Intron Length Polymorphism Markers Research Articles

Assessment of genetic diversity and population structure of carrot accessions with DcSto Intron Length Polymorphism markers

Assessment of genetic diversity and population structure of carrot accessions with <i>DcSto</i> Intron Length Polymorphism markers

Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research.

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

Sui Generis Development of Molecular Markers from Transcriptome of Crotalaria juncea, a Fabaceae Family Bast Fiber Crop

ABSTRACT Lack of genomic resources in sunn hemp (Crotalaria juncea L.), a leguminous bast fiber crop, is one of the major constraints for germplasm characterization and genetic analysis. We identified non-redundant sets of a variety of molecular markers from the de novo flower-bud transcriptome of sunn hemp. These molecular markers included 1683 simple sequence repeats, 4759 intron length polymorphism markers primarily from protein-coding genes, and 3309 single nucleotide polymorphism allele-specific markers. All of these marker resources have been integrated into a simple database for easy access. To evaluate marker efficiency and polymorphism information content of the sampled primers, a subset of SSR and intron length polymorphism markers were validated in sunn hemp germplasm accessions to obtain average PIC values of 0.26–0.32. The SSR genotyping profiles also aided in the genetic distance matrix-based clustering of sunn hemp germplasm accessions. The ability of these molecular markers to successfully amplify in cross-genera bast fiber crops further demonstrated their effectiveness in genotyping. Thus, the current study describes the identification of previously undisclosed endogenous molecular markers in sunn hemp, which have potential applications in genetic studies and breeding in sunn hemp for bast fiber improvement.

Discrimination of Curcuma species from Asia using intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase

Recently, Curcuma rhizome-related foods with claimed health benefits have been used worldwide; however, correct identification and quality assessment have not been conducted. Due to the wide distribution and morphological similarities of Curcuma species, the classification of some species is debated and nomenclature is inconsistent among countries. In this study, to elucidate specific molecular markers of medicinally used Curcuma species in Asia, and to solve the confusion on the reported botanical origin of crude drugs, molecular analysis based on the intron length polymorphism (ILP) in genes encoding diketide-CoA synthase and curcumin synthase and the trnK intron sequences was performed using 59 plant specimens and 42 crude drug samples from 13 Curcuma species, obtained from Asian countries. The ILP patterns of the respective species from both plant specimens and crude drug samples revealed high consistency in C. aromatica, C. zedoaria, C. phaeocaulis, C. aeruginosa, C. wenyujin, and C. zanthorrhiza, but showed intraspecies polymorphism in C. longa, C. kwangsiensis, C. amada, C. mangga and C. comosa. The C. longa specimens and samples were separated into three subgroups which were highly consistent with their geographical origins. Based on the ILP markers and the trnK intron sequences, the botanical origins of “Khamin oi” from Thailand were correctly determined to be C. longa or a hybrid between C. longa and other species, and “Wan narn kum” from Thailand and “Kasturi manjal” from India were correctly determined to be C. zanthorrhiza.

Identification of Candidate Gene-Based Markers for Girth Growth in Rubber Trees.

Girth growth is an important factor in both latex and timber production of the rubber tree. In this study, we performed candidate gene association mapping for girth growth in rubber trees using intron length polymorphism markers (ILP) in identifying the candidate genes responsible for girth growth. The COBL064_1 marker developed from the candidate gene (COBL4) regulating cellulose deposition and oriented cell expansion in the plant cell wall showed the strongest association with girth growth across two seasons in the Amazonian population and was validated in the breeding lines. We then applied single molecule real-time (SMRT) circular consensus sequencing (CCS) to analyze a wider gene region of the COBL4 to pinpoint the single nucleotide polymorphism (SNP) that best explains the association with the traits. A SNP in the 3’ UTR showing linkage disequilibrium with the COBL064_1 most associated with girth growth. This study showed that the cost-effective method of ILP gene-based markers can assist in identification of SNPs in the candidate gene associated with girth growth. The SNP markers identified in this study added useful markers for the improvement of girth growth in rubber tree breeding programs.

Exploring the basis of 2-propenyl and 3-butenyl glucosinolate synthesis by QTL mapping and RNA-sequencing in Brassica juncea.

Brassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. Joint QTL mapping and RNA-sequencing analyses reveal one candidate gene of IIL1 (LOC106416451) for GSL metabolism in B. juncea. These results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea.

Диференціація генотипів сосни звичайної за поліморфізмом довжини інтронів генів дефензинів

Наведено результати дослідження генотипів сосни звичайної (Pinus sylvestris L.) із використанням молекулярних маркерів на підстаі генетичного поліморфізму довжини інтронів генів дефензинів PsDef1-4. Виявлено високу інформативність маркерів на підстаі інтрону гена PsDef2 (IPL-PsDef2) для дослідження внутрішньовидового поліморфізму в сосни звичайної. Встановлено межі осередка інфекції кореневої губки (Heterobasidion annosum (Fr.) Bref.) в сосновому насадженні за допомогою полімеразно-ланцюгової реакції (ПЛР) із використанням видоспецифічних праймерів. Проведено ДНК-профілювання модельних дерев способом електрофоретичного розділення ампліконів IPL-PsDef2 маркерів. За результатами поліморфізму довжини інтронів генів дефензинів здійснено кластеризацію генотипів сосни звичайної із використанням алгоритму UPGMA. на підстаі дендрограми виділено два кластери генотипів зі 100 % бутстреп підтримкою, які відрізняються за стійкістю до кореневої губки. Методом К-середніх визначено значущість кожного з ампліконів для диференціації генотипів сосни на групи. Виявлено амплікони, які можуть бути генетичними маркерами для оцінки потенційної стійкості генотипів сосни звичайної до кореневої губки. Отримані дані щодо поліморфізму локусів генів дефензинів, а також результати кластерного аналізу вказують на перспективність використання IPL-PsDef2 маркерів для генотипування сосни звичайної.

Genome-wide analysis of SSR and ILP markers in trees: diversity profiling, alternate distribution, and applications in duplication

Molecular markers are efficient tools for breeding and genetic studies. However, despite their ecological and economic importance, their development and application have long been hampered. In this study, we identified 524,170 simple sequence repeat (SSR), 267,636 intron length polymorphism (ILP), and 11,872 potential intron polymorphism (PIP) markers from 16 tree species based on recently available genome sequences. Larger motifs, including hexamers and heptamers, accounted for most of the seven different types of SSR loci. Within these loci, A/T bases comprised a significantly larger proportion of sequence than G/C. SSR and ILP markers exhibited an alternative distribution pattern. Most SSRs were monomorphic markers, and the proportions of polymorphic markers were positively correlated with genome size. By verifying with all 16 tree species, 54 SSR, 418 ILP, and four PIP universal markers were obtained, and their efficiency was examined by PCR. A combination of five SSR and six ILP markers were used for the phylogenetic analysis of 30 willow samples, revealing a positive correlation between genetic diversity and geographic distance. We also found that SSRs can be used as tools for duplication analysis. Our findings provide important foundations for the development of breeding and genetic studies in tree species.

Genetic Diversity in Aus Rice Genotypes using ILP Markers

Assessment of genetic diversity is essential for germplasm characterization, utilization and conservation. Genetic diversity of 31 Aus rice genotypes of Bangladesh was assessed using 11 ILP (intron length polymorphism) markers. A total of 28 alleles were detected and the number of alleles per locus varied from 2 (RI01779, RI05751, RI05304, RI03205, RI00299, RI05407) to 4 (RI05559). The PIC values ranged from 0.06 (RI05407) to 0.57 (RI05559) with an average of 0.33. PIC value revealed that RI05559 was the best ILP markers for the studied 31 Aus rice genotypes. The dendrogram from unweighted pair-group method with arithmetic average clustering classified the genotypes into five groups at a coefficient of 0.57. Two dimensional graphical views of Principal Coordinate Analysis (PCoA) revealed that the genotypes Kuchmuch, Kalo dhan, Aus dhan, Sadey aus, Chaina and Dighi bawalia were found far away from the centroid of the cluster and can be seslected as parents for further breeding programmes. Parangi and V3, Adubali and H1-2, Begunbichi and Hashikalmi had closest distance (0.000) in the distance matrix might have same genetic background. This information will be useful for the selection of genetically diversed parents and assist in trait development using genotypes in rice breeding programmes in future. The results provided some useful implications for establishment of sovereignty of Bangladeshi rice gene pool. It was also suggested that ILP markers could be very useful for the genetic study and breeding in rice.Bangladesh Rice j. 2016, 20(2): 13-19

Miniature Inverted Repeat Transposable Element Insertions Provide a Source of Intron Length Polymorphism Markers in the Carrot (Daucus carota L.).

The prevalence of non-autonomous class II transposable elements (TEs) in plant genomes may serve as a tool for relatively rapid and low-cost development of gene-associated molecular markers. Miniature inverted-repeat transposable element (MITE) copies inserted within introns can be exploited as potential intron length polymorphism (ILP) markers. ILPs can be detected by PCR with primers anchored in exon sequences flanking the target introns. Here, we designed primers for 209 DcSto (Daucus carota Stowaway-like) MITE insertion sites within introns along the carrot genome and validated them as candidate ILP markers in order to develop a set of markers for genotyping the carrot. As a proof of concept, 90 biallelic DcS-ILP markers were selected and used to assess genetic diversity of 27 accessions comprising wild Daucus carota and cultivated carrot of different root shape. The number of effective alleles was 1.56, mean polymorphism informative content was 0.27, while the average observed and expected heterozygosity was 0.24 and 0.34, respectively. Sixty-seven loci showed positive values of Wright's fixation index. Using Bayesian approach, two clusters comprising four wild and 23 cultivated accessions, respectively, were distinguished. Within the cultivated carrot gene pool, four subclusters representing accessions from Chantenay, Danvers, Imperator, and Paris Market types were revealed. It is the first molecular evidence for root-type associated diversity structure in western cultivated carrot. DcS-ILPs detected substantial genetic diversity among the studied accessions and, showing considerable discrimination power, may be exploited as a tool for germplasm characterization and analysis of genome relationships. The developed set of DcS-ILP markers is an easily accessible molecular marker genotyping system based on TE insertion polymorphism.

Genome-wide development and deployment of informative intron-spanning and intron-length polymorphism markers for genomics-assisted breeding applications in chickpea

The discovery and large-scale genotyping of informative gene-based markers is essential for rapid delineation of genes/QTLs governing stress tolerance and yield component traits in order to drive genetic enhancement in chickpea. A genome-wide 119169 and 110491 ISM (intron-spanning markers) from 23129 desi and 20386 kabuli protein-coding genes and 7454 in silico InDel (insertion-deletion) (1–45-bp)-based ILP (intron-length polymorphism) markers from 3283 genes were developed that were structurally and functionally annotated on eight chromosomes and unanchored scaffolds of chickpea. A much higher amplification efficiency (83%) and intra-specific polymorphic potential (86%) detected by these markers than that of other sequence-based genetic markers among desi and kabuli chickpea accessions was apparent even by a cost-effective agarose gel-based assay. The genome-wide physically mapped 1718 ILP markers assayed a wider level of functional genetic diversity (19–81%) and well-defined phylogenetics among domesticated chickpea accessions. The gene-derived 1424 ILP markers were anchored on a high-density (inter-marker distance: 0.65cM) desi intra-specific genetic linkage map/functional transcript map (ICC 4958×ICC 2263) of chickpea. This reference genetic map identified six major genomic regions harbouring six robust QTLs mapped on five chromosomes, which explained 11–23% seed weight trait variation (7.6–10.5 LOD) in chickpea. The integration of high-resolution QTL mapping with differential expression profiling detected six including one potential serine carboxypeptidase gene with ILP markers (linked tightly to the major seed weight QTLs) exhibiting seed-specific expression as well as pronounced up-regulation especially in seeds of high (ICC 4958) as compared to low (ICC 2263) seed weight mapping parental accessions. The marker information generated in the present study was made publicly accessible through a user-friendly web-resource, “Chickpea ISM-ILP Marker Database”. The designing of multiple ISM and ILP markers (2–5 markers/gene) from an individual gene (transcription factor) with numerous aforementioned desirable genetic attributes can widen the user-preference to select suitable primer combination for simultaneous large-scale assaying of functional allelic variation, natural allelic diversity, molecular mapping and expression profiling of genes among chickpea accessions. This will essentially accelerate the identification of functionally relevant molecular tags regulating vital agronomic traits for genomics-assisted crop improvement by optimal resource expenses in chickpea.

Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice.

We developed genome-wide 84634 ISM (intron-spanning marker) and 16510 InDel-fragment length polymorphism-based ILP (intron-length polymorphism) markers from genes physically mapped on 12 rice chromosomes. These genic markers revealed much higher amplification-efficiency (80%) and polymorphic-potential (66%) among rice accessions even by a cost-effective agarose gel-based assay. A wider level of functional molecular diversity (17–79%) and well-defined precise admixed genetic structure was assayed by 3052 genome-wide markers in a structured population of indica, japonica, aromatic and wild rice. Six major grain weight QTLs (11.9–21.6% phenotypic variation explained) were mapped on five rice chromosomes of a high-density (inter-marker distance: 0.98 cM) genetic linkage map (IR 64 x Sonasal) anchored with 2785 known/candidate gene-derived ISM and ILP markers. The designing of multiple ISM and ILP markers (2 to 4 markers/gene) in an individual gene will broaden the user-preference to select suitable primer combination for efficient assaying of functional allelic variation/diversity and realistic estimation of differential gene expression profiles among rice accessions. The genomic information generated in our study is made publicly accessible through a user-friendly web-resource, “Oryza ISM-ILP marker” database. The known/candidate gene-derived ISM and ILP markers can be enormously deployed to identify functionally relevant trait-associated molecular tags by optimal-resource expenses, leading towards genomics-assisted crop improvement in rice.

Development and utility of PCR-based intron polymorphism markers in sorghum [Sorghum bicolor (L.) Moench

Sequence variations involving DNA insertions and deletions within introns provide suitable targets for the development of PCR-based markers. In this study, 37861 potential introns were identified in sorghum and primers were designed to develop intron length polymorphism (ILP) markers. Drastic variation was observed in the number of ILP markers on each chromosome with a range of 1498 (chr. 5) - 7290 (chr. 1), which was also reflected in the fluctuations in their density. About 200 ILP markers were assessed for their potential as PCR-based markers in 24 sorghum genotypes representing different races. Of these, 172 gave clear and robust amplification without multiple amplicons. Forty-eight of these markers were polymorphic yielding 122 alleles with an average of 2.5 alleles per marker. The number of alleles ranged from 2 to 6 while the PIC value ranged from 0.14 to 0.69. Cluster analysis grouped the genotypes into two major clusters. These markers would form an important addition to the existing DNA marker resources in sorghum and could be highly useful for genetic diversity analysis, construction of linkage maps and comparative genomics studies.

Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species

Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma.

Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content.

BackgroundAlliinase is an important enzyme occurring in Allium species that converts precursors of sulfuric compounds, cysteine sulfoxides into a biologically active substance termed allicin. Allicin facilitates garlic defense against pests and produces health-promoting compounds. Alliinase is encoded by members of a multigene family that has not yet been sufficiently characterized, namely with regard to the copy numbers occurring within the genome and the polymorphisms among the family members.ResultsWe cloned 45 full-length alliinase amplicons of cultivar (cv.) Jovan. Sequence analyses revealed nine different sequence variants (SVs), confirming the multilocus nature of this gene family. Several mutations in exons, mainly occurring in the first exon coding for vacuolar signal peptide, were found. These results enabled us to identify sequences with putatively modified vacuole-targeting abilities. We found additional sequence variants using partial amplicons. We estimated that the minimum number of gene copies in the diploid genome of the investigated cultivar was fourteen. We obtained similar results for another three cultivars, which differed in bolting type and place of origin. The further identification of high degree of polymorphisms in the intron regions allowed us to develop a specific polymerase chain reaction assay capable to capture intron length polymorphism (ILP). This assay was used to screen 131 additional accessions. Polymorphic data were used for cluster analysis, which separated the bolting and non-bolting garlic types and those with high cysteine-sulfoxide contents in a similar way as AFLP analysis in previous study. These newly developed markers can be further applied for the selection of desirable garlic genotypes.ConclusionsDetailed analysis of sequences confirmed multigenic nature of garlic alliinase. Intron and exon polymorphism analysis generated similar results as whole genome variability assessed previously by AFLP. Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes. ILP markers capable to detect intron polymorphisms were newly developed. Developed markers could be applied in garlic breeding. Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0214-z) contains supplementary material, which is available to authorized users.

Development of SSR and ILP markers in horsegram (Macrotyloma uniflorum), their characterization, cross-transferability and relevance for mapping

Simple sequence repeat (SSR) and intron length polymorphism (ILP) primers were developed and characterized from public sequence data in horsegram. All characterized primers amplified alleles in a range of 1–8 with an average of 2.64 alleles per primer. Polymorphism information content ranged from 0.048 for primers MUMS-24, MUMST-21, MUMST-26 and MUMST-29 to 0.755 in MUMS-10 with an average of 0.360. Dendrogram and principal component analysis showed that newly characterized SSR and ILP markers developed in this study distinguished horsegram germplasm lines into two groups and revealed two genetic stocks for characterized germplasm. Cross-amplification of these developed markers was tested across 12 related legume species. Nineteen primers pairs showed a maximum of 100 % cross-transferability, while a minimum of 8.3 % transferability was shown by 27 primers. As no genomic resources are available in horsegram, these newly developed markers can advance molecular breeding research in this crop species and can be helpful in germplasm characterization, diversity studies, mapping and in comparative genomic studies.

A simple approach based on intron length polymorphism (ILP) for chromosomal localization of GeneWAG-2

Intron polymorphism can be used to develop potential intron polymorphism (PIP) markers. Intron length polymorphism (ILP) is one of the most easily identified PIP markers. WAG-2, a class C MADS-box gene, had been isolated from the wheat expressed sequence tag (EST) database. Rich polymorphisms, such as intron allele variation, and length polymorphism occur in WAG-2 gene introns. Especially, a significant length polymorphism is shared by intron IV. In present study, based on intron IV length polymorphism, using wheat nullisomic-tetrasomic (NT) and ditelosomic (DT) lines, a pair of ILP primers were designed in exons flanking introns IV and the WAG-2 gene was located on the short arm of homologous group 3 chromosome in the wheat genome. The gene location approach by using ILP markers combining with aneuploidy is convenient, reliable, specific, codominant, and stable. It is expected that ILP markers will play important roles in the genetic studies and breeding of wheat.

Genome‐wide identification of candidate phosphate starvation responsive genes and the development of intron length polymorphism markers in maize

AbstractTo develop genetic markers associated with tolerance to low phosphorus, we identified candidate phosphate starvation responsive (PSR) genes and developed their intron length polymorphism (ILP) markers in maize on a genome‐wide scale. Based on the known plant PSR genes, 161 candidate PSR genes were identified. Of these genes, 138 genes contained at least one intron and were then used to develop 606 PSR‐ILP markers by designing PCR primers to the intron‐flanking exonic regions. PCR evaluation was performed on 43 randomly selected PSR‐ILP markers in 30 maize inbred lines. Of the primers, 88.4% amplified stable and pure products in all maize inbred lines, and 53.5% of the markers showed ILP, with PIC values ranging from 0.06 to 0.80. The result of clustering analysis of the 30 maize inbred lines, based on the polymorphism of 23 ILP markers, suggests that the ILP markers developed in this study are not only efficient for genetic diversity analysis but also potentially useful for marker assisted selection for tolerance to low phosphorus.

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.

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.