Discovery and use of single nucleotide polymorphic (SNP) markers in Jatropha curcas L.

Various programs for genetic improvement in oil yield of the biofuel plant Jatropha curcas L. are currently in progress worldwide. In order to develop strategies for genetic improvement, it is important to estimate the degree of diversity at the genetic level among various genotypes of J. curcas. High-throughput sequencing of complexity-reduced nuclear genomic DNA of J. curcas coupled with computational analysis discovered 2,482 informative single nucleotide polymorphisms (SNPs). Genotyping of selective SNPs among 148 global collections of J. curcas lines and further diversity analysis through NTSYS-pc, DARwin and Structure?2.0 software revealed that a narrow level of genetic diversity existed among the indigenous genotypes as compared to the exotic genotypes of J. curcas. The level of marker informativeness along with distance-based and Bayesian clustering revealed grouping of the accession from Togo (Africa) with various Indian accessions at K?=?4 and K?=?5 values (where K represents the number of populations). The diverse accessions identified in the study will be of further use in genetic improvement of J. curcas through quantitative trait loci and association mapping.     

Molecular characterization and identification of markers for toxic and non-toxic varieties of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using RAPD,AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).     

Isolation of novel microsatellites using FIASCO by dual probe enrichment from Jatropha curcas L. and study on genetic equilibrium and diversity of Indian population revealed by isolated microsatellites

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petrodiesel. Very few attempts were made to isolate novel microsatellite markers and assessment of the extent of genetic equilibrium and diversity that exists in J. curcas. Therefore, the present investigation was undertaken to isolate the novel microsatellites and access genetic equilibrium, diversity that exists among 44 diverse germplasm collected from distinct geographical areas in India using isolated microsatellites. The overall efficiency of the enrichment of microsatellite by dual probe in the present study found to be 54% and among the sequences obtained the percentage of sequences having suitable flanking regions for the primer designing was found to be 89.58%. The mean co-efficient of genetic similarity (CGS) was found to be 0.97. The overall diversity obtained by microsatellites was found to be low in comparison with the diversity reported by multilocus markers systems observed in earlier studies; however, the good allele polymorphism was observed. The overall dendrogram of microsatellite analysis resulted in random clustering of germplasm and not in accordance to geographical area of collection. The present study, diversity analysis using microsatellite markers concludes the low genetic diversity and genetic disequlibrium of J. curcas in India and will provide pavement for further intra-population studies on narrow geographical areas to understand the population genetic structure, phylogeography and molecular ecological studies. The germplasm characterized, and the microsatellite markers isolated and characterized in the present study can be employed efficiently in breeding programs for genetic improvement of the species through marker assisted selection and QTL analysis, for further genetic resource management and help in making the J. curcas as potential crop with superior agronomical traits.     

Development of EST-SSR markers through data mining and their use for genetic diversity study in Indian accessions of Jatropha curcas L.: a potential energy crop

Jatropha curcas L. is gaining importance as a potential energy crop. However, lack of sufficient numbers of molecular markers hinder current research on crop improvement in Jatropha. The expressed sequences tags (EST) sequences deposited in public databases, offers an excellent opportunity to identify simple sequence repeats (SSRs) through data mining, for further research on molecular breeding. In the present study 42,477 ESTs of J. curcas were screened, out of which 5,673 SSRs were identified with 48.8 % simple (excluding mononucleotide repeats) and 52.2 % compound repeat motifs. Amongst these repeat motifs, dinucleotide repeats were abundant (26.5 %), followed by trinucleotide (23.1 %) and tetranucleotide repeats (0.8 %). From these microsatellites, 32 EST-SSR (genic microsatellite) primer pairs were designed. These primers were used to analyze the genetic diversity among 42 accessions collected from different parts of India. Out of the 32 EST-SSR primers, 24 primer pairs exhibited polymorphism among the genotypes, with amplicons varying from one to eight, giving an average of 2.33 alleles per polymorphic marker. Polymorphic information content value ranged from 0.02 to 0.5 with an average of 0.402 indicating moderate level of informativeness within these EST-SSRs markers. The EST-SSR markers developed here will serve as a valuable resource for genetic studies, like linkage mapping, diversity analysis, quantitative trait locus/association mapping, and molecular breeding. The current study also revealed low diversity in the screened Indian Jatropha germplasm. Therefore, the future efforts must be made to broaden the gene pool of Jatropha for the creation of genetic diversity that can be further used for crop improvement through breeding.     

Jatropha curcas: a review on biotechnological status and challenges

Plant tissue culture and molecular biology techniques are powerful tools of biotechnology that can complement conventional breeding, expedite crop improvement and meet the demand for availability of uniform clones in large numbers. Jatropha curcas Linn., a non-edible, eco-friendly, non-toxic, biodegradable fuel-producing plant has attracted worldwide attention as an alternate sustainable energy source for the future. This review presents a consolidated account of biotechnological interventions made in J. curcas over the decades and focuses on contemporary information and trends of future research.     

SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis

Background  

The oil palm (Elaeis guineensis Jacq.) is a perennial monocotyledonous tropical crop species that is now the world's number one source of edible vegetable oil, and the richest dietary source of provitamin A. While new elite genotypes from traditional breeding programs provide steady yield increases, the long selection cycle (10-12 years) and the large areas required to cultivate oil palm make genetic improvement slow and labor intensive. Molecular breeding programs have the potential to make significant impacts on the rate of genetic improvement but the limited molecular resources, in particular the lack of molecular markers for agronomic traits of interest, restrict the application of molecular breeding schemes for oil palm.     

Sequence Analysis of the Genome of an Oil-Bearing Tree, Jatropha curcas L.

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ∼95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.     

SNP Discovery with EST and NextGen Sequencing in Switchgrass (Panicum virgatum L.)

Although yield trials for switchgrass (Panicum virgatum L.), a potentially high value biofuel feedstock crop, are currently underway throughout North America, the genetic tools for crop improvement in this species are still in the early stages of development. Identification of high-density molecular markers, such as single nucleotide polymorphisms (SNPs), that are amenable to high-throughput genotyping approaches, is the first step in a quantitative genetics study of this model biofuel crop species. We generated and sequenced expressed sequence tag (EST) libraries from thirteen diverse switchgrass cultivars representing both upland and lowland ecotypes, as well as tetraploid and octoploid genomes. We followed this with reduced genomic library preparation and massively parallel sequencing of the same samples using the Illumina Genome Analyzer technology platform. EST libraries were used to generate unigene clusters and establish a gene-space reference sequence, thus providing a framework for assembly of the short sequence reads. SNPs were identified utilizing these scaffolds. We used a custom software program for alignment and SNP detection and identified over 149,000 SNPs across the 13 short-read sequencing libraries (SRSLs). Approximately 25,000 additional SNPs were identified from the entire EST collection available for the species. This sequencing effort generated data that are suitable for marker development and for estimation of population genetic parameters, such as nucleotide diversity and linkage disequilibrium. Based on these data, we assessed the feasibility of genome wide association mapping and genomic selection applications in switchgrass. Overall, the SNP markers discovered in this study will help facilitate quantitative genetics experiments and greatly enhance breeding efforts that target improvement of key biofuel traits and development of new switchgrass cultivars.     

Genetic Diversity Among Jatropha and Jatropha-Related Species Based on ISSR Markers

Jatropha curcas (jatropha) is a potential biodiesel crop. A major limitation in production is that jatropha remains wild with low genetic variation. Related species/genera in the Euphorbiaceae can potentially be used for its genetic improvement. In this study, we employed inter-simple sequence repeats (ISSRs) to assess genetic variation among 30 accessions of jatropha, two accessions of bellyache bush (Jatropha gossypifolia), two accessions of spicy jatropha (Jatropha integerrima), two accessions of bottleplant shrub (Jatropha podagrica), and three accessions of castor bean hybrids. Genetic relationships were evaluated using 27 of 86 ISSR markers, yielding 307 polymorphic bands with polymorphism contents ranging from 0.76 to 0.95 for IMPN 1 and UBC 807 markers, respectively. Dice’s genetic similarity coefficient ranged from 0.39 to 0.99, which clearly separated the plant samples into seven groups at the coefficient of 0.48. The first group comprised J. curcas from Mexico, the second group comprised J. curcas from China and Vietnam, the third group comprised J. curcas from Thailand, the fourth group was J. integerrima, the fifth group was J. gossypifolia, the sixth group was J. podagrica, and the last and most distinct group was Ricinus communis. Analysis of molecular variance revealed that 63% of the variability was attributable to variation among groups, while 37% was due to variation within groups. Based on Nei’s genetic distance, the population from G2 (J. curcas from China) and G4 (J. curcas from Vietnam) had the least ISSR variability (0.0668), whereas G8 (R. communis) and Jatropha spp. displayed the highest distance (0.6005–0.7211).     

Development and characterization of EST-SSR markers from Jatropha curcas EST database and their transferability across jatropha-related species/genus

Jatropha curcas (jatropha) is a multipurpose plant with potential as a raw material for biofuel. In the present study, a total of 43,349 expressed sequence tags (ESTs) from J. curcas were searched for type and frequency of simple sequence repeat (SSR) markers. Five thousand one hundred and seventy-five sequences were indentified to contain 6,108 SSRs with 90.8% simple and 9.2% compound repeat motifs. One hundred and sixty-three EST-SSRs were developed and used to evaluate the transferability and genetic relatedness among 4 accessions of J. curcas from China, Mexico, Thailand and Vietnam; 5 accessions of congeneric species, viz. J. gossypiifolia, dwarf J. integerrima, normal J. integerrima, J. multifida, J. podagrica; and Ricinus communis. The polymorphic markers showed 75.56–85.19% transferability among four species of Jatropha and 26.67% transferability across genera in Ricinus communis. Investigation of genetic relatedness showed that J. curcas and J. integerrima are closely related. EST-SSRs used in this study demonstrate a high efficiency of cross species/genera amplification and are useful for identifying genetic diversity of jatropha and its close taxa and to choose the desired related species for wide crossing to improve new varieties of jatropha. The markers can also be further exploited for genetic resource management and genetic improvement of related species/genera through marker-assisted breeding programs.     

Cross species amplification ability of novel microsatellites isolated from <Emphasis Type="Italic">Jatropha curcas</Emphasis> and genetic relationship with sister taxa

The present investigation was undertaken with an aim to check the ability of cross species amplification of microsatellite markers isolated from Jatropha curcas—a renewable source of biodiesel to deduce the generic relationship with its six sister taxa (J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis). Out of the 49 markers checked 31 markers showed cross species amplification in all the species studied. JCDS-30, JCDS-69, JCDS-26, JCMS-13 and JCMS-21 amplified in J. curcas. However, these markers did not show any cross species amplification. Overall percentage of polymorphism (PP) among the species studied was 38% and the mean genetic similarity (GS) was found to be 0.86. The highest PP (24) and least GS (0.76) was found between J. curcas/J. podagrica and J. curcas/J. multifida and least PP (4.44) and highest GS (0.96) was found between J. integerrima/J. tanjorensis. Dendrogram analysis showed good congruence to RAPD and AFLP than nrDNA ITS data reported earlier. The characterized microsatellites will pave way for intraspecies molecular characterization which can be further utilized in species differentiation, molecular identification, characterization of interspecific hybrids, exploitation of genetic resource management and genetic improvement of the species through marker assisted breeding for economically important traits.     

Application of whole genome re-sequencing data in the development of diagnostic DNA markers tightly linked to a disease-resistance locus for marker-assisted selection in lupin (Lupinus angustifolius)

Background

Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding.

Results

Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding.

Conclusions

We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1878-5) contains supplementary material, which is available to authorized users.     

Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant

The family Euphorbiaceae includes some of the most efficient biomass accumulators. Whole genome sequencing and the development of genetic maps of these species are important components in molecular breeding and genetic improvement. Here we report the draft genome of physic nut (Jatropha curcas L.), a biodiesel plant. The assembled genome has a total length of 320.5 Mbp and contains 27 172 putative protein‐coding genes. We established a linkage map containing 1208 markers and anchored the genome assembly (81.7%) to this map to produce 11 pseudochromosomes. After gene family clustering, 15 268 families were identified, of which 13 887 existed in the castor bean genome. Analysis of the genome highlighted specific expansion and contraction of a number of gene families during the evolution of this species, including the ribosome‐inactivating proteins and oil biosynthesis pathway enzymes. The genomic sequence and linkage map provide a valuable resource not only for fundamental and applied research on physic nut but also for evolutionary and comparative genomics analysis, particularly in the Euphorbiaceae.