Molecular markers for genetic diversity studies in Jatropha ( Jatropha curcas L . )

The molecular markers namely random amplified polymorphic DNA, amplified fragment length polymorphism, sequence-characterized amplified region, inter simple sequence repeats, simple sequence repeats and single-nucleotide polymorphism, etc. have been successfully used for genetic diversity studies in J. curcas. The assessment of genetic variations among Jatropha germplasms using molecular markers show the presence of high genetic diversity for the Central and South American regions and insignificant genetic variation from Asia and Africa. The use of molecular markers for the assessment of phylogenetic relationships among different Jatropha species has been restricted only to the well adapted and acclimatized species in India. The significant genetic variability in the genus Jatropha for vegetative and floral traits for seed oil content, productivity, toxicity (phorbol esters, curcin), fatty acid profiles, etc. has been studied in the past decade using molecular markers. Genetic enhancement of Jatropha through conventional breeding and interspecific gene transfer can be attempted by exploiting the diverse genetic resource form J. curcas and their wild species. This review focuses on the importance and use of molecular markers towards studying diversity analysis in J. curcas growing at different areas.


Introduction
Jatropha, commonly called as physic nut, belongs to family Euphorbiaceae and comprises of 172 species of which the most primitive species is Jatropha curcas L. (2n = 22) [1].Jatropha curcas has been recognized as an important crop for biodiesel production.The Jatropha species is perennial shrub and serves as an important wasteland crop facilitating soil conservation by protecting soil against erosion via wind and animals interference.It is widely distributed in Central and South America, Africa, India and South East Asia.The J. curcas originated in Central and South America, and distributed to other tropical countries [2].It is mainly evolved for dryland and adapt well in limited water source.Several characteristics of J. curcas such as drought resistance, speedy growth, easy propagation and wide adaptation to soil conditions have led to the wide spread of J. curcas [3][4][5].It has acclimatized in different agro-climatic regions because of its strong adaptation at morphological and physiological level.J. curcas is considered as the best source for restoration of ruined lands and rural development.The J. curcas seeds have semi-drying oil (32-35%) and yield biodiesel of European (EN 14214) and American (ASTM D6751) standards [3,6] via trans-esterification.J. curcas seed oil used in the production of biodiesel is eco-friendly, biodegradable, non-toxic and renewable to petroleum diesel [7,8].It can be used as a traditional vegetable oil or trans-esterified to produce biodiesel for use in standard diesel engines.These properties of J. curcas have fascinated globally to develop a sustainable alternative feedstock for biodiesel production [9,10].After oil extraction, the remaining seed cake can be used as organic fertilizer.Seed cake can be also used for direct combustion or charcoal conversion [11].J. curcas has been used for the isolation and production of pharmaceutical and pesticides in China [12].Anti-viral (against herpesvirus I and II) and antibacterial (Staphylococcus aureus and Monilia albicans) skin disinfectors are commercialized [13].Additional products from J. curcas are A2-Jetfuel kerosene and polyol biodegradable foam for use in packaging and insulation industry, paint from the bark [14].
DNA (or molecular) markers are used predominantly due to their abundance, plentiful in number and are unaltered by environmental factors and/or the plant developmental stage.Molecular markers develop from different classes of DNA mutations such as rearrangements (insertions or deletions), substitution (point mutations), or errors in replication of tandemly repeated DNA.Molecular markers have several applications in plant breeding such as (i) evaluation and selection of breeding materials for genetic diversity, parental selection, cultivar identity and assessment of cultivar purity, (ii) trait pyramiding, and (iii) backcrossing.
Molecular markers have played an important role in the detection of unique alleles between two or more species which can be applied in the species characterization and improvement.Molecular markers have been abundantly used to evaluate biodiversity, phylogenetic relationships, construction of genetic linkage maps and in tagging and mapping of useful traits.Currently, genetic diversity studies in the genus Jatropha are focused on J. curcas and few wild species that are commonly distributed in India.Different types of single and multi-locus molecular markers such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), sequence-characterized amplified region (SCAR), inter simple sequence repeats (ISSRs), simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNPs), etc. were used for assessment of genetic diversity in the available germplasm.This review mainly focus on the different studies carried out on genetic variability present in the Jatropha species other than Central and South-American and African regions.

Amplified fragment length polymorphism (AFLP)
The AFLP is trustworthy, high throughput and less expansive.Many researchers from India, China, Brazil and Mexico used AFLP markers for genetic diversity analysis in J. curcas accessions and reported the presence of low to high genetic variability.Some researchers reported that germplasms from Mexico and Central America have high genetic diversity by using AFLP markers (Table 1).The different accessions from Mexico and Central America regions with high oil content and other characters are shown link with its productivity.These genetically contrary germplasm may provide critical resources for future genetic improvement of J. curcas.Zhang et al. [15] studied the genetic diversity within 240 samples by AFLP from three Asian countries, two African countries and different geographical regions in China.The germplasm of J. curcas showed a narrow genetic diversity in China and Southeast Asia with molecular polymorphism of 14.8%.Genotypic and phenotypic evaluation of a total of 182 accessions from Asia (91), Africa (35), South America ( 9) and Central America (47) was carried out to estimate the genetic variation.A very large genetic variation by DNA-marker in the pool of Central American accessions was observed as compared from other regions.According to Luis et al. [16] the Central American accessions can be considered as the most important source for plant breeding as they have highest phenotypic variation.Further, it is also stated that Mexico and Central America have highest genetic diversity in J. curcas than in other parts of the world because the Mexico and Central America (Mesoamerican region) may be a centre of origin and diversity of J. curcas [17].The genetic diversity study consisted of 114 accessions from 15 populations of 4 different species: J. curcas, J. costaricensis, J. gossypifolia and J. stevensii collected from Costa Rica were analysed by AFLP.The results showed that the species that obtained the highest average of polymorphic loci was J. curcas, followed by J. gossypifolia, J. costaricensis, and J. stevensii [18].A broad genetic base of 48 J. curcas germplasm from six different states in India was reported [19].Genetic and fatty acid variability in four datasets of accessions and pre-breeding lines of J. curcas was analysed.The results revealed that both genetic as well as fatty acid variability was significantly higher in the newly created pre-breeding lines and the level of fatty acid variability in these datasets was different and highly correlated to the level of their genetic variability as revealed by AFLP markers [20].

Randomly amplified polymorphic DNA (RAPD)
The RAPD technique is simple, rapid, low-cost and can be deployed even in the absence of any prior information related the genome or DNA of the plant.The RAPD technique has been used for assessment of genetic diversity for J. curcas (Table 2) and has revealed that low to moderate level of genetic diversity of Indian germplasms.Assessment of genetic diversity among 43 germplasm collected from different regions in India showed moderate level of genetic variation, however, wide genetic variation was observed between the Indian and Mexican genotypes [21].Hybrid conformity using RAPD markers was carried out for a backcross involving J. curcas and J. integerrima, thereby, indicating the potential of employing the RAPD analysis for pre-breeding and genetic enhancement of J. curcas through interspecific hybridization [22].A narrow genetic diversity in 192 J. curcas accessions of Brazil was revealed by using RAPD markers [23].

AIMS Environmental Science
Volume 5, Issue 5, 340-352.The genetic diversity and genetic structure analysis of 160 individuals from eight Kenya populations, suggested that the Kenya germplasms have a broad genetic base and are therefore, important and useful for breeding programmes of Jatropha [4].

Inter simple sequence repeats (ISSRs)
The ISSR analysis, is technically simple, it has been used for evaluating the genetic variation and genetic relatedness of the Jatropha species and high level of genetic variation is predicted in Jatropha by using ISSR markers (Table 3).The genetic variability among 332 J. curcas cultivated accessions from Brazil were investigated using ISSR primers revealed high genetic diversity of J. curcas at species level [24].A high level of genetic diversity in 224 J. curcas accessions in China was reported based on ISSR molecular profiles [25].Genetic diversity analysis of J. curcas accessions from Mexico revealed a high genetic diversity in germplasm [26].Characterization of Jatropha species occurring in India is using nuclear and organelle specific primers for supporting interspecific gene transfer.The J. tanjorensis is considered as a natural hybrid between J. gossypifolia and J. curcas via ISSR markers [22].

Simple sequence repeats (SSRs)
The SSRs (microsatellites), with their larger number and immense potential for variation, form an indispensable component of genetic diversity studies.There is a wide scope and utility for these SSR markers towards diversity estimation and marker assisted breeding of J. curcas (Table 4).A total of 182 accessions were evaluated at genetic and phenotypic level using SSRs for genetic diversity assessment revealed moderate diversity [16].Low genetic diversity was recorded among 192 J. curcas accessions collected from different geographical regions throughout Brazil using SSR markers [23].Very low genetic diversity was observed among the 58 accessions of J. curcas collected across China as out 17 genomic SSRs, only one was polymorphic [27].Pamidimarri DVNS et al. [28] analysed 12 microsatellite markers, and found seven polymorphic markers between the toxic and non-toxic varieties at molecular level.

Expressed sequence tag (EST)-SSRs
The EST-SSRs markers are important to investigate genetic diversity of J. curcas.The SSR (including EST-SSRs) have predicted lower genetic diversity of J. curcas germplasm as compared to AFLP, RAPD and ISSR makers in several research reports (Table 5).This is SSR markers identify the variation in repeat regions only whereas entire genome is used by AFLP, RAPD and ISSR makers for diversity analysis.Moderate level of genetic diversity was observed among 45 J. curcas accessions using EST-SSRs [29].The genetic relationships determined by EST-SSRs among 25 J. curcas accessions were grouped into three main clusters which reveals low genetic diversity among accessions [30].Out of 432 EST-SSR primer pairs, 269 were polymorphic among the Jatropha and Jatropha-related species [31].

Single-nucleotide polymorphisms (SNPs)
In recent years, the employment of SNP markers towards assessing population genetic structure in different species have increased.The unavailability of DNA sequence information restricts the use of SNPs for assessing the population genetic structure of the diverse J. curcas accessions.As SNPs show the most abundant source of genetic polymorphism, there is an immediate need to develop SNP markers and use them in various studies in J. curcas (Table 6).A narrow level of genetic diversity was observed among 148 global collections of J. curcas lines using SNPs [32].The genetic diversity analysis among 273 J. curcas accessions using SNP markers revealed the presence of low genetic diversity [33].Twenty candidate SNPs were identified from J. curcas accessions having low and high phorbol esters (PEs), and interestingly, one gene involved PE biosynthesis pathway was also identified [31].

Conclusions
The review suggests that the analysis of J. curcas germplasm via different molecular markers have led to identification of low genetic diversity of accessions in the Asian region and also a close clustering of African and Asian accessions highlighting the existence of a common ancestor.The low genetic base of the African and Asian accessions could be attributed to less introduction from other areas, the prevelance of asexual reproduction and/or occurrence of apomix.According to the [2], J. curcas distribution and spread at the tropical belt was via the Cape Verde Islands, which was further confirmed by analysing diversity employing various molecular markers.The South American, Mexican and Meso-American regions show the existence of rich allelic diversity with useful and novel genes.These accessions can be an important resource for improving genetic base of J. curcas.Although molecular markers suggest variations, however, only limited quantitative genetic variability is predicted [27].Hence, detailed evaluation of molecular diversity is the need of the hour, to identify divergent material for molecular breeding, construction of linkage maps, diversity analysis and QTL/association mapping for J. curcas.

Contributions by authors
All authors have equally contributed towards writing and have read and finalized the MS.

Table 1 .
Genetic Diversity of J. curcas by using AFLP Marker.

Table 2 .
Genetic Diversity of J. curcas by using RAPD Marker.

Table 3 .
Genetic Diversity of J. curcas by using ISSR Marker.

Table 4 .
Genetic Diversity of J. curcas by using SSR Marker.

Table 5 .
Genetic Diversity of J. curcas by using EST-SSR Marker.

Table 6 .
Genetic Diversity of J. curcas by using SNP Marker.