Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity

Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F₂ mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F₂ plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker‐assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.     

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).     

金沙江干热河谷地区麻疯树单粒种子核糖体失活蛋白含量及其与种子性状的相关性

对金沙江干热河谷地区麻疯树(Jatropha curcas L.)种子中具有抗肿瘤活性的核糖体失活蛋白curcin含量及其生物学性状进行研究。结果表明,用酶联免疫吸附法测定麻疯树单颗种子的curcin含量为0.275～3.183 mg g-1,平均(1.369±0.055)mg g-1。Pearson相关性分析表明:单粒麻疯树种子的curcin含量与种子其他性状的相关性均未达显著水平,但与种子质量、种仁质量及含油量存在一定程度的负相关,而与出仁率和可溶性蛋白含量存在一定的正相关,且与可溶性蛋白含量的正相关系数(0.190)和与含油率的负相关系数(–0.177)较高。这表明可在金沙江干热河谷地区开展高curcin含量麻疯树种质资源收集和选育工作,以及培育高含油量、高curcin含量的双高品种,以提高麻疯树生物燃油产业的综合效益。     

Molecular characterization of Jatropha genetic resources through inter-simple sequence repeat (ISSR) markers

The genetic diversity among eight Jatropha species and three Jatropha curcas accessions were analyzed using ISSR-PCR. Nine ISSR primers generated reproducible amplification banding pattern of 61 polymorphic bands out of 64 scored accounting for 98.14% polymorphism across the species. The ISSR primers viz., I1, I2, I3, I4, I5, I6, I7 and I10 generated 100% polymorphic patterns. Jaccard’s coefficient of similarity varied from 0.346 to 0.807, indicative of high level of genetic variation among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L (TNMJ1, TNMJ 22 and TNMJ 23), while second included four species viz., J. tanjorensis J. L. Ellis et Saroja., J. gossypifolia L., J. podagrica Hook and J. maheshwarii Subrum and M.P. Nayer and the third cluster included another four species viz., J. villosa Wight J. multifida L., J. integerrima Jacq and J. glandulifera Roxb. The overall grouping pattern of clustering corresponds well with principal component analysis (PCA) confirming patterns of genetic diversity observed among the species. So far, there are no reports on the molecular diversity of the Jatropha species through ISSR marker. This study provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources and also for further breeding programme towards biodiesel production.     

Molecular characterization and genetic diversity analysis of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. in India using RAPD and AFLP analysis

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America and widely distributed in South and Central America, attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petro-diesel. Very few attempts were made to understand the extent of genetic diversity that exists in J. curcas. Therefore, the present investigation was undertaken to asses the genetic diversity among 28 diverse germplasm collected from distinct geographical areas in India. The overall percentage of polymorphism (PP) was found to be 50.70 and 60.95 by RAPD and AFLP, respectively. The mean PP was found to be 9.72 and 20.57 by RAPD and AFLP, respectively. The mean genetic similarity was observed to be 0.89 by RAPD and 0.88 by AFLP. Among the germplasm JCI20 found to be the most diverged one. The dendrogram analysis of RAPD and AFLP data showed good congruence, but better resolution and more polymorphism was observed with AFLP. When the dendrogram of RAPD was compared with AFLP dendrogram, the major clustering pattern was found to be similar; however, changes in minor grouping were observed. In both RAPD and AFLP analysis clustering of germplasm did not show any correlation with the geographical area of collection. Low genetic diversity observed in J. curcas and the clustering pattern indicates that the distribution of species might have happened through anthropogenic activity and warrants the need for widening the genetic base. The present study will provide pavement for further intra-population studies on narrow geographical areas, to understand the population genetic structure, phylogeography, molecular ecological studies. The marker information and the characterized germplasm help in further improvement of the species through marker assisted breeding programs.     

Efficacy of karanjin and phorbol ester fraction against termites (Odontotermes obesus)

The non-edible oil seeds of Jatropha curcas (physic nut) and Pongamia pinnata (karanja) contain some toxic components (phorbol esters in J. curcas and karanjin in P. pinnata), which may be used as biopesticides. In this study, the active components of J. curcas and P. pinnata oil were extracted and their efficacy against the termites Odontotermes obesus (Rambur), was tested. The phorbol ester fraction of J. curcas and karanjin of P. pinnata oil were found to be effective against termites. A mortality rate of 100% was achieved in 6 h with karanjin and in 12 h with phorbol ester fraction. The LC₅₀ levels of karanjin and phorbol esters fractions were 0.038 and 0.071 g ml⁻¹, respectively, after 24 h at a 95% (0.05) confidence limit.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.     

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).     

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.     

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.     

Understanding invasion history: genetic structure and diversity of two globally invasive plants and implications for their management

Aim Resolving the origin of invasive plant species is important for understanding the introduction histories of successful invaders and aiding strategies aimed at their management. This study aimed to infer the number and origin(s) of introduction for the globally invasive species, Macfadyena unguis‐cati and Jatropha gossypiifolia using molecular data. Location Native range: Neotropics; Invaded range: North America, Africa, Europe, Asia, Pacific Islands and Australia. Methods We used chloroplast microsatellites (cpSSRs) to elucidate the origin(s) of introduced populations and calculated the genetic diversity in native and introduced regions. Results Strong genetic structure was found within the native range of M. unguis‐cati, but no genetic structuring was evident in the native range of J. gossypiifolia. Overall, 27 haplotypes were found in the native range of M. unguis‐cati. Only four haplotypes were found in the introduced range, with more than 96% of introduced specimens matching a haplotype from Paraguay. In contrast, 15 haplotypes were found in the introduced range of J. gossypiifolia, with all invasive populations, except New Caledonia, comprising multiple haplotypes. Main conclusions These data show that two invasive plant species from the same native range have had vastly different introduction histories in their non‐native ranges. Invasive populations of M. unguis‐cati probably came from a single or few independent introductions, whereas most invasive J. gossypiifolia populations arose from multiple introductions or alternatively from a representative sample of genetic diversity from a panmictic native range. As introduced M. unguis‐cati populations are dominated by a single haplotype, locally adapted natural enemies should make the best control agents. However, invasive populations of J. gossypiifolia are genetically diverse and the selection of bio‐control agents will be considerably more complex.     

小桐子糖原合成激酶基因JcGSK的克隆与表达分析

以小桐子(Jatropha curcas L.)cDNA为模版,克隆了JcGSK基因的CDS序列。序列分析表明,JcGSK基因包含1 230bp完全阅读框(ORF),编码409个氨基酸。预测其编码蛋白质的相对分子量为46.33kD,理论等电点为8.58。Blast搜索结果及进化分析结果表明,JcGSK蛋白与巴西橡胶树GSK蛋白的氨基酸序列一致性最高(94%)且亲缘关系最近;JcGSK基因编码的蛋白具有一个蛋白激酶特有的结构域。组织表达结果显示,JcGSK基因在小桐子根、茎、叶、花、果皮和种子中都有表达,且在根中表达量最高。小桐子幼苗在NaCl、ABA、PEG、低温和机械损伤处理后JcGSK基因表达量有不同程度的上调,推测其参与小桐子非生物胁迫响应和信号传导过程。JcGSK基因在种子中也有较高表达,在种子发育过程中表达量的变化与种子生长发育趋势基本一致,推测JcGSK基因也参与调控小桐子种子的生长发育。     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO₂ in C₄ and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C₃-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.     

Osmoconditioning prevents the onset of microtubular cytoskeleton and activation of cell cycle and is detrimental for germination of Jatropha curcas L. seeds

• Jatropha curcas is an oilseed crop renowned for its tolerance to a diverse range of environmental stresses. In Brazil, this species is grown in semiarid regions where crop establishment requires a better understanding of the mechanisms underlying appropriate seed, seedling and plant behaviour under water restriction conditions. In this context, the objective of this study was to investigate the physiological and cytological profiles of J. curcas seeds in response to imbibition in water (control) and in polyethylene glycol solution (osmoticum).
• Seed germinability and reactivation of cell cycle events were assessed by means of different germination parameters and immunohistochemical detection of tubulin and microtubules, i.e. tubulin accumulation and microtubular cytoskeleton configurations in water imbibed seeds (control) and in seeds imbibed in the osmoticum.
• Immunohistochemical analysis revealed increasing accumulation of tubulin and appearance of microtubular cytoskeleton in seed embryo radicles imbibed in water from 48 h onwards. Mitotic microtubules were only visible in seeds imbibed in water, after radicle protrusion, as an indication of cell cycle reactivation and cell proliferation, with subsequent root development. Imbibition in osmoticum prevented accumulation of microtubules, i.e. activation of cell cycle, therefore germination could not be resumed.
• Osmoconditioned seeds were able to survive re‐drying and could resume germination after re‐imbibition in water, however, with lower germination performance, possibly due to acquisition of secondary dormancy. This study provides important insights into understanding of the physiological aspects of J. curcas seed germination in response to water restriction conditions.

     

High efficiency and reliability of inter-simple sequence repeats (ISSR) markers for evaluation of genetic diversity in Brazilian cultivated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. accessions

Jatropha curcas L. is found in all tropical regions and has garnered lot of attention for its potential as a source of biodiesel. As J. curcas is a plant that is still in the process of being domesticated, interest in improving its agronomic traits has increased in an attempt to select more productive varieties, aiming at sustainable utilization of this plant for biodiesel production. Therefore, the study of genetic diversity in different accessions of J. curcas in Brazil constitutes a necessary first step in genetic programs designed to improve this species. In this study we have used ISSR markers to assess the genetic variability of 332 accessions from eight states in Brazil that produce J. curcas seeds for commercialization. Seven ISSR primers amplified a total of 21,253 bands, of which 19,472 bands (91%) showed polymorphism. Among the polymorphic bands 275 rare bands were identified (present in fewer than 15% of the accessions). Polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.26, 17.86 and 19.87 per primer, respectively, showing the high efficiency and reliability of the markers used. ISSR markers analyses as number of polymorphic loci, genetic diversity and accession relationships through UPGMA-phenogram and MDS showed that Brazilian accessions are closely related but have a higher level of genetic diversity than accessions from other countries, and the accessions from Natal (RN) are the most diverse, having high value as a source of genetic diversity for breeding programs of J. curcas in the world.     

Mapping QTL controlling fatty acid composition in a doubled haploid rapeseed population segregating for oil content

Increasing oil content and improving the fatty acid composition in the seed oil are important breeding goals for rapeseed (Brassica napus L.). The objective of the study was to investigate a possible relationship between fatty acid composition and oil content in an oilseed rape doubled haploid (DH) population. The DH population was derived from a cross between the German cultivar Sollux and the Chinese cultivar Gaoyou, both having a high erucic acid and a very high oil content. In total, 282 DH lines were evaluated in replicated field experiments in four environments, two each in Germany and in China. Fatty acid composition of the seed oil was analyzed by gas liquid chromatography and oil content was determined by NIRS. Quantitative trait loci (QTL) for fatty acid contents were mapped and their additive main effects were determined by a mixed model approach using the program QTLMapper. For all fatty acids large and highly significant genetic variations among the genotypes were observed. High heritabilities were determined for oil content and for all fatty acids (h ² = 0.82 to 0.94), except for stearic acid content (h ²= 0.38). Significant correlations were found between the contents of all individual fatty acids and oil content. Closest genetic correlations were found between oil content and the sum of polyunsaturated fatty acids (18:2 + 18:3; r _G = −0.46), the sum of monounsaturated fatty acids (18:1 + 20:1 + 22:1; r _G = 0.46) and palmitic acid (16:0; r _G = −0.34), respectively. Between one and eight QTL for the contents of the different fatty acids were detected. Together, their additive main effects explained between 28% and 65% of the genetic variance for the individual fatty acids. Ten QTL for fatty acid contents mapped within a distance of 0 to 10 cM to QTL for oil content, which were previously identified in this DH population. QTL mapped within this distance to each other are likely to be identical. The results indicate a close interrelationship between fatty acid composition and oil content, which should be considered when breeding for increased oil content or improved oil composition in rapeseed.