Development, characterization, genetic diversity and cross-species/genera transferability of ILP markers in rubber tree (Hevea brasiliensis)

To construct a linkage map enriched with tapping panel dryness (TPD)-related markers, we firstly utilized rubber tree ESTs associated with TPD to develop intron length polymorphism (ILP) markers. In this study, 52 new ILP markers were further developed. Together with the ILP markers previously reported, 102 ILP markers developed from TPD-related ESTs were analyzed within 39 Hevea germplasm in detail. The PCR success rate and polymorphism rate of ILP markers was 97.06 and 61.62 %, respectively. The results based on PCR amplification and sequence analyses provided the evidences on cross-species/genera transferability of rubber tree ILP markers. The average polymorphic information content (PIC) values of 39 Hevea germplasm were about 0.1719, indicating that the genetic base of Hevea germplasm selected in this study was very narrow. Among 39 Hevea germplasm, the PIC value of wild rubber tree accessions was the highest, followed by Hevea species and cultivated rubber tree clones. Based on the similarity coefficient of ILP markers, 39 Hevea germplasm were divided into three groups including cultivated clones, wild accessions and Hevea species, suggesting that the classification was generally related to the characterization of Hevea germplasm. The ILP markers developed in this study further enriches the number of molecular marker in rubber tree, and the ILP markers will have a wide application in DNA fingerprinting, genetic diversity, marker-assisted selection and genetic mapping, etc. Moreover, the ILP markers transferred cross-euphorbiaceae family might be utilized in cassava, castor bean and physic nut.     

Development of EST-PCR markers for <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis> chromosome 2Ai#2 and their application in characterization of novel wheat-grass recombinants

A series of expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers specific to chromosome 2Ai#2 from Thinopyrum intermedium were developed in this study using a new integrative approach. The target alien chromosome confers high resistance to barley yellow dwarf virus (BYDV), which is a severe virus disease in wheat. To generate markers evenly distributed on 2Ai#2, a total of 105 primer pairs were designed based on mapped ESTs from 8 bins of wheat chromosome 2B with intron-prediction by aligning ESTs with genomic sequences of the new model plant Brachypodium distachyon. Eight and seven polymorphic markers on the short arm and the long arm of chromosome 2Ai#2, respectively, were obtained with a polymorphism rate of 14.3%. These chromosome 2Ai#2-specific EST-PCR markers were then used in tracing and exploring the structural variation of the alien chromosome in the population derived from the immature embryo culture of the cross between N452, a 2Ai#2(2D) substitution line, and common wheat CB037. Two centric fusion of translocations involving 2Ai#2 short or long arm with wheat chromosome 2D and some new genetic stocks including telosomes with the alien chromosome short or long arm were identified in the SC₃ generations, which provided basic materials to further study the mechanism of the BYDV resistance. BYDV tests in two field seasons suggest that the BYDV resistance was mainly conferred by the short arm, gene interaction on both arms of the alien chromosome was discussed.     

ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

Background  

In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.     

Development of EST-SSR markers in castor bean (Ricinus communis L.) and their utilization for genetic purity testing of hybrids

Expressed sequence tag (EST) databases offer opportunity for the rapid development of simple sequence repeat (SSR) markers in crops. Sequence assembly and clustering of 57?895 ESTs of castor bean resulted in the identification of 10?960 unigenes (6459 singletons and 4501 contigs) having 7429 SSRs. On an average, the unigenes contained 1 SSR for every 1.23?kb of unigene sequence. The identified SSRs mostly consisted of dinucleotide (62.4%) and trinucleotide (33.5%) repeats. The AG class was the most common among the dinucleotide motifs (68.9%), whereas the AAG class (25.9%) was predominant among the trinucleotide motifs. A total of 611 primer pairs were designed for the SSRs, having repeat length more than or equal to 20 nucleotides, of which a set of 130 markers were tested and 92 of these yielding robust amplicons were analyzed for their utility in genetic purity assessment of castor bean hybrids. Nine markers were able to detect polymorphism between the parental lines of nine commercial castor bean hybrids (DCH-32, DCH-177, DCH-519, GCH-2, GCH-4, GCH-5, GCH-6, GCH-7, and RHC-1), and their utility in genetic purity testing was demonstrated. These novel EST-SSR markers would be a valuable addition to the growing molecular marker resources that could be used in genetic improvement programmes of castor bean.     

Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed ‘intron-flanking‘ EST–PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST–PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron. Electronic Supplementary Material Supplementary material is available for this article at     

Novel Chloroplast Microsatellite (cpSSR) Markers for Genetic Diversity Assessment of Cultivated and Wild Hevea Rubber

Understanding the genetic diversity and population structure of Hevea is of great importance for managing its conservation and for utilization of rubber genetic resources. In this study, we investigated the genetic diversity and population structure of eight populations of Hevea rubber genotypes from Malaysia (MY), India (IN), Sri Lanka (LK), Indonesia (ID), France (FR), Thailand (TH), Brazil (BR), and China (CN), in addition to individual primary clones, using 10 nuclear and 11 polymorphic novel chloroplast microsatellite markers (nSSRs and cpSSRs, respectively). The BR population exhibited the greatest genetic diversity (H _e ) for both nSSR (0.841) and haploid genetic diversity (cpSSR; 0.207) markers. Bayesian analysis was applied to infer genetic structure based on nSSR data using STRUCTURE software, and a neighbor-joining tree was used to construct an unrooted phylogram based on Nei’s genetic distance that clustered these Hevea genotypes into three groups. Six polymorphic cpSSR markers produced 13 alleles and eight chlorotypes. Seven chlorotypes, A, B, C, D, E, F, and H were detected among Brazilian populations from Acre (AC), Rondônia (RO), and Mato Grosso (MT) locations. The G and H chlorotypes were found in Asiatic genotypes or Wickham clones and only one genotype, 15 AC-F-7 38-62 from AC location. These results provide valuable data for in situ or ex situ conservation and utilization of germplasm collections for breeding programs.

     

Genetic and epigenetic uniformity of polyembryony derived multiple seedlings of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

Hevea brasiliensis Muell. Arg (Para rubber tree) is a tropical tree species of Amazonian origin widely cultivated in several parts of the world for natural rubber, a highly priced commodity inevitable for the world rubber industry. Large, tree to tree variation in growth and latex yield among individual plants of high yielding Hevea clones is a common phenomenon observed in mature rubber plantations. The genetic heterogeneity of the seedlings which are used as rootstocks for propagation through budgrafting is considered as a major factor  responsible for this variation. In order to minimize this variation, attempts were made to develop highly uniform rootstock material via an in vitro technique by inducing zygotic polyembryony in Hevea. Immature open pollinated fruits of a high yielding clone RRII 105 were cultured by half ovulo embryo culture technique. Multiple embryos were induced from the 8–10-week-old zygote with a novel combination of gibberellic acid (GA₃), kinetin, and zeatin. Plantlets were successfully generated from the multiple embryos and raised in the field post hardening. Screening using genetic and epigenetic molecular markers revealed that the multiple seedlings developed are highly uniform and are of single zygotic origin. Development of plants having genetic and epigenetic uniformity suggests that this technique is ideal for raising uniform rootstock material in Hevea which may significantly reduce intraclonal variations. Moreover, these plants could serve as ideal material for physiological and molecular investigations towards the understanding of  stock–scion interaction process in rubber.     

Discovery, validation, and in silico functional characterization of EST-SSR markers in Eucalyptus globulus

Eucalyptus globulus is the most commonly planted hardwood species for pulpwood in temperate regions. We aimed to develop and characterize functional molecular markers for population genetic analyses and molecular breeding in this model tree species. Public expressed sequence tag (EST) databases were screened for nonredundant sequences to predict putative gene functions and to discover simple sequence repeats (EST-SSRs), which were then validated in E. globulus and six other Eucalyptus species. A total of 4,924 nonredundant sequences were identified from 12,690 updated E. globulus ESTs. Approximately 19.3% (952) were unigenes and contained 1,140 EST-SSR markers, which were mainly trimeric (58.6%). A set of 979 primers for putative SSR markers was designed after bioinformatic analysis. The predicted functions of these ESTs containing SSR were classified according to their gene ontology (GO) categories (biological process, molecular function, and cellular component). GO categories were assigned to 226 ESTs (30.2%). Most ESTs containing SSR (78.7%) had significant matches (E ≤ 10⁻⁵) with the nonredundant protein database using BLASTX. From a set of 56 random primer pairs, 37 could be validated in eight E. globulus genotypes and were also tested for cross-transferability to other six Eucalyptus species (Eucalyptus grandis, Eucalyptus saligna, Eucalyptus dunnii, Eucalyptus viminalis, Eucalyptus camaldulensis, Eucalyptus tereticornis). Seventeen polymorphic EST-SSR markers for E. globulus were evaluated in 60 unrelated trees, being representative of the species’ natural distribution. As a result, six highly informative markers were proposed for genetic diversity analyses, fingerprinting, and comparative population studies, between different species of E. globulus.     

Identification and characterization of genes associated with tapping panel dryness from <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>latex using suppression subtractive hybridization

Background  

Tapping panel dryness (TPD) is one of the most serious threats to natural rubber production. Although a great deal of effort has been made to study TPD in rubber tree, the molecular mechanisms underlying TPD remain poorly understood. Identification and systematical analyses of the genes associated with TPD are the prerequisites for elucidating the molecular mechanisms involved in TPD. The present study is undertaken to generate information about the genes related to TPD in rubber tree.     

Exploitation of conserved intron scanning as a tool for molecular marker development in the Saccharum complex

This study investigated the potential of the conserved intron scanning approach to develop molecular markers for genes involved in lignin biosynthesis among members of the Saccharum complex and a distantly related Imperata species. Five intron-flanking primer sets targeting genes encoding five lignin biosynthetic enzymes??phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4-CL), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR) and peroxidase (POX)??were designed based on the sequence analysis between sugarcane expressed sequence tags (ESTs) and Sorghum bicolor orthologs. Nucleotide sequence analyses of the amplicons of PAL, 4-CL and CCoAOMT orthologs revealed the presence of single nucleotide polymorphisms (SNPs) and insertions?Cdeletions (INDELs) in the target introns as well as (CT)-simple sequence repeats (SSRs) in CCoAOMT orthologs. The SSR marker screening against fifty-nine accessions of the Saccharum complex and an Imperata species confirmed that the identified SSR markers were highly polymorphic among Saccharum and Erianthus species. PCR-restriction fragment length polymorphism (PCR?CRFLP) and cleaved amplified polymorphic sequence (CAPS) marker screening of 4-CL and CCoAOMT orthologs developed genus-specific molecular markers that confirmed the intergeneric hybridization status of Saccharum Fiji hybrids. The current study showed that the conserved intron scanning strategy is applicable to multiple copy genes of polyploid monocots. The conserved intron scanning approach provides a novel way of investigating DNA polymorphisms among species within the Saccharum complex and has the potential to help in the development of marker-assisted selection in intergeneric hybrids.     

Localization of anchor loci representing five hundred annotated rice genes to wheat chromosomes using PLUG markers

PCR-based Landmark Unique Gene (PLUG) markers are EST-PCR markers developed based on the orthologous gene conservation between rice and wheat, and on the intron polymorphisms among the three orthologous genes derived from the A, B and D genomes of wheat. We designed a total of 960 primer sets from wheat ESTs that showed high similarity with 951 single-copy rice genes. When genomic DNA of Chinese Spring wheat was used as a template, 872 primer sets amplified one to five distinct products. Out of these 872 PLUG markers, 531 were assigned to one or more chromosomes by nullisomic-tetrasomic analysis. For each wheat chromosome, the number of loci detected ranged from 32 for chromosome 6A to 73 for chromosome 7D, with an average of 48 loci per chromosome. Several novel synteny perturbations were identified using deletion bin-mapping of markers. Furthermore, we demonstrated that PLUG markers can be used as probes to simultaneously identify BAC clones that contain homoeologous regions from all three genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Development and Leaf Consumption by Spodoptera cosmioides (Walker) (Lepidoptera: Noctuidae) Reared on Leaves of Agroenergy Crops

Spodoptera cosmioides (Walker) (Lepidoptera: Noctuidae) is a polyphagous pest that threatens more than 24 species of crop plants including those used for biodiesel production such as Ricinus communis (castor bean), Jatropha curcas (Barbados nut), and Aleurites fordii (tung oil tree). The development and leaf consumption by S. cosmioides reared on leaves of these three species were studied under controlled laboratory conditions. The egg-to-adult development time of S. cosmioides was shortest when reared on castor bean leaves and longest when reared on tung oil tree leaves. Larvae reared on castor bean and Barbados nut leaves had seven instars, whereas those reared on tung oil tree leaves had eight. Females originating from larvae reared on castor bean and Barbados nut leaves showed greater fecundity than did females originating from larvae reared on tung oil tree leaves. Insects fed on castor bean leaves had shorter life spans than those fed on tung oil tree and Barbados nut leaves although the oviposition period did not differ significantly. The intrinsic and finite rates of increase were highest for females reared on castor bean leaves. Total leaf consumption was highest for larvae reared on tung oil tree leaves and lowest for those reared on Barbados nut leaves. We conclude that castor bean is a more appropriate host plant for the development of S. cosmioides than are Barbados nut and tung oil tree.     

A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.     

Developing and characterising Ricinus communis SSR markers by data mining of whole-genome sequences

Ricinus communis is a versatile industrial oil crop that is cultivated worldwide. Genetic improvement and marker-assisted breeding of castor bean have been slowed owing to the lack of abundant and efficient molecular markers. As co-dominant markers, simple sequence repeats (SSRs) are useful for genetic evaluation and molecular breeding. The recently released whole-genome sequence of castor bean provides useful genomic resources for developing markers on a genome-wide scale. In the present study, the distribution and frequency of microsatellites in the castor bean genome were characterised and numerous SSR markers were developed using genomic data mining. In total, 18,647 SSR loci at a density of one SSR per 18.89 Kb in the castor bean genome sequence (representing approximately 352.27 Mb) were identified. Dinucleotide repeats were the most frequently observed microsatellites, although the AAT repeat motif was also prevalent. Using six cultivars as screening samples, 670 polymorphic SSR markers from 1,435 primer pairs (46.7 %) were developed. Trinucleotide motif loci contained a higher proportion of polymorphisms (48.5 %) than dinucleotide motif loci (39.2 %). The polymorphism level in the SSR loci was positively correlated with the increasing number of repeat units in the microsatellites. The phylogenetic relationship among 32 varieties was evaluated using the developed SSR markers. Cultivars developed at the same institute clustered together, suggesting that these cultivars have a narrow genetic background. The large number of SSR markers developed in this study will be useful for genetic mapping and for breeding improved castor-oil plants. These markers will also facilitate genetic and genomic studies of Euphorbiaceae.