Yellow lupin (Lupinus luteus L.) transcriptome sequencing: molecular marker development and comparative studies
© Parra-González et al.; licensee BioMed Central Ltd. 2012
Received: 9 May 2012
Accepted: 13 August 2012
Published: 24 August 2012
Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species.
Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession’s origin.
L. luteus deep transcriptome sequencing will facilitate the further development of genomic tools and lupin germplasm. Massive sequencing of cDNA libraries will continue to produce raw materials for gene discovery, identification of polymorphisms (SNPs, EST-SSRs, INDELs, etc.) for marker development, anchoring sequences for genome comparisons and putative gene candidates for QTL detection.
L. luteus is a member of the genistoid clade of the Fabaceae family (2n = 52), which is the third largest flowering plant family with over 700 genera and 20,000 species . The genus Lupinus comprises more than 200 annual and perennial herbaceous species of which several are cultivated and used as human food or animal feed . Some of them show high levels of tolerance to biotic and abiotic stresses. For instance, L. hispanicus, a wild relative of L. luteus, has high tolerance to diseases and good adaptation to poor soils, but high levels of bitter alkaloids and low agronomic yields . Lupins are considered to be of polyploid origin which probably played a crucial role in the evolution of their ancestral genomes [4, 5]. The major cultivated species are the old world lupin L. albus (white lupin), L. angustifolius (narrow-leafed lupin), L. luteus (yellow lupin), and the new world species L. mutabilis (pearl lupin or tarwii) .
L. luteus is widely distributed across the Mediterranean region, has shallow soil requirements, and cultivated accessions have variable seed yields in Mediterranean environments . In addition, yellow lupin seeds have the highest protein content and twice the cysteine and methionine content of most lupins [8, 9]. However, despite its highly nutritional qualities, there is a lack of genetic and molecular tools to aid the genetic breeding of this species.
EST sequencing has accelerated gene discovery when genome sequences are not available, facilitating gene family identification and development of molecular markers. Next-generation sequencing has generated enormous amount of expressed sequence data for a wide number of plant species, specially minor or orphan crops . For example, EST and genome sequencing of lentil and chickpea would not have been feasible without next-generation sequencing [11, 12]. The lower cost and greater sequence yield has allowed the identification of candidate genes, even when they are expressed at low levels [13, 14].
Research on plants, animals and fungi has shown that sequences of expressed genes are often widely transferable among species, and even genera, allowing wide genome comparative mapping studies [15, 16]. For instance, the combination of orphan crop EST sequences with model plant genetic and genomic resources, such as Lotus japonicus (Japanese trefoil) and Medicago truncatula (barrel medic), has identified macro- and micro-scale synteny, discovered new genes and alleles, and provided insights into genome evolution and duplication [17, 18]. Comparisons between ESTs and gene sequences among several legume species have allowed comparative genome studies between L. albus and M. truncatula, and L. angustifolius and Lotus japonicus.
Several molecular markers have been developed for Lupinus species, including RFLPs, ITAPs (Intron targeted amplified polymorphic sequences), and AFLPs, which have been used to build genetic linkage maps in L. albus and L. angustifolius[20, 21]. So far, a limited number of SSRs have been developed for Lupinus species, and very few of these are EST-SSRs i.e. SSRs that are found in expressed sequences [21–23]. Genomic and EST-SSRs have been widely used for the improvement of major crop plants, but their initial development with traditional methods requires significant research investment. Now, an almost unlimited number of genomic and EST-SSRs can be readily developed from next-generation sequencing approaches within most crop species, including orphan crops such as lupin [24–28]. The expressed nature of EST-SSRs allows the annotation of these markers with putative functions by sequence homology and potentially reduces the genetic distance between marker and causal gene to 0 cM. [29, 30]. For instance, the length of a dinucleotide SSR at the 5’ UTR of a waxy gene has been associated with amylase content in rice [31, 32]. EST-SSRs have also been associated with several disease resistant genes in wheat and rice [33, 34] and a number of agronomically important traits in cotton, maize and narrow-leafed lupin [35–37].
In this study, we constructed 454-EST libraries, carried out comparative studies between L. luteus and model legume species, and mapped L. luteus expressed sequences on the M. truncatula chromosomes. Alignments between our putative L. luteus genes and their homologs in M. truncatula, coupled with amplifications of intergenic regions provided evidence of microscale synteny between both species. In addition, we developed EST-SSR markers and illustrated their utility within diverse accessions of yellow lupin. Finally, because these EST-SSR markers are gene-based, they are also likely conserved among different species of lupin. We evaluated EST-SSR utility in the other Lupinus species, L. mutabilis and L. hispanicus.
Library construction and 454 sequencing
cDNA libraries were constructed from mRNA isolated from two tissue pools. Pool 1 (L1) included young leaves, buds and flowers, and pool 2 (L2), seeds in different developmental stages. RNA from pool 1 and 2 was isolated separately according to the guanidine hydrochloride method . Both RNAs were assessed for quality by inspecting rRNA bands on an Agilent Bioanalyzer (Agilent Technologies, CA, USA).
cDNAs libraries were normalized and prepared using procedures for Roche 454 Titanium sequencing (Roche, Branford, CT, USA). cDNAs from L1 and L2 were synthesized using the stratagene AccuScript High Fidelity RT-PCR System (Agilent Technologies, CA, USA) and 5’ specific adaptors from Clontech. A cDNA normalization was used to improve coding sequence coverage, avoid AT homopolymer artifacts, and reduce excessive 3’ end transcript sequence . cDNAs from both libraries were amplified using the Clontech Advantage HF system (Clontech Laboratories, Inc) and normalized utilizing the Evrogen Trimmer cDNA Normalization kit (Axxora, LLC). These un-cloned, normalized cDNA libraries were prepared for pyrosequencing according to the manufacturers specifications. One 454 run of sequencing was performed for each EST library (454 Life Sciences, Roche).
Separate transcriptome assemblies of L1 and L2 libraries were created using Newbler (de novo sequence assembly software of Roche 454 Life Sciences) and the cDNA option. A third assembly (L1L2) was completed using the reads from both libraries to avoid sequence redundancy when developing SSR markers. Reads were initially assembled into contigs and contigs into isotigs, which are equivalent to splice transcriptional variants. Sequence read EST data for L1 and L2 are available through the Sequence Read Archive (SRA055806).
EST annotation, function and comparative genomics to other species
Comparing isotigs from the combined assembly (L1L2) to the curated non-redundant protein database (nr, http://www.ncbi.nlm.nih.gov; blastx, e value ≤ 1e-10) provided a functional annotation for each isotig. Alignments of translated-isotigs and proteins with an e-value ≤ 1e-40 were considered to have significant homology. Annotations of the aligned proteins were extrapolated to annotate our putative isotig sequence using Blast2GO (http://www.blast2go.org). To directly compare the lupin isotigs to the genes of other crops, blast searches were also used to compare isotig translations to Arabidopsis thaliana, Glycine max, Medicago truncatula and Lotus japonicus Gene Indices (tblastx, e-value ≤ 1e-10). Isotigs were also annotated using Gene Ontology (GO) annotations from InterProScan (http://www.ebi.ac.uk).
In silico lupin EST mapping and microsynteny
Blast was used to compare lupin EST isotigs to the Medicago genome 3.0 release (≤ 1e-20, HSP identity 60% and HSP length > 50 bp.) The Blast results were visualized using GBrowse where positive matches were displayed as featured tracks on GBrowse 2.13 . The presence of microsynteny was evaluated by PCR amplification of putatively conserved chromosome blocks between L. luteus and M. truncatula. Where alignments between yellow lupin and M. truncatula were identified, specific primer pairs were designed to amplify intergenic regions (Additional file 1). These targeted, intergenic regions were PCR amplified from two L. luteus and one L. hispanicus accessions using 100 ng of genomic DNA in 20 ul reactions containing 100 ng of genomic DNA, 0.2 mM dNTPs, 2 mM MgCl2, 1X PCR buffer, 2.5% DMSO, 1 U taq polymerase (Agilent Technologies, Santa Clara, CA) and 5 pmoles of each forward-reverse primer pair. PCR reactions were carried out following a touchdown protocol on a peltier thermalcycler (MJ Research, Inc.) 94°C for 5 min; 5 cycles of 1 min at 94°C, 1 min at 55-65°C decreasing 1°C per cycle, 2 min at 72°C followed by 35 cycles of 1 min at 94°C, 1 min at 50-60°C and 2 min at 72°C. Amplicons were purified from agarose gels and sequenced. These amplified, intergenic sequences were mapped onto the M. truncatula genome and visualized within a local implementation of GBrowse (Additional file 1). Positive PCR microsynteny set of primers were additionally tested against a screening panel consisting of six diverse accessions of L. luteus to search for polymorphisms among yellow lupin genotypes (Additional file 2).
Identification of EST-SSRs
SSR containing lupin isotigs were identified using the software MISA (MIcroSAtellite, http://www.pgrc.ipk-gatersleben.de/misa). SSR search criteria changed according to repeat types. Di-, and tri-repeats were selected with a minimum length of 12 and 15 nucleotides, respectively. For tetra-, penta- and hexa-repeats, the minimum length was 20 nucleotides. Mononucleotide repeats were not considered due to the possibility of 454 homopolymer sequencing errors associated with this technology. To estimate the amount of SSRs included in coding regions, L1L2 sequences were analyzed using ESTScan (http://www.ch.embnet.org/software/ESTScan.html). ORFs discovery was carried out using default parameters and putative cd sequences scanned for SSR motifs using MISA.
From all selected-SSR containing isotigs, only sequences with a motif of at least 7 repeat units were considered for primer design. Flanking primer pairs were designed using the Primer3 software available at NCBI v.3.12 with expected amplicon lengths between 150 - 500 bp. Oligonucleotides were synthesized by IDT (Integrated DNA Technologies, Inc.).
Evaluation and utility of EST-SSRs
EST-SSR polymorphisms and transferability were evaluated on the germplasm screening panel previously mentioned, and one accession each of L. hispanicus and L. mutabilis.
DNAs were extracted following standard procedures , quantified using a synergy HT Multimode Microplate Reader (Biotek Instruments, Winooski, VT), and diluted to 50 ng/ul in TE buffer (10 Mm TRIS, 1 mM EDTA pH 7.5). DNA amplification was carried out in 20ul PCR reactions as described above.
PCR products were separated on 6% denaturing polyacrylamide gels, run in TBE buffer at 60 watts for 3–4 hours and visualized using silver stain procedures. DNA amplicons of six EST-SSR primer-pairs used in the polymorphism screening were purified from agarose gels and sequenced in an Applied Biosystems 3730xl DNA Analyzer sequencer (Applied Biosystems, Carlsbad, CA). Amplicon sequences from each EST-SSR primer-pairs were aligned using Geneious version 126.96.36.199 (Biomatters Ltd., using default parameters).
Characteristics of 50 EST-SSR primers developed in L. luteus. Shown for each primer pair are the library specificity, repeat motif, forward and reverse sequence, allele range size (bp), number of alleles, amplification in other Lupin species, and annotation
Forward primer (5′-3′)
Reverse primer (5′-3′)
No of alleles
Pollen-specific protein SF3
Delta-8 sphingolipid desaturase
18S ribosomal RNA gene
BSD domain-containing protein
Alphavirus core protein family
LPA2 (low psii accumulation2)
Lipase class 3 family protein
f-box family protein
Small nuclear ribonucleoprotein
Ser/thr-protein kinase AFC2
Zinc finger, Transcription factor
Seed and leaf-flower EST libraries
cDNA 454 assembly statistics of L1, L2 and L1L2 L. luteus libraries
Number of sequenced bases
Number of reads
Number of reads assembled
Read average length
Number of contigs
Contig average size
Number of isotigs
Isotig average size
Number of isogroups
Isogroup average size
Average number of reads by contig
Gbrowse mapped sequences
Functional classification and in silico comparative genomics
In silico mapping of lupin ESTs on M. Truncatula chromosomes
Alignment of L. luteus isotig sequences to the M. truncatula genome (Blastn; ≤1e-20; MT3) was used to identify local genomic variability between our ESTs and a related, well-annotated reference genome sequence. The alignments were visualized using GBrowse (v. 2.13) with the Blast matches displayed as feature tracks. A total of 25,400 sequences (46%) from L1L2 had a positive match with MT3 and were distributed heterogeneously on the M. truncatula chromosomes. Chromosomes 3 and 1 had the highest (34,636) and lowest (16,055) number of matches, respectively. Each L. luteus sequence was mapped to an average of 3.7 positions on the Medicago genome.
When these markers were evaluated on the screening panel of diverse germplasm accessions, 10 had length polymorphism for these intergenic regions (Additional file 1). In addition to EST-SSRs, this new Conserved Microsynteny (CMS) marker could be valuable resource for crop improvement with molecular markers.
Identification of EST-SSRs
Features of EST-SSRs identified in assembled L1L2 L. luteus library
Total number of examined sequences
Estimated transcriptome screened (kbp)
Number of sequences containing SSRs
Number of identified SSR
Number of EST-SSRs in coding regions
Number of sequences containing more than 1 SSRs
Number of SSRs present in compound formation
Frequency of SSR in transcriptome
Distribution of repeat types and number of repeats within the L1L2 L. luteus library
Number of repeat units
Evaluation of EST-SSRs within yellow lupin and other lupin species
Next-generation sequencing has reduced the existing gap between major crop genomic platforms and the limited resources that are currently available for orphan crops . Complete transcriptome sequencing has generated species specific molecular markers, in silico expression analyses, gene discovery, and phylogenetic relationships [43, 44].
In this research, we used 454 cDNA sequences to assemble transcriptomes of two tissues (L1 and L2) of yellow lupin. We recovered a large number of previously unknown and uncharacterized yellow lupin gene sequences (Table 2). The total number of sequences for the combined library was mostly additive from L1 and L2. The L1 library favored the inclusion of longer 3’UTR regions, and thus, reducing the amount of coding sequences needed to assemble longer combined contigs (L1L2). As a consequence, two or more sequences belonging to the same transcript may not be assembled together, causing an overestimation of expressed sequences. The larger amount of 3’UTR regions for L1 is also in agreement with the lower GC content, condition typically associated with untranslated regions [45, 46]. Undoubtedly, a number of expressed sequences are tissue specific and will not assemble into combined contigs. For instance, several genes related to seed dormancy and germination are not expressed in vegetative and floral tissues [47, 48]. The same specificity was observed in a number of tissues and plant species [49–51]. The assembly of L1L2 generated 55,309 isotigs of which 30,811 had similarity to putative proteins found in other plant species. Comparative studies carried out against L. japonicus, M. truncatula and G. max showed a total of 31,520 lupin sequences similar to at least one of the model legume databases and 22,219 were similar to all of them. Lotus and Medicago belong to the Galegoid subclade, which includes mostly temperate legume species . Glycine is a member of the Phaseoloid subclade which comprises mostly tropical species . Lupins belong to the Genistoid subclade, which is sister (and distant) to most of the described Papilionoid subclades; especially those containing most domesticated species .
Although micro-repeat motifs are frequent in plant genomes and their respective transcriptomes, the frequency of SSR discovery depends on the search criteria [42, 54–56]. We analyzed 55,309 lupin isotig sequences using MISA and identified 2,796 SSR motifs with an average frequency of one SSR per 17.75 kbp. Tri-nucleotide repeats were the motifs most frequently found in L. luteus expressed sequences. Similar results have been reported in numerous plant species [26, 28, 54, 55, 57]. The abundance of trimeric EST-SSRs has been attributed to the absence of frameshift mutations when there is length variation in these SSRs . Indeed, 1,435 EST-SSRs were discovered within coding regions of the gene. Among tri-nucleotide repeats, AT-rich motifs were the most predominant ones (74.5%), which have also been observed in soybean, Citrus and Arabidopsis [54, 57]. For di-nucleotide repeats, AT was the most frequently observed motif, contrasting with results from Arabidopsis, soybean, maize, rice, wheat and barley where AC/GT were the most frequent repeats [26, 28, 54, 55, 57]. The high proportion of untranslated sequences (specifically 3’UTR), mainly contributed from the L1, could explain the bias toward A/T-rich repeat sequences observed in yellow lupin. There were no CG repeats in the lupin sequences, similar to results obtained in barrel medic , rice, corn, soybean , wheat , Sorghum , Arabidopsis, apricot and peach .
We used GBrowse to visualize lupin ESTs aligned to the M. truncatula chromosomes (Figure 3). This approach potentially identifies paralogs sequences and allows color-coded alignment by BLAST significance . A total of 25,400 L. luteus contigs were localized and found to be distributed across the entire Medicago genome with chromosomes Mt1 and Mt3 having the highest number of gene matches. Each yellow lupin sequence was mapped to an average of 3.7 locations, which may correspond in part to rounds of genome duplications previously described for the Medicago genome . Understanding syntenic relationships among species is essential to exploit the available tools developed for comparative genomic analysis. Using this approach, we created a new method of developing molecular markers, markers that are based on conserved microsynteny (CMS) between orphan and model species. Genome comparisons among M. truncatula, G. max and L. japonicus have shown that, in general, most genes in Papilionoid legume species are likely to be found within a relatively long syntenic region of any other Papilioniod species . Positive amplification and sequencing of L. luteus intergenic regions, based on PCR primers located on M. truncatula adjacent genes, suggested the existence of microscale synteny between these legume species. Roughly 40% of the targeted intergenic L. luteus regions amplified, points out the usefulness of conserved legume chromosome blocks for genomic studies of orphan crops. Although some primer pairs failed to amplify, poor amplification could be a consequence of non-synteny, but also other technical limitations could also explain negative PCR results. For instance it is known that non-coding DNA regions are highly variable among species [63, 64], and negative PCR amplifications could easily due to excessively long L. luteus intergenic regions.
Few studies have reported the use of EST-SSRs in Lupinus species [19, 21, 22]. Most efforts have focused on genetic linkage mapping and in diversity studies in L. angustifolius, L. albus and L. luteus. To validate our L. luteus polymorphic markers we tested 50 EST-SSRs on a population of 64 genotypes of L. luteus. An analysis of genotypic diversity illustrated the existence of several clusters within L. luteus germplasm. The lack of a clear pattern following the geographical accession origin (country) could be explained by three reasons. 1) The number of accessions may not have been large enough to allow a clear pattern to emerge. 2) L. luteus is widely distributed across the Mediterranean region, mainly due to human introductions . This situation could have homogenized natural genetic distinctiveness, leaving mostly population subdivisions based on breeding histories. 3) Finally, it is possible some accessions could have been misclassified; and thus, obscuring an existing geographical clustering pattern.
We observed that a number of high yellow lupin EST-SSR amplified fragments in two other lupin species, L. hispanicus and L. mutabilis (Table 1). The high number of transferable markers between L. luteus and L. hispanicus confirmed their closer genetic relationship [5, 65] than L. luteus and L. mutabilis. The two closely related species have the same chromosome number (2n = 52) and are still interfertile, generating a natural hybrid called hispanicoluteus. Phylogenetic studies have placed new and old world lupins into two different clades [5, 65, 67]. Thus, most EST-SSRs amplified in L. mutabilis (2n = 48), the only cultivated new world lupin , should have high transferability rates to other lupin species, such as L. albus and L. angustifolius. The understanding of the genetic diversity among other close relative lupin species will facilitate the transfer of favorable variation into cultivated species. For instance, L. hispanicus has been suggested as a reservoir of favorable variation for a number of biotic and abiotic stresses currently affecting L. luteus[68, 69].
L. luteus deep transcriptome sequencing will facilitate the further development of genomic tools and lupin germplasm. Massive sequencing of cDNA libraries will continue to produce raw materials for gene discoveries, identification of polymorphisms (SNPs, EST-SSRs, INDELs, etc.) for marker development, anchoring sequences for genome comparison studies and putative gene candidates for QTL detection. We are also exploiting the microsyntenic regions observed among L. luteus and legume model species to saturate yellow lupin linkage maps by amplifying conserved regions across legume species. The utilization of these tools will allow transforming L. luteus into a valid temperate legume crop alternative.
This research was funded by the National Commission for Scientific & Technological Research (FONDECYT Project No.1090759) and CONICYT Regional/GORE Araucanía/CGNA/R10C1001, Chile. We thank Héctor Urbina for his assistance on L. luteus sequence assemblies.
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