Developmenrt of EST-SSR and genomic-SSR markers to assess genetic diversity in Jatropha Curcas L.
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 28 January 2010
Accepted: 24 February 2010
Published: 24 February 2010
Jatropha curcas L. has attracted a great deal of attention worldwide, regarding its potential as a new biodiesel crop. However, the understanding of this crop remains very limited and little genomic research has been done. We used simple sequence repeat (SSR) markers that could be transferred from Manihot esculenta (cassava) to analyze the genetic relationships among 45 accessions of J. curcas from our germplasm collection.
In total, 187 out of 419 expressed sequence tag (EST)-SSR and 54 out of 182 genomic (G)-SSR markers from cassava were polymorphic among the J. curcas accessions. The EST-SSR markers comprised 26.20% dinucleotide repeats, 57.75% trinucleotide repeats, 7.49% tetranucleotide repeats, and 8.56% pentanucleotide repeats, whereas the majority of the G-SSR markers were dinucleotide repeats (62.96%). The 187 EST-SSRs resided in genes that are involved mainly in biological and metabolic processes. Thirty-six EST-SSRs and 20 G-SSRs were chosen to analyze the genetic diversity among 45 J. curcas accessions. A total of 183 polymorphic alleles were detected. On the basis of the distribution of these polymorphic alleles, the 45 accessions were classified into six groups, in which the genotype showed a correlation with geographic origin. The estimated mean genetic diversity index was 0.5572, which suggests that our J. curcas germplasm collection has a high level of genetic diversity. This should facilitate subsequent studies on genetic mapping and molecular breeding.
We identified 241 novel EST-SSR and G-SSR markers in J. curcas, which should be useful for genetic mapping and quantitative trait loci analysis of important agronomic traits. By using these markers, we found that the intergroup gene diversity of J. curcas was greater than the intragroup diversity, and that the domestication of the species probably occurred partly in America and partly in Hainan, China.
Jatropha curcas L. is a perennial, monoecious shrub that belongs to the Euphorbiaceae family. The species is native to America but is distributed widely in the tropics [1, 2]. Wild or semicultivated types of J. curcas can grow well under all unfavorable climatic and soil conditions . The seeds of J. curcas contain 40-45% oil [4–6] with a high percentage of monounsaturated oleic and polyunsaturated linoleic acid. The seed oils can be classified as semi-drying . In recent years, the economic importance of J. curcas for the production of biodiesel fuel has been increasingly recognized .
To use J. curcas for producing biofuel, it is crucial to develop varieties with a high seed yield and a high oil content that are adapted well to varied conditions . In recent years, J. curcas germplasm has been collected and analyzed in Brazil, India, Indonesia, and China [10, 11]. J. curcas has a heterozygous genome, so conventional breeding programs for its improvement might not be effective. Hence, it is likely that genomics-based breeding strategies need to be used. However, the genetic map of J. curcas is not well-developed and very limited information is available with respect to molecular markers. The traditional methods of developing simple sequence repeat (SSR) markers are usually time-consuming and labor-intensive. However, an alternative strategy has been developed that uses comparative genomics to identify SSR markers. This strategy has been used successfully in barley  and Brassica .
In the study reported herein, we selected 419 expressed sequence tag (EST)-SSR and 182 genomic (G)-SSR primer pairs that had been developed for Manihot esculenta (cassava), which also belongs to the Euphorbiaceae family, and investigated whether they could be transferred to J. curcas. Firstly, these primer pairs were tested using five accessions of J. curcas. The primer pairs that produced specific amplicons were then tested further. Finally, we used the transferable markers to analyze the genetic diversity of our collection of J. curcas accessions.
Identification of EST-SSRs and G-SSRs for use in J. curcas
Characteristics of the EST-SSR and G-SSR markers and relevant genes in J. curcas
Structural characteristics of the ES T-SSR and G-SSR markers in J. curcas
Number and percentage
Number and percentage
Assessment of genetic diversity in J. curcas
Thirty-six EST-SSRs and 20 G-SSRs were used to estimate the genetic diversity of 45 accessions of J. curcas. A total of 216 alleles were identified, and 183 (84.72%) of them were polymorphic. The sizes of the amplicons for the EST-SSRs and G-SSRs ranged from 120 to 600 bp. The 36 EST-SSRs generated 152 alleles, of which 128 (84%) were polymorphic, whereas the 20 G-SSRs produced 64 alleles, of which 55 (86%) were polymorphic. The number of alleles for each G-SSR ranged from one to six with a mean of 3.20; in contrast, the number of alleles for each EST-SSR ranged from one to nine with a mean of 4.22. Therefore, more alleles were obtained with the EST-SSRs than with the G-SSRs.
Parameters of intra- and intergroup genetic diversity among the five populations of J. curcas
Development of molecular markers in J. curcas
It was crucial to develop a sufficient number of species-specific markers to enable the fingerprinting and genetic mapping of J. curcas; in rice and soybean, for example, more than 10,000 SSR and SNP markers are available [14, 15]. Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers have been used to evaluate genetic diversity and for fingerprinting in accessions of J. curcas and related species [11, 16–18]. Up to now, only 12 SSR markers had been developed for Jatropha (they are polymorphic in six species of Jatropha) , and no genetic map of Jatropha has been reported. The transferable SSRs identified in the present study should enable the genetic diversity, elite clones, and evolution of J. curcas to be assessed, and a linkage map to be constructed.
Transferability of EST-SSRs and G-SSRs from cassava
The development of EST-SSR markers was particularly attractive because they represent coding regions of the genome. It has been reported that EST-derived SSRs and G-SSRs show a considerable degree of transferability to related species [20–24]. In the present study, we observed a high level of transferability from cassava to J. curcas; the level of transferability was higher for EST-SSRs (44.63%) than for G-SSRs (29.67%). This result was consistent with that reported for wheat and related species . In several studies, the level of transferability of G-SSRs was found to be only 20-30% [26–29]. The higher levels of transferability of EST-SSRs than of G-SSRs reflect the conserved nature of coding sequences as compared with non-coding genomic DNA, and the fact that the mutation frequency of EST sequences is lower than that of genomic DNA sequences. These results demonstrate the potential value of EST-SSR markers for the development of genetic maps, assessment of genetic diversity, and marker-assisted selection (MAS) breeding in J. curcas, all of which would benefit comparative mapping and analysis of the comparative functions of genes among the economic species in the Euphorbiaceae family. Our finding that the majority of transferable EST-SSRs were trinucleotide repeats agreed with previous studies [21, 30–32], and can be explained by the suppression of non-trimeric SSRs in coding regions due to the risk of frameshift mutations, which might occur with non-trimeric microsatellites [33–36].
Collection of germplasm and evaluation of genetic diversity
Several projects to collect germplasm have been carried out in Brazil , India , and China , but systematic work on the collection of germplasm and its evaluation is still in its infancy. The low genetic variability found among accessions of J. curcas from Africa and Asia [11, 16, 17] has demonstrated the need for new sources of genetic variation in J. curcas that could be used in breeding programs. Such sources of genetic variation have been identified in Latin America, especially in Guatemala . In the present study, high levels of genetic diversity were revealed in the 45 Jatropha accessions analyzed with the 56 EST-SSR and G-SSR primer pairs that were utilized. The accessions from South America, Yunnan (China), and Indonesia showed higher levels of genetic variation than the other two geographic regions (Grenada and Hainan, China). In particular, the collections from Yunnan (China) could be used to enrich the genetic background of J. curcas for breeding. Gst and Nm showed that some differentiation occurred in each geographic group.
We developed a set of 241 SSR primer pairs for use in J. curcas. Of these markers, 187 EST-SSRs should be useful for genetic mapping and quantitative trait loci analysis of important agronomic traits. Fifty-six EST-SSRs and G-SSRs were used successfully to analyze genetic diversity in 45 accessions of J. curcas. The accessions analyzed showed a broad genetic background with an average genetic diversity index of 0.5572. The intergroup genetic diversity was larger than the intragroup diversity, and domestication had taken place in both America and Hainan, China.
The J. curcas accessions and their origins
District of collection
District of collection
District of collection
Design of EST-SSR and G-SSR primers and validation in J. curcas
A total of 419 EST-SSR primers that had been developed for cassava in our laboratory  and 182 G-SSR primers  were synthesized and used. Amplification by PCR was performed in a 20-μl reaction mixture that contained 50 ng template DNA, 1× PCR buffer (20 mM Tris pH 9.0, 100 mM KCl, 3.0 mM MgCl2), 400 μM of each of the four dNTPs, 0.4 μM of each of the forward and reverse primers, and one unit of Taq DNA polymerase. The following PCR conditions were used: 94°C for 1 min, followed by 35 cycles of 94°C for 1 min, 45-57°C for 1 min, 72°C for 1 min, and 10 min at 72°C for the final extension. PCR products were separated on a 2% agarose gel, and visualized using SYBR Green http://www.Genecopoeia.com.
Putative functional annotation of EST-SSRs
To assess the putative function of the EST-SSRs developed here, a BLASTX search of the GenBank nonredundant database http://www.ncbi.nlm.nih.gov/BLAST was performed using the 187 ESTs that contained polymorphic microsatellites. The threshold for a significant BLAST hit was set at a bit score greater than 80 bits. Functional categories were assigned on the basis of the BLAST searches using a specially formatted database of the Eukaryotic Orthologous Groups of proteins (KOG).
Analysis of genetic diversity
The polymorphic alleles obtained with each primer pair were scored for their presence (1) or absence (0). From the data matrix, a dendrogram was constructed using the unweighted pair group method using arithmetic averages (UPGMA), the similarity coefficient, and the software NTSYS-pc2.1 . The binary data were also subjected to PCA to investigate the structure of our collection. The genetic diversity parameters of each geographic group, which included the percentage of polymorphic loci (PLP), observed number of alleles per locus (na), effective number of alleles per locus (ne), Nei's gene diversity (h), Shannon's information index (I), Gst, and Nm, were calculated by POPGENE 32 .
This work was supported by the Cooperative Research Foundation of China and Brazil (2008KR0395) and the Basic Scientific Research Foundation of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science (ITBBZX0843). The authors wish to thank M Fregene of the International Center for Tropical Agriculture (CIAT) for assistance with the development of the cassava G-SSR primers and Ruqiang Xu of the Virginia Commonwealth University for excellent suggestions and discussions about the paper. Finally, the authors wish to thank Cathel Kerr, Lucy Colegrove, and Chris Wright of Genedits http://www.genedits.com for editing.
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