Intron Polymorphism Markers Research Articles

Development of chromosome-specific potential intron polymorphism markers in watermelon

Development of chromosome-specific potential intron polymorphism markers in watermelon

Chromosome-specific potential intron polymorphism markers for large-scale genotyping applications in pomegranate.

Despite the availability of whole genome assemblies, the identification and utilization of gene-based marker systems has been limited in pomegranate. In the present study, we performed a genome-wide survey of intron length (IL) markers in the 36,524 annotated genes of the Tunisia genome. We identified and designed a total of 8,812 potential intron polymorphism (PIP) markers specific to 3,445 (13.40%) gene models that span 8 Tunisia chromosomes. The ePCR validation of all these PIP markers on the Tunisia genome revealed single-locus amplification for 1,233 (14%) markers corresponding to 958 (27.80%) genes. The markers yielding single amplicons were then mapped onto Tunisia chromosomes to develop a saturated linkage map. The functional categorization of 958 genes revealed them to be a part of the nucleus and the cytoplasm having protein binding and catalytic activity, and these genes are mainly involved in the metabolic process, including photosynthesis. Further, through ePCR, 1,233 PIP markers were assayed on multiple genomes, which resulted in the identification of 886 polymorphic markers with an average PIC value of 0.62. In silico comparative mapping based on physically mapped PIP markers indicates a higher synteny of Tunisia with the Dabenzi and Taishanhong genomes (>98%) in comparison with the AG2017 genome (95%). We then performed experimental validation of a subset of 100 PIP primers on eight pomegranate genotypes and identified 76 polymorphic markers, with 15 having PIC values ≥0.50. We demonstrated the potential utility of the developed markers by analyzing the genetic diversity of 31 pomegranate genotypes using 24 PIP markers. This study reports for the first time large-scale development of gene-based and chromosome-specific PIP markers, which would serve as a rich marker resource for genetic variation studies, functional gene discovery, and genomics-assisted breeding of pomegranate.

Development of Intron Polymorphism Markers and Their Association With Fatty Acid Component Variation in Oil Palm.

Oil palm (Elaeis guineensis Jacq.) is a tropical woody oil crop of the palm family and is known as “the oil king of the world,” but its palm oil contains about 50% palmitic acid, which is considered unhealthy for humans. Intron polymorphisms (IP) are highly efficient and easily examined molecular markers located adjacent to exon regions of functional genes, thus may be associated with targeted trait variation. In order to speed up the breeding of oil palm fatty acid composition, the current study identified a total of 310 introns located within 52 candidate genes involved in fatty acid biosynthesis in the oil palm genome. Based on the intron sequences, 205 primer pairs were designed, 64 of which showed polymorphism among 70 oil palm individuals. Phenotypic variation of fatty acid content in the 70 oil palm individuals was also investigated. Association analysis revealed that 13 IP markers were significantly associated with fatty acid content variation, and these IP markers were located on chromosomes 2, 5, 6, 8, 9, and 10 of oil palm. The development of such IP markers may be useful for the genetic improvement of fatty acid composition in oil palm.

Relationship and genetic diversity analysis of Brassica juncea and U tringle species using intron polymorphic markers

Intron Polymorphism (IP) markers were used to unravel the genetic variation and relationship among 26 genotypes representing six cultivated Brassica species described in the classical U triangle. One hundred and twenty-five Arabidopsis thaliana-derived IP markers were assayed and 90 to 100% cross-transferability was observed in the six Brassica species suggesting that IP markers were highly conserved during the evolution of different Brassica species. The number of alleles observed in species at each locus ranged from one to ten with an average of 2.89 alleles per primer pair and there was no consensus between the number of alleles amplified in diploid and tetraploid species. The size range of amplified alleles was 120-1250bp, which reflects enormous deletions/insertions in different alleles. In B. juncea, 100% cross-transferability had been obtained and 121 IP markers resulted in polymorphic amplicons with PIC value of 0.04 to 0.48. The dendrogram divided all the 26 genotypes into two groups composed of B. napus/B. rapa/B. oleracea and B. carinata/B. nigra/B. juncea. A-genome present in B. juncea and B. napus/B. rapa seems distinct from each other and hence provides a great opportunity for generating diversity through resynthesizing amphidiploids from different available sources of Agenome. The A and B genomes are more similar in comparison to C genome in tetra-diploid species.The evolutionary relationship established between various Brassica species would support in formulating suitable breeding approaches for widening the genetic base of Brassica amphidiploids by exploiting the genetic diversity found in diploid progenitor gene pools.

Genetics of white rust resistance in Indian mustard (Brassica juncea L.) and its validation on using molecular markers

White rust resistance loci (AcB1-A4.1 and AcB1-A5.1) associated with intron polymorphic (IP) markers i.e. At5g41560 and At2g36360, respectively, were used for validation of P1, P2, F1, F2, BC1F1 and BC2F1 generations. The donor parents namely, Bio-YSR and BEC-144 produced desired banding pattern of 430 and 750 bp while recipients viz., NRCHB 101 and DRMR-150-35 exhibited different pattern from donors confirming white rust resistance loci 4.1 and 5.1 with marker At5g41560 and At2g36360, respectively. Confirmation of these set of two IP markers in the parents and F1s lead us to further screening of selected F2, BC1F1 and BC2F1 populations. Available data on white rust reaction in different generations under study revealed that single dominant gene is responsible for white rust resistance. Potential of molecular markers in developing white rust resistant genotypes is proved under present study.

Molecular mapping of a novel early leaf-senescence gene Els2 in common wheat by SNP genotyping arrays

Early leaf senescence in wheat (Triticum aestivum L.) is one of the limiting factors for developing high yield potential. In this study, a stably inherited, early leaf-senescence mutant LF2099 was initially identified in an M2 population of the common wheat accession H261 after ethyl methanesulfonate (EMS) mutagenesis. Early leaf senescence was observed in the LF2099 mutant during the three-leaf-stage, and then the etiolated area of the wheat leaf increased gradually from the bottom to the top throughout development. Compared with H261, the chlorophyll (Chl a, Chl b) and carotenoid contents and photosynthetic capacity of the mutant were significantly decreased. All of its yield-related traits except for spike length were also significantly reduced. Dissolved cytoplasm, abnormal chloroplast structure, dissolved chloroplast membrane, abnormal thylakoid development, and more plastoglobules were observed in the senescent leaf region of the mutant by transmission electronic microscope. Genetic analysis indicated that the early leaf-senescence phenotype is controlled by an incomplete-dominance nuclear gene, here designated Els2. Using single nucleotide polymorphisms and bulked segregant analysis, the els2 gene was anchored in a region on chromosome 2BL between simple sequence repeat (SSR) markers gpw4043 and wmc149. Six new polymorphic SSR markers were developed from the Chinese Spring 2BL shotgun survey sequence contigs. By means of comparative genomics analyses, the collinearity genomic regions of the els2 locus on wheat 2BL were identified in Brachypodium distachyon chromosome 5, rice (Oryza sativa) chromosome 4 and sorghum (Sorghum bicolor) chromosome 6. Five intron polymorphism (IP) markers were further developed from this collinearity genomic region. Ultimately, Els2 was mapped in a genetic interval of 0.95 cM flanked by IP markers 2BIP09 and 2BIP14. The co-segregating IP markers 2BIP12 and 2BIP17 provide a starting point for the fine mapping and map-based cloning of Els2.

Genetic diversity of Rhodiola sp. in Altai as assessed by intron polymorphism markers

Genetic diversity of Rhodiola sp. in Altai as assessed by intron polymorphism markers

Identification of genic SSRs and construction of a SSR-based linkage map in Brassica juncea

Search for genic SSRs from transcriptome sequence data of Brassica juncea and B. nigra and from unigene databank of B. rapa resulted in identification of 20,529 SSRs from 330,827 gene sequences. Identified SSRs (named as unigene microsatellite; UGM) were characterized based on their number of repeats, type of motif and base composition. Primers were designed for 2118 UGMs after in silico polymorphism survey and were tested across 11 different genotypes of B. juncea and B. rapa for PCR amplification and polymorphism. It resulted in clear-cut amplification from 1993 UGMs of which 1319 UGMs showed polymorphism. It was observed that a priori in silico polymorphism survey helped in increasing the polymorphism percentage (6.1–13.2) by PCR amplification. We also report here the development of first SSR-based linkage map of Brassica juncea using a DH mapping population derived from F1 of the cross between the lines EH-2 and Pusajaikisan. The map consisted of 860 markers comprising 462 UGMs, 157 BAC-derived SSRs and 241 intron polymorphic markers and covered a total genetic length of 2073.6 cM. The study also reports an interesting observation of appearance of novel non-parental bands in the mapping population from 18 SSRs and 13 IP markers. Search of Arabidopsis genes orthologous to their primer sequences showed that 11 of such Arabidopsis genes code for proteins involved in various abiotic stresses. The highly transferable genic SSRs developed in the study would serve as valuable resources for comparative genome mapping, mapping of qualitative and quantitative traits, gene cloning and marker-assisted breeding in Brassica species.

Development of intron polymorphism markers in major latex-like protein gene for locality-level and cultivar identification of Salvia miltiorrhiza.

Background Salvia miltiorrhiza (Danshen) is one of the most widely used medicinal herbs in traditional Chinese medicine. Locality-level and cultivar identification is of great importance not only for protecting highest therapeutic effectiveness of Daodi Danshen, but also for the genetic conservation and utilization of existing S. miltiorrhiza populations.ResultsIntron polymorphisms including SNPs (single nucleotide polymorphisms) and indels were exploited in major latex-like protein (MLP) gene. Based on these markers, genetic relationships among S. miltiorrhiza cultivar and populations in different locations were evaluated by constructing a dendrogram. Moreover, S. miltiorrhiza specimens from Laiwu region were geographically distinguishable by the developed SNP marker. A 204 bp-indel marker was exploited for the first space breeding cultivar Luyuan Danshen-1 (LD-1), and an effective real-time PCR assay was successfully developed for fast screening of LD-1 among local landraces.Conclusions MLP intron is a valuable DNA barcode for intra-specific study of S. miltiorrhiza populations, and the developed markers can serve as a useful tool for molecular identification of LD-1 cultivar and geographically distinct populations of S. miltiorrhiza.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3611-5) contains supplementary material, which is available to authorized users.

Fine mapping of a dominant thermo-sensitive genic male sterility gene (BntsMs) in rapeseed (Brassica napus) with AFLP- and Brassica rapa-derived PCR markers.

A new thermo-sensitive dominant genic male sterility (TSDGMS) line of Brassica napus was found and mapped in this paper. Our result will greatly accelerate the map-based cloning of the BntsMs gene. TE5A is a thermo-sensitive dominant genic male sterility line originating from spontaneous mutation of the inbred line TE5 in Brassica napus and provides a promising system for the development of hybrid cultivars. Genetic analysis has revealed that the BntsMs mutant is controlled by a single, dominant gene. Here, we describe the fine mapping of BntsMs using amplified fragment length polymorphism (AFLP) and intron polymorphism (IP) methodologies. We screened 1,024 primer combinations and then identified five AFLP markers linked to the BntsMs gene, two of which were successfully converted into sequence-characterised amplified region (SCAR) markers. The linkage of the markers was identified by analysing a large BC2 population of 700 recessive-fertility individuals. Two SCAR markers were found in the flanking region of the BntsMs gene at distance of 3.5 and 4.8 cm. Based on sequence information from the previously screened AFLP markers and on genome organisation comparisons of the A genome of Brassica rapa and Arabidopsis, seven IP markers linked to the BntsMs gene were developed. By analysing the 700 recessive-fertility individuals, two IP markers, IP004 and IP470, were localised to the flanking region of the BntsMs gene at a distance of 0.3 and 0.2 cm, respectively. A comparison of the B. rapa and Arabidopsis genomes revealed 27 genes of B. rapa in the flanking region of these two IP markers. It is likely that the molecular markers developed from these investigations will greatly accelerate the positional cloning of the BntsMs gene.

Mapping of BnMs4 and BnRf to a common microsyntenic region of Arabidopsis thaliana chromosome 3 using intron polymorphism markers

A recessive epistatic genic male sterile two-type line, 7365AB (Bnms3ms3ms4msRrfRf/BnMs3ms3ms4ms4RfRf), combined with the fertile interim-maintainer 7365C (Bnms3ms3ms4ms4rfrf) is an effective pollination control system in hybrid rapeseed production. We report an effective strategy used to fine map BnMs4 and BnRf. The two genes were both defined to a common microsyntenic region with Arabidopsis chromosome 3 using intron polymorphism (IP) markers developed according to Arabidopsis genome information and published genome organization of the A genome. The near-isogenic lines 7365AC (Bnms3ms3ms4ms4Rfrf/Bnms3ms3ms4ms4rfrf) of BnRf and 736512AB (Bnms3ms3Ms4ms4RfRf/Bnms3ms3ms4ms4RfRf) of BnMs4 were constructed to screen developed markers and create genetic linkage maps. Nine polymorphic IP markers (P1-P9) were identified. Of these, P2, P3, P4, and P6 were linked to both BnMs4 and BnRf with genetic distances <0.6 cM. Three simple sequence repeat markers, SR2, SR3, and SR5, were also identified by using public information. Subsequently, all markers linked to the two genes were used to compare the micro-collinearity of the regions flanking the two genes with Brassica rapa and Arabidopsis. The flanking regions showed rearrangements and inversion with fragments of different Arabidopsis chromosomes, but a high collinearity with B. rapa. This collinearity provided extremely valuable reference for map-based cloning in polyploid Brassica species. These IP markers could be exploited for comparative genomic studies within and between Brassica species, providing an economically feasible approach for molecular marker-assisted selection breeding, accelerating the process of gene cloning, and providing more direct evidence for the presence of multiple alleles between BnMs4 and BnRf.

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.

Development of a high density integrated reference genetic linkage map for the multinational Brassica rapa Genome Sequencing ProjectThis article is one of a selection of papers from the conference “Exploiting Genome-wide Association in Oilseed Brassicas: a model for genetic improvement of major OECD crops for sustainable farming”.

We constructed a high-density Brassica rapa integrated linkage map by combining a reference genetic map of 78 doubled haploid lines derived from Chiifu-401-42× Kenshin (CKDH) and a new map of 190 F2 lines derived from Chiifu-401-42× rapid cycling B. rapa (CRF2). The integrated map contains 1017 markers and covers 1262.0 cM of the B. rapa genome, with an average interlocus distance of 1.24 cM. High similarity of marker order and position was observed among the linkage groups of the maps with few short-distance inversions. In total, 155 simple sequence repeat (SSR) markers, anchored to 102 new bacterial artificial chromosomes (BACs) and 146 intron polymorphic (IP) markers were mapped in the integrated map, which would be helpful to align the sequenced BACs in the ongoing multinational Brassica rapa Genome Sequencing Project (BrGSP). Further, comparison of the B. rapa consensus map with the 10 B. juncea A-genome linkage groups by using 98 common IP markers showed high-degree colinearity between the A-genome linkage groups, except for few markers showing inversion or translocation. Suggesting that chromosomes are highly conserved between these Brassica species, although they evolved independently after divergence. The sequence information coming out of BrGSP would be useful for B. juncea breeding. and the identified Arabidopsis chromosomal blocks and known quantitative trait loci (QTL) information of B. juncea could be applied to improve other Brassica crops including B. rapa.