A full-length enriched cDNA library and expressed sequence tag analysis of the parasitic weed, Striga hermonthica
© Yoshida et al; licensee BioMed Central Ltd. 2010
Received: 2 December 2009
Accepted: 30 March 2010
Published: 30 March 2010
The obligate parasitic plant witchweed (Striga hermonthica) infects major cereal crops such as sorghum, maize, and millet, and is the most devastating weed pest in Africa. An understanding of the nature of its parasitism would contribute to the development of more sophisticated management methods. However, the molecular and genomic resources currently available for the study of S. hermonthica are limited.
We constructed a full-length enriched cDNA library of S. hermonthica, sequenced 37,710 clones from the library, and obtained 67,814 expressed sequence tag (EST) sequences. The ESTs were assembled into 17,317 unigenes that included 10,319 contigs and 6,818 singletons. The S. hermonthica unigene dataset was subjected to a comparative analysis with other plant genomes or ESTs. Approximately 80% of the unigenes have homologs in other dicotyledonous plants including Arabidopsis, poplar, and grape. We found that 589 unigenes are conserved in the hemiparasitic Triphysaria species but not in other plant species. These are good candidates for genes specifically involved in plant parasitism. Furthermore, we found 1,445 putative simple sequence repeats (SSRs) in the S. hermonthica unigene dataset. We tested 64 pairs of PCR primers flanking the SSRs to develop genetic markers for the detection of polymorphisms. Most primer sets amplified polymorphicbands from individual plants collected at a single location, indicating high genetic diversity in S. hermonthica. We selected 10 primer pairs to analyze S. hermonthica harvested in the field from different host species and geographic locations. A clustering analysis suggests that genetic distances are not correlated with host specificity.
Our data provide the first extensive set of molecular resources for studying S. hermonthica, and include EST sequences, a comparative analysis with other plant genomes, and useful genetic markers. All the data are stored in a web-based database and freely available. These resources will be useful for genome annotation, gene discovery, functional analysis, molecular breeding, epidemiological studies, and studies of plant evolution.
Striga hermonthica is an obligate root parasite belonging to the family Orobanchaceae, and is a major constraint of crop production in sub-Saharan Africa. S. hermonthica infests economically important crops such as sorghum, maize, millet, and upland rice, and the yield losses caused by this species have been estimated to cost as much as US$ 7 billion annually . However, methods for controlling S. hermonthica are not well established. Despite its agricultural importance, the molecular mechanisms controlling the establishment of parasitism are poorly understood.
The S. hermonthica life cycle is unique and well adapted to its parasitic lifestyle. The seeds need to be exposed to germination stimulants exudated from the host roots, such as strigolactones and ethylene; otherwise they can remain dormant in the soil for several decades . The seeds are tiny and possess limited amounts of nutrients, and this restricts their growth without a host connection. When a potential host is recognized through the sensing of strigolactones or other germination stimulants, the seeds that are close to the host roots (within 5 mm) can germinate. The germinated seedlings form haustoria, which are round shaped organs specialized in host attachment and penetration . The formation of haustoria also requires host-derived signal compounds. The haustoria penetrate the host roots and finally connect with the vasculature to rob the host plant of water and nutrients. This dramatic developmental transition from an autotrophic to a heterotrophic lifestyle occurs within several days.
Intensive efforts in the scientific community, mainly in the United States during the 1960s, lead to the identification of some germination stimulants. This was followed by the development of a "suicidal germination" strategy to eradicate Striga weeds . By this strategy, a germination stimulant (in this case ethylene) is mixed in the soil to trigger germination in the absence of the hosts. This approach was used successfully to eradicate Striga asiatica infestations in North Carolina. Although suicidal germination was effective for controlling S. asiatica, this approach was not applicable for African farmers due to the high cost of the strategy and the much larger scale of infestation.
Whole genome sequencing is a valuable approach to understanding an organism. The genome sequences of growing numbers of model and crop plant species have been published in recent years, providing new insights in plant biology. The development of new generation sequencing technologies has dramatically accelerated the speed of large-scale sequencing. However, the de novo sequencing of the whole genome of a non-model plant is still a challenging and laborious task . Expressed sequence tags (ESTs) are a less expensive alternative for gaining information about the expressed genes of an organism . In particular, the ESTs from a full-length enriched cDNA library provide the complete sequences of functional proteins .
This study aims to provide genome scale molecular resources for understanding the parasitic processes of the obligate parasite, S. hermonthica. We constructed a full-length enriched cDNA library from S. hermonthica and generated a large-scale EST dataset by reading the sequences of individual clones from both ends. The only other genus from the family Orobanchaceae with publically available EST data is Triphysaria . Triphysaria spp. are facultative hemiparasites, which are able to complete their life cycles without hosts. The comparison of our S. hermonthica EST dataset with those of Triphysaria and other non-parasitic plantspecies enabled us to identify the potentially parasite specific genes. Furthermore, our results provide the tools to analyze genetic diversity within S. hermonthica. We found 1,445 putative simple sequence repeats (SSRs) that could be useful as markers. We amplified the genomic regions flanking some of these SSRs from S. hermonthica individuals that were collected in different fields in Africa. The results revealed high sequence divergence in the S. hermonthica genomes. All the sequences and the annotation results are freely available on the internet .
Results and Discussion
Genome size of S. hermonthica
Full-length enriched cDNA library construction
RNA samples used for the S. hermonthica full-length enriched cDNA library construction.
Growth stage or treatment
At 3 d after strigol treamtment
At 3 d after co-incubation with rice roots
Leaves and stems
From mature plants parasitized on rice
Roots (secondary haustoria)
From mature plants parasitized on rice in rhizotron
From mature plants parasitized on rice
Axenically grown plants
Grown axenically for 1 month
Distribution of insert lengths in the S. hermonthica full-length enriched cDNA library.
To assess the proportion of the library containing full-length cDNA clones, we randomly picked 90 clones and sequenced them from both the 5' and 3' ends. These DNA sequences were analyzed against the Arabidopsis genome database using the blastx program. Of the 90 clones, 79 contained sequences similar to those of Arabidopsis genes (e_value < e-10), while the insert sequences of the other 11 clones did not show any similarity. The 5'- and 3'- sequences of the 79 clones were aligned with the homologous Arabidopsis cDNAs. The 5'-sequences of 62 clones contained ATG start codons at similar positions to those in the corresponding Arabidopsis homologs, and 59 possessed stop codons at the equivalent positions. Therefore, we estimated that approximately 75% of the clones in the S. hermonthica library encode full-length cDNAs. Among the 59 sequenced full-length clones, the average lengths of the 5'- and 3'-untranslated regions (UTRs) were 127 bp and 203 bp, respectively, and the longest 5'-and 3' -UTRs were 486 bp and 480 bp, respectively.
EST sequencing and statistical analysis
Summary of the S. hermonthica EST sequence analysis
Number of independent clones
Number of raw sequences
Number of high quality sequences
Number of unigenes
Average unigene length
Minimum unigene length
Maximum unigene length
Average number of ESTs per unigene
Maximum number of ESTs per contig
Number of superunigenes
with more than one unigene
with one unigene
Number of putative SNPs (pSNPs)
Number of putative SSRs (pSSRs)
Functional annotation of the unigene sequences
Comparative analysis with other plant genes
The S. hermonthica unigenes were compared with genes in other plant genomes, including A. thaliana, poplar (Populus trichocarpa), grape (Vitis vinifera), soybean (Glycine max), rice (Oryza sativa), sorghum (Sorghum bicolor), a moss (Physcomitrella patens), and an algae (Chlamydomonas reindardtii) [22–26]. Seventy-seven to seventy-nine percent of the S. hermonthica unigenes showed similarities with genes from other dicotyledonous plants (Arabidopsis, grape, soybean, and poplar), as detected by blastx (e_value < e-10). Approximately 75% of the unigenes have homologs in monocotyledonous plants (rice and sorghum), and approximately 65% and 38% showed blastx hits in the P. patens and C. reindardtii databases, respectively. These lower percentages of blast hits are consistent with the greater evolutionary distances from those organisms.
Summary of blast search results using S. hermonthica unigenes.
Number of hits
Of these 662 sequences, 73 show similarities to sequences in other databases such as rice, sorghum, soybean, Physcomitrella, UniRef90 or nr (the non-redundant peptide database from NCBI). We found no other homologs for the remaining 589 unigenes (Additional file 2). Since T. pusilla and T. versicolor are hemiparasitic plants, these 589 might include genes specific to parasitic plants. The ongoing project to sequence the genome of Mimulus spp. may help to narrow down the number of candidate genes that are involved in parasitism, because Mimulus spp. are non-parasitic members of the family Scrophulariaceae, which is taxonomically close to Orobanchaceae. The 2,389 unigenes (14%) that did not show significant hits with any known peptide sequences in the tested databases (including nr) are also listed in Additional file 2. These unigenes may include sequences that are specific to Striga.
Genetic diversity of the S. hermonthicasequences
S. hermonthica is an obligate outcrossing plant with high levels of morphological and genetic variation . The EST2uni program detected 9,299 putative single nucleotide polymorphisms (SNPs) among the S. hermonthica unigenes. To exclude the misidentification of sequencing errors as SNPs, only polymorphisms confirmed by at least 2 independent sequences were counted, although there is still the possibility that those polymorphisms occurred during cDNA synthesis. The average frequency of SNPs in the unigene sequences is 0.67%, or approximately 1 SNP per 1.5 kbp. Although these SNPs will need to be confirmed, these data will be useful for developing EST-SNP markers for S. hermonthica .
We found 1,445 di-, tri- or tetra-nucleotide microsatellites (or SSRs) among the S. hermonthica unigenes. The most frequent of these are the tri-nucleotide repeats (Additional file 3), which is in agreement with previous studies of other plant species [31–33]. The most frequent individual microsatellite repeat is AG (including TC, GA, and TC) (283, 19.6%) and the second most frequent is AC (including TG, CA, and GT) (218, 15.1%). The most frequent tri-nucleotide repeat is ATC (including TCA and CAT) (157, 11.0%) (Additional file 4).
The EST-SSR sequences are good candidates for genetic markers, which can be used for molecular diagnosis, for biotyping weeds, and for investigating the genetic diversity and population structures of S. hermonthica. To investigate whether the SSRs that we identified can be used as such markers, we designed primers using sequences flanking the putative SSRs and looked for polymorphisms by PCR. First, we pooled DNA samples extracted from the leaves of several plants in the same field and used the DNA pools as PCR templates. Of the 64 primer sets tested, 44 successfully amplified DNA bands. However, 26 primer sets (59%) produced smears or multiple bands that were not countable and only 18 primer pairs (41%) amplified clear separate bands (Additional file 5). The smeared bands may indicate heterozygosity and genetic diversity among the individual plants harvested from the same field. Therefore, we tested the individual plants for polymorphisms. Several markers that showed smear patterns from the pooled DNA templates actually amplified clear polymorphic bands from individual plants in the same population (Additional file 6). These data verify that S. hermonthica is a highly adaptable weed that has maintained a high degree of genetic variation and plasticity, to survive in various ecosystems .
Genetic distances among S. hermonthicapopulations with different hosts
Genetic diversity among S. hermonthica populations collected from various locations and host plants.
No of repeats
No of alleles
The results of the sequencing and analysis of the S. hermonthica ESTs are freely available online from our web-based database . The web interface was based on the original EST2uni web site . The database contains features for complex query searches and a blast search. A page for each unigene consists of its sequence, contig images, results of blast similarity searches, lists of detected SSRs and SNPs, and GO categorizations. In addition, the homologs of each unigene are linked to outside databases such as The Arabidopsis Information Resource (TAIR) . This web-based database will be a powerful tool for the detailed analysis of S. hermonthica genes.
This paper provides large scale EST information about S. hermonthica, which can be used in studies of parasitic plants, plant-plant interactions, weed management, and plant evolution. Comparative analyses between S. hermonthica and other plant genomes should allow us to identify genes responsible for plant parasitism. These genes are of particular interest as potential targets for future pest management strategies against noxious parasitic weeds. Our analysis also highlights the intra-species genetic diversity of S. hermonthica. A more detailed analysis might contribute to future breeding programs to develop resistant crops, since genetic variation in the weed population could be the main factor allowing the quick breakdown of resistance. In summary, our study provides powerful analytical tools for the molecular analysis of the parasitic weed S. hermonthica. Our data will alsocontribute to the annotation of genes identified by the on-going genome-scale sequencing of the parasitic genera from Orobanchaceae.
Plant materials and growth conditions
S. hermonthica seeds collected from a sorghum field in 1994 in Kenya were provided by Dr. A. G. Babiker (Univ. of Sudan, Khartoum, Sudan). Rice seeds (Oryza sativa L. subspecies japonica, cultivar Koshihikari) were originally obtained from the National Institute of Agricultural Sciences (NIAS, Tsukuba, Japan). S. hermonthica plants parasitizing rice were grown in rhizotrons as described previously  or in soil (1:1 mixture of vermiculite: clay). For the axenic culture of S. hermonthica, seeds were sterilized with 20% bleach solution (approx. 6% NaOCl) for 5 min and washed thoroughly with sterile water. The sterile seeds were preconditioned on MS medium with 1% sucrose and 0.5% phytagel (Sigma) at 26°C for 7 to 10 days in the dark and germination was stimulated by the exogenous application of 5 μl 1 μM Strigol per plate. Sterile S. hermonthica plants were grown on the same medium at 26°C with a 16-h photoperiod, and the medium was renewed every 3 weeks.
Determination of nuclear DNA content
The nuclear DNA content was analyzed with a flow cytometer (Partec PA, Tokyo, Japan). Soil-grown S. hermonthica (host: rice) leaves were chopped with a razor blade into small pieces and analyzed according to the previously published method . Leaves of Arabidopsis (ecotype Col -0) were used as the control.
The S. hermonthica tissues and developmental stages used for RNA extraction are listed in Table 1. S. hermonthica RNAs were extracted using a modified cetyl trimethylammonium bromide (CTAB) method. Briefly, plant tissues were ground under liquid nitrogen and suspended in 5 × volumes of CTAB solution (2% CTAB, 2% polybinylpyrrolidone (PVP), 25 mM ethylenediaminetetraacetic acid(EDTA), 2 M NaCl, 1% beta-mercaptoethanol, 100 mM Tris-HCl (pH 8.0)) and phenol:chloroform (5:1, pH 4.7, Sigma). The mixtures were shaken at 55°C for 5 min. After 10 min centrifugation, the aqueous phase was extracted with an equal volume of phenol:chloroform, and subsequently with chloroform. The RNAs were precipitated by adding 0.25 volumes of 10 M LiCl. The RNA pellet was washed with 70% ethanol and then dissolved in nuclease-free water. Samples were subsequently purified using the PureLink RNA mini kit (Invitrogen) according to the manufacture's instructions. To obtain mRNA for library construction, total RNAs from each tissue and developmental stage were mixed and purified using an mRNA purification kit (GE) according to the manufacture's instructions. The quality and quantity of the total RNA and the mRNA were assessed by measurements of OD230, OD260, and OD280, followed by visual checking by electrophoresis.
Library construction and EST sequencing
The construction of the normalized, full-length enriched library was carried out in Evrogen (Russia). The cDNA normalization was conducted using a Duplex-specific nuclease (DSN)-based method, and full-length cDNAs were enriched using the SMART™ technology (Clontech). Each cDNA was inserted into the pAL17.3 vector. Sequencing of randomly picked clones was performed in the Genome Center at Washington University using the ABI3730 capillary sequencer.
The EST sequences were automatically trimmed, clustered and annotated using the EST2uni analysis pipeline . Sequence assembly was performed using the CAP3 program with the default parameter settings . Blast searches were performed with NCBI blast program against the databases shown in Table 4. The S. hermonthica online database was constructed based on the EST2uni web program with slight modifications.
SSR markers and genetic diversity analysis
where n is the number of populations sampled, p lu is the frequency of uth allele at the lth locus, and f is the inbreeding coefficient (association between alleles) at the lth locus. The Polymorphism Information Content (PIC) was estimated as , where the p lv is the frequency of the vth allele at the lth locus. The phylogenetic UPGMA tree was generated based on a matrix of the frequencies and distances using the LogSharedAllele algorithm with the PowerMarker v.3.25 program. Bootstrap analysis was performed using the software package WINBOOT .
We thank Dr K. Mochida for advice on bioinformatics, K. Akiyama and T. Sakurai for web-server maintenance, and Dr A. G. Babiker for providing the S. hermonthica seeds. This work was funded by grants from the Gatsby Charitable Foundation, the RIKEN president fund, and KAKENHI (19780040 and 21780044 to SY and 19678001 to KS). JKI is supported by the MEXT scholarship program.
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