analysis (PCoA) methods. Genotyping by 12 SSR and 16 RGA markers amplified a total of 172 loci in the study population. The RGA markers generated higher number of alleles per marker (8.2) as compared to SSR (3.4), but exhibited lower heterozygosity in the population. Genetic variance and heterozygosity in roselle population for both marker systems were lower than in kenaf. RGA markers revealed higher variation among populations. Bayesian structure as well as PCoA analysis using RGA marker revealed distinct cluster for roselle, while SSR-based classification revealed high admixture. Results indicate geographic isolation and natural selection for adaptive RGA alleles in kenaf. The Indian kenaf landraces were distinct from the exotic kenaf accessions, suggesting separate lineage formation by geographic separation. Possible origin and domestication of roselle in South India is proposed.

Morpho-anatomical and SSR diversity in mutant gene pool of jute (Corchorus olitorius L.)

Characterization of induced mutants is gaining importance for increasing efficiency of mutation assisted plant breeding. In this study, phenotypic and genetic diversity of a set of 60 mutants of Corchorus olitorius (tossa jute), an important fibre yielding crop species were characterized using morphological traits related to fibre and biomass productivity, anatomical traits related to fibre distribution and development in the bark tissues of jute and simple sequence repeat (SSR) polymorphism. Morpho-anatomical diversity revealed high variability in the population, identifying mutant genotypes with superior fibre recovery and fibre anatomy traits. Ten of these mutants exhibited higher fibre recovery than a popular cultivar, JRO 204. A good number of mutants exhibited superior fibre anatomy characters like fibre wedge length, fibre wedge breadth and number of fibre cells per bundle than the cultivars. Twenty three SSR markers amplified a total of 53 alleles with polymorphism information content of 0.53. Six markers, MJM 006, MJM 432, MJM 489, MJM 566, MJM 569 and MJM 618 exhibited high resolving power (= 0.5). SSR marker based cluster analysis identified more genetic groups than morphogenetic classification, but association between genetic and phenotypic classification was low. For selection of parents for mutation assisted genetic improvement in jute, judicious use of both diversity measures for selection of parents would be more useful.

Transcriptome profiling uncovers β-galactosidases of diverse domain classes influencing hypocotyl development in jute (Corchorus capsularis L.)

Enzyme β-galactosidase (EC 3.2.1.23) is known to influence vascular differentiation during early vegetative growth of plants, but its role in hypocotyl development is not yet fully understood. We generated the hypocotyl transcriptome data of a hypocotyl-defect jute (Corchorus capsularis L.) mutant (52,393 unigenes) and its wild-type (WT) cv. JRC-212 (44,720 unigenes) by paired-end RNA-seq and identified 11 isoforms of β-galactosidase, using a combination of sequence annotation, domain identification and structural-homology modeling. Phylogenetic analysis classified the jute β-galactosidases into six subfamilies of glycoside hydrolase-35 family, which are closely related to homologs from Malvaceous species. We also report here the expression of a β-galactosidase of glycoside hydrolase-2 family that was earlier considered to be absent in higher plants. Comparative analysis of domain structure allowed us to propose a domain-centric evolution of the five classes of plant β-galactosidases. Further, we observed 1.8-12.2-fold higher expression of nine β-galactosidase isoforms in the mutant hypocotyl, which was characterized by slower growth, undulated shape and deformed cell wall. In vitro and in vivo β-galactosidase activities were also higher in the mutant hypocotyl. Phenotypic analysis supported a significant (P ≤ 0.01) positive correlation between enzyme activity and undulated hypocotyl. Taken together, our study identifies the complete set of β-galactosidases expressed in the jute hypocotyl, and provides compelling evidence that they may be involved in cell wall degradation during hypocotyl development.

Plant Biotechnology and Crop Improvement

Abstract The impact on plant biotechnology is multifarious and distributed in different frontiers. It is, therefore, difficult to have a comprehensive estimate of the contribution of various biotechnological tools on crop improvement. Nevertheless, several glorious examples exist to support that biotechnology is indispensable for genetic improvement of crop species, understand basic scientific principles related to crop biology and physiology, and to utilize plants as industrial factories. Past commercial successes and recent scientific developments in this field should, therefore, be integrated in the strategic planning for achieving the millennium and sustainable development goals. Here, we have discussed some of the well-established successes in the area of transgenic crop development, DNA marker and genomics-assisted molecular breeding, nutritional improvement and industrial production of metabolites in plants, and new research and developments having potential for large-scale application in crop improvement in near future.

New Generation Markers for Fingerprinting and Structural Analysis of Fungal Community

Fungi are one of the major components of global biodiversity playing a crucial role in ecological dynamics. More than a million fungal species make the global fungal diversity. Conventional methods of fungal identification and community analysis largely relied on morphometric studies, sequencing internal transcribed spacer (ITS) region and first generation molecular markers like RAPD and RFLP. Although, identification of fungal species or strains is done routinely on the basis of ITS region but use of new markers based on histone genes, elongation factors, mating type genes or start codon regions are also becoming quite handy in identification and diagnostics. With the advent of molecular biology and sequencing technology, new molecular tools like denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, amplified ribosomal DNA restriction analysis, amplified ribosomal intergenic spacer restriction analysis are being increasingly used for fungal community analysis and ecological studies due to their higher resolution, speed and accuracy. Next generation sequencing techniques have also enabled us to understand the actual population structure of a particular habitat through metagenome sequencing. These next generation molecular markers and tools will surely make us understand the fungal diversity and community structure in a far better way and enable us to conserve this biological treasure.