Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP,SSAP, SAMPL and SSR markers

In this study we evaluate the informativeness and efficiency of Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), Selectively Amplified Microsatellite Polymorphic Loci (SAMPL) and Simple Sequence Repeat (SSR) markers for genetic diversity, phylogenetic relationship among the Citrus species and mapping ability of the marker system. The SSR exhibited relatively higher level of polymorphism information content in terms of the expected heterozygosity, than that of the AFLPs, SSAPs and SAMPLs. For each marker system, average level of the discriminating potential was very close to the actual discriminating potential. Similarity matrices showed weak, yet significant correlations when Mantel's test was applied. The highest positive (0.72) correlation was found between the AFLP and SSAP markers. The SSR and SAMPL markers were poorly correlated. The dendrogram topology among the four marker systems had high similarity. Taken together, the SSAP and SAMPL were highly efficient in detecting genetic similarity in Citrus, while the SSR may be more useful for segregation studies and genome mapping in Citrus. The SSAP and SAMPL markers could be useful for Citrus genome mapping in combination with AFLP and SSR markers. To our knowledge, this was the first detail report of a comparison of performances among AFLP, SSR and retrotrasposon based molecular marker technique on a set of samples of Citrus. Our result provides guidance for future efficient use of these molecular methods in genetic analysis of Citrus sp. and its relatives.     

High throughput,high resolution selection of polymorphic microsatellite loci for multiplex analysis

Background  

Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers.     

A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

A number of molecular marker technologies have allowed important advances in the understanding of the genetics and evolution of Eucalyptus, a genus that includes over 700 species, some of which are used worldwide in plantation forestry. Nevertheless, the average marker density achieved with current technologies remains at the level of a few hundred markers per population. Furthermore, the transferability of markers produced with most existing technology across species and pedigrees is usually very limited. High throughput, combined with wide genome coverage and high transferability are necessary to increase the resolution, speed and utility of molecular marker technology in eucalypts. We report the development of a high-density DArT genome profiling resource and demonstrate its potential for genome-wide diversity analysis and linkage mapping in several species of Eucalyptus.     

A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors

Background

The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs). This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge.

Results

We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes.

Conclusion

The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques. We show that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles.     

A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta

Background  

Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems.     

A novel fluorescent pH probe for expression in plants

Background  

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.     

Association analysis of fruit traits in mulberry species (Morus L.)

Improvement of fruit traits is an important objective in current mulberry breeding programs. In this study, 93 mulberry accessions of diverse origin were genotyped using 15 ISSR markers to identify marker–trait associations with fruit traits. Fifteen ISSR primers generated a total of 104 amplification products, of which 94 were polymorphic, revealing 90.38% polymorphism; the mean PIC value was 0.2698. UPGMA cluster analysis showed clear genetic relationships between the 93 mulberry cultivars, and the major clusters were related to known pedigree relationships and their ecotype. The mean r² value for all intra-chromosomal loci pairs was 0.0210. Marker–trait associations were investigated using the unified mixed-model approach, considering both population structure (Q) and kinship (K). In total, 24 marker–trait associations (p < 0.01) were identified using different ISSR markers. The results suggest that association mapping in mulberry is a viable alternative to quantitative trait loci mapping, and detection of associations between markers and mulberry fruit traits will also provide important information for marker-assisted breeding.     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

Genome-wide identification of microsatellites in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) using FIASCO and phpSSRMiner

Background  

Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.     

Linkage map saturation,construction, and comparison in four populations of Prunus

Summary

One of the objectives of the ISAFRUIT Project was to perform genetic analyses in four populations of Prunus, two of peach (P. persica) and two of apricot (P. armeniaca), in order to identify major genes and quantitative trait loci (QTLs) for characters related to fruit quality. This required the construction of saturated marker maps in each of these populations. Marker maps were available for an intra-specific peach × peach F₂, a BC₂ peach × P. davidiana (using peach as the recurrent parent), and an apricot × apricot F₁. We have further saturated these maps mainly with SSR (simple sequence repeat) markers. A new map, constructed uniquely from SSRs was prepared for a fourth apricot × apricot F₁ population. Using anchor markers, we compared these four maps with the reference Prunus map, constructed using an almond × peach F₂ population. As previously observed, conservation of synteny and co-linearity were the general rule, providing additional evidence of the high level of similarity between all Prunus genomes. Comparisons of genetic distances between the maps suggested that those involving similar genomes had higher levels of recombination than those with more distant genomes, particularly the inter-specific crosses.     

Genetic diversity analysis in date palm (Phoenix dactylifera L.): a comparative assessment using ISSR and RAPD marker assays

SUMMARY

A comparative study was conducted to evaluate genetic diversity in 45 genotypes of date palm (Phoenix dactylifera L.), including both male and female plants, employing RAPD and ISSR marker systems. The data were analysed to calculate the total number of bands, the number of polymorphic bands, the percentage polymorphism, the average number of bands per primer, the effective multiplex ratio (EMR), the polymorphic information content (PIC), the marker index (MI), and genetic similarity coefficients. The 37 RAPD and 53 ISSR primers used generated 363 and 608 scorable amplified products, respectively, of which 95.0% and 90.9% were polymorphic. The ISSR markers produced more information than the RAPD markers due to their higher EMR and MI values. Jaccard similarity values among male plants, female plants, and between all male and all female plants varied between 0.72 – 0.80. The results indicate the effectiveness of these two marker systems for demonstrating genetic relationships among date palm genotypes.     

Are rhododendron hybrids distinguishable on the basis of morphology and microsatellite polymorphism?

Sequence Tagged Microsatellite Sites (STMSs) and morphological trait markers were used to evaluate 33 rhododendron germplasm for genetic diversity assessment and discrimination power. The average genetic diversity estimates were 0.724 (morphological traits) and 0.174 (STMSs) marker datasets. The Shannon index was higher for morphological traits (1.797) than STMS (0.302). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the two markers, showed that estimated values of relationships given for morphological and STMS were not significantly related (p > 0.05). The dataset from STMS, supported by the total probability of identity (1.13 × 10⁻⁹) and total paternity exclusion probability (0.9999), allowed all accessions to be uniquely identified. In summary, STMS marker proved to be an efficient tool in assessing the genetic variability among old broad leaf rhododendron genotypes. The pattern of variation appeared to be consistent, and it can be used for germplasm conservation and management for restoration of historical genetic resources.     

EST-PCR markers developed for highbush blueberry are also useful for genetic fingerprinting and relationship studies in rabbiteye blueberry

The pedigrees of most rabbiteye blueberry (Vaccinium virgatum) cultivars can be traced back to four wild selections, ‘Ethel’, ‘Clara’, ‘Myers’, and ‘Black Giant’; thus, they result from a very narrow germplasm base and are highly related. Until now randomly amplified polymorphic DNA (RAPD) has been the only type of molecular marker used in rabbiteye blueberry. Here we have tested whether a type of sequence-tagged site (STS) marker which utilizes specific ∼20-mer primers from expressed sequence tags (ESTs) of highbush blueberry (V. corymbosum), called EST-PCR markers, are useful for genetic fingerprinting and relationship studies in rabbiteye blueberry. Of 44 EST-PCR primer pairs, from an assortment of genes expressed in flower buds of cold acclimated and non-acclimated plants, and shown to amplify polymorphic fragments among a collection of highbush genotypes, 40 (91%) resulted in successful amplification, and 33 of those (83%) amplified polymorphic fragments among the rabbiteye genotypes. The average number of scorable bands per primer pair was two. A dendrogram constructed from genetic similarity values, based on the EST-PCR marker data, tended to group siblings and parent/progeny together, generally agreeing with pedigree information. A group of 20 markers from five EST-PCR primer pairs distinguished all the genotypes in this study. These markers are as easy to generate and as affordable as RAPDs, but are based on actual gene sequences, and should have general utility for DNA fingerprinting, genetic diversity, and mapping studies.     

Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

Background  

The isolation of green fluorescent protein (GFP) and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types.     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

The lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e.g., Arabidopsis) and grass (e.g., Oryza) model species.     

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

Background  

The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.     

Genetic diversity of Dianthus accessions as assessed using two molecular marker systems (SRAPs and ISSRs) and morphological traits

Dianthus chinensis, Dianthus barbatus and Dianthus superbus are members of the Caryophyllaceae and are grown widely as ornamental plants. Information about relative genetic relationships can facilitate breeding programs. Here, we have compared two polymerase chain reaction (PCR)-based systems (sequence-related amplified polymorphisms (SRAPs) and inter-simple sequence repeats (ISSRs)) and morpological trait measurements for their relative effectiveness in estimating the genetic diversity found between 22 Chinese pink (D. chinensis) inbred-lines, one accession of D. barbatus and one accession of D. superbus. Interspecific differences were readily detected but the markers were less reliable in distinguishing the accessions according to their region of origin or in separating the wild species from the cultivars. Morphological traits were found to be the least effective genetic markers. The relative effectiveness of the three systems as markers for genetic diversity was concluded to be SRAP > ISSR > morphological traits, but the combined data from ISSR + SRAP analyses was superior to all three. The information generated by the SRAP marker system correlated more closely with morphological variability than did the ISSR marker system. The morphological markers of plant height/crown size ratio, lower leaf length, ovary shape index and calyx length showed strong correlations with the genetic diversity index (GD_ij, PPB(II) and PSB) as generated by the percentage of polymorphic bands and percentage of special bands of the PCR-based markers.     

Identification of quantitative trait loci for bolting and flowering times in Chinese kale (Brassica oleracea var. alboglabra) based on SSR and SRAP markers

SUMMARY

Expressed sequence tag-simple sequence repeat (EST-SSR), simple sequence repeat (SSR), and sequence-related amplified polymorphism (SRAP) markers were used to construct the first intra-specific genetic linkage map for B. oleracea var. alboglabra. In addition, QTL locations were determined for bolting and flowering traits. A total of 189 polymorphic marker loci were detected in the two parents, with 45 marker loci not located on any linkage group (LG). The remaining 144 loci defined ten LGs and included 69 EST-SSR loci, three SSR loci, and 72 SRAP loci. The length of the map was 1,173.8 cM, the average distance between loci was 8.15 cM, and the proportion of segregation distortion loci was 25.7%. Three QTLs controlling bolting time and two QTLs controlling flowering time were detected in the F2:3 generation, which explained 37.07% and 18.44% of the phenotypic variation, respectively. Two QTLs controlling bolting time and two QTLs controlling flowering time were detected in the F2 generation, which explained 27.90% and 25.59% of the phenotypic variation, respectively. In both generations, QTLs ftB.1 and ftA.1 were located on LG5, and the distance between the two QTLs was only 1.0 cM, suggesting that they could be at an identical locus. These results provide a useful reference for future QTL studies on bolting and flowering time in B. oleracea var. alboglabra, and can be used to identify markers linked to these QTLs for marker-assisted selection in commercial breeding programmes.     

Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

Background  

We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.