Monika Rakoczy-Trojanowska

DArT markers for the rye genome - genetic diversity and mapping

BackgroundImplementation of molecular breeding in rye (Secale cereale L.) improvement programs depends on the availability of high-density molecular linkage maps. However, the number of sequence-specific PCR-based markers available for the species is limited. Diversity Arrays Technology (DArT) is a microarray-based method allowing for detection of DNA polymorphism at several thousand loci in a single assay without relying on DNA sequence information. The objective of this study was the development and application of Diversity Arrays technology for rye.ResultsUsing the Pst I/Taq I method of complexity reduction we created a rye diversity panel from DNA of 16 rye varieties and 15 rye inbred lines, including parents of a mapping population consisting of 82 recombinant inbred lines. The usefulness of a wheat diversity panel for identification of DArT markers for rye was also demonstrated. We identified 1022 clones that were polymorphic in the genotyped ILs and varieties and 1965 clones that differentiated the parental lines L318 and L9 and segregated in the mapping population. Hierarchical clustering and ordination analysis were performed based on the 1022 DArT markers to reveal genetic relationships between the rye varieties and inbred lines included in the study. Chromosomal location of 1872 DArT markers was determined using wheat-rye addition lines and 1818 DArT markers (among them 1181 unique, non-cosegregating) were placed on a genetic linkage map of the cross L318 × L9, providing an average density of one unique marker every 2.68 cM. This is the most saturated rye linkage map based solely on transferable markers available at the moment, providing rye breeders and researches with a better choice of markers and a higher probability of finding polymorphic markers in the region of interest.ConclusionThe Diversity Arrays Technology can be efficiently and effectively used for rye genome analyses - assessment of genetic similarity and linkage mapping. The 11520-clone rye genotyping panel with several thousand markers with determined chromosomal location and accessible through an inexpensive genotyping service is a valuable resource for studies on rye genome organization and in molecular breeding of the species.

A High Density Consensus Map of Rye (Secale cereale L.) Based on DArT Markers

Background Rye (Secale cereale L.) is an economically important crop, exhibiting unique features such as outstanding resistance to biotic and abiotic stresses and high nutrient use efficiency. This species presents a challenge to geneticists and breeders due to its large genome containing a high proportion of repetitive sequences, self incompatibility, severe inbreeding depression and tissue culture recalcitrance. The genomic resources currently available for rye are underdeveloped in comparison with other crops of similar economic importance. The aim of this study was to create a highly saturated, multilocus linkage map of rye via consensus mapping, based on Diversity Arrays Technology (DArT) markers. Methodology/Principal Findings Recombinant inbred lines (RILs) from 5 populations (564 in total) were genotyped using DArT markers and subjected to linkage analysis using Join Map 4.0 and Multipoint Consensus 2.2 software. A consensus map was constructed using a total of 9703 segregating markers. The average chromosome map length ranged from 199.9 cM (2R) to 251.4 cM (4R) and the average map density was 1.1 cM. The integrated map comprised 4048 loci with the number of markers per chromosome ranging from 454 for 7R to 805 for 4R. In comparison with previously published studies on rye, this represents an eight-fold increase in the number of loci placed on a consensus map and a more than two-fold increase in the number of genetically mapped DArT markers. Conclusions/Significance Through the careful choice of marker type, mapping populations and the use of software packages implementing powerful algorithms for map order optimization, we produced a valuable resource for rye and triticale genomics and breeding, which provides an excellent starting point for more in-depth studies on rye genome organization.

The identification of QTLs associated with the in vitro response of rye (Secale cereale L.).

This study was conducted in order to identify quantitative trait loci (QTLs) for the in vitro culture response of winter rye (Secale cereale L.) immature embryos and immature inflorescences. A genetic linkage map comprising 67 SSRs, 9 ISSRs, 13 SAMPLs, 7 RAPDs, 2 SCARs and one EST marker was created based on the analyses of 102 recombinant inbred lines from the cross between lines L318 (which has a good response in tissue cultures) and L9 (which is unable to regenerate plants from somatic tissues and anthers). The map spans 979.2 cM, and the average distance between markers is 9.9 cM. Two characteristics were evaluated: callus induction (CI) and somatic embryogenesis ability (SE). They were expressed as the percentage of immature embryos/inflorescences producing callus (designated ECI/ICI) and the percentage of explants producing somatic embryos (ESE/ISE). All the analysed traits showed continuous variation in the mapping population but a non-normal frequency distribution. We identified nine putative QTLs controlling the tissue culture response of rye, explaining up to 41.6% of the total phenotypic variation: two QTLs for ECI — eci-1, eci-2; 4 for ESE — ece-1, ese-2, ese-3, ese-4; 2 for ICI — ici-1, ici2; and 1 for ISE — ise-1. They were detected on chromosomes 1R, 4R, 5R, 6R and 7R.

Efficiency of different PCR-based marker systems in assessing genetic diversity among winter rye (Secale cereale L.) inbred lines

SummaryThe aim of this study was to investigate the efficiency of ISSR, SSR, and SAMPL marker systems in detecting genetic polymorphism among 30 winter rye inbred lines and to compare the results of cluster analysis performed on data from these marker systems using different statistical methods and coefficients. Each marker system was able to discriminate among the materials analyzed with the lowest value of average genetic similarity (GS) obtained with ISSR markers (0.2888) and the highest with SAMPLs (0.5381). EST-derived SSRs turned out to be less efficient in detecting genetic diversity than those from genomic libraries (average GS values 0.3814 and 0.3221, respectively). The average GS value for combined SSR data was 0.3569. The lack of correlations between similarity and cophenetic matrices obtained with various methods systems suggests that different marker systems should be used simultaneously for a genetic diversity study to exploit as many sources of polymorphisms as possible.

Genetic Mapping of QTLs for Tissue-Culture Response in Plants

SummaryPlant regeneration from cultured tissues has been shown to be under genetic control in a number of plant species. Using quantitative trait loci (QTL) mapping technology, it has become possible to estimate the number of loci controlling genetic variation and to characterize their map position in the genome. With the use of different types of mapping populations such as recombinant inbred lines (RIL), F2 or doubled haploids (DH) it was possible to detect QTLs for tissue culture response (TCR). For the evaluation of TCR numerous parameters describing induction and regeneration steps were applied. However, the lack of consistency with respect to parameters used by different researchers sometimes makes comparisons between QTL studies complicated. Another problem frequently present in works dealing with QTLs for TCR is skewed distributions of the traits used for evaluating tissue culture capability, usually interpreted as the indication of a presence of major genes. The majority of QTL analyses of TCR-traits were conducted with monocots – rice, barley and maize. The number of QTLs detected for a particular characteristic of tissue culture capability ranged from 1 to 8. The percentage of phenotypic variation explained by a single QTL varied from 2.7 to 65.4%, with the values between ca. 6 and ca. 26 % being more common. Determination of molecular markers linked to a QTL of TCR provides valuable tools for biotechnological approaches aimed at improving plant regeneration capability.

The influence of genotype and medium on rye (Secale cereale L.) anther culture

Anthers of three rye inbred lines - L9, L318, Dw28, one synthetic hybrid - F1(5) and one variety, Dańkowskie Zote (DZ), were cultured on two media based on N6, and two based on P2 components. The induction rate significantly depended on genotype - the best results were obtained for line L318 and the lowest percentage of responding anthers was noticed in line L9. There was no universal medium for all tested genotypes. The highest induction rate (IR) for lines L318, L9 and hybrid F1(5) was obtained on medium CI (11.94, 0.71 and 1.75 respectively). For DZ, the P2I medium was better than the others while for Dw28 CIP turned to be as suitable as CI. A highly significant interaction between genotype and medium was proved. Single anthers of DZ, L318 and L9 produced embryos on CI, CIP and P2I media. They did not develop into plants but after transferring them to CS1.7 medium, a secondary, embryogenic callus was obtained. Such a reaction has not been described in rye until now. In spite of a relatively high IR, plant regeneration was rather poor. An elaboration of this step in haploid production is needed. Most of the analyzed calluses and microspore derived regenerants proved to be haploids, according to flow cytometry analysis. Plants treated with colchicine were doubled haploids.

Genome-wide characterization of genetic diversity and population structure in Secale

BackgroundNumerous rye accessions are stored in ex situ genebanks worldwide. Little is known about the extent of genetic diversity contained in any of them and its relation to contemporary varieties, since to date rye genetic diversity studies had a very limited scope, analyzing few loci and/ or few accessions. Development of high throughput genotyping methods for rye opened the possibility for genome wide characterizations of large accessions sets. In this study we used 1054 Diversity Array Technology (DArT) markers with defined chromosomal location to characterize genetic diversity and population structure in a collection of 379 rye accessions including wild species, landraces, cultivated materials, historical and contemporary rye varieties.ResultsAverage genetic similarity (GS) coefficients and average polymorphic information content (PIC) values varied among chromosomes. Comparison of chromosome specific average GS within and between germplasm sub-groups indicated regions of chromosomes 1R and 4R as being targeted by selection in current breeding programs. Bayesian clustering, principal coordinate analysis and Neighbor Joining clustering demonstrated that source and improvement status contributed significantly to the structure observed in the analyzed set of Secale germplasm. We revealed a relatively limited diversity in improved rye accessions, both historical and contemporary, as well as lack of correlation between clustering of improved accessions and geographic origin, suggesting common genetic background of rye accessions from diverse geographic regions and extensive germplasm exchange. Moreover, contemporary varieties were distinct from the remaining accessions.ConclusionsOur results point to an influence of reproduction methods on the observed diversity patterns and indicate potential of ex situ collections for broadening the genetic diversity in rye breeding programs. Obtained data show that DArT markers provide a realistic picture of the genetic diversity and population structure present in the collection of 379 rye accessions and are an effective platform for rye germplasm characterization and association mapping studies.

Erratum to: Structural characteristics of ScBx genes controlling the biosynthesis of hydroxamic acids in rye (Secale cereale L.)

Benzoxazinoids (BX) are major secondary metabolites of gramineous plants that play an important role in disease resistance and allelopathy. They also have many other unique properties including anti-bacterial and anti-fungal activity, and the ability to reduce alfa–amylase activity. The biosynthesis and modification of BX are controlled by the genes Bx1 ÷ Bx10, GT and glu, and the majority of these Bx genes have been mapped in maize, wheat and rye. However, the genetic basis of BX biosynthesis remains largely uncharacterized apart from some data from maize and wheat. The aim of this study was to isolate, sequence and characterize five genes (ScBx1, ScBx2, ScBx3, ScBx4 and ScBx5) encoding enzymes involved in the synthesis of DIBOA, an important defense compound of rye. Using a modified 3D procedure of BAC library screening, seven BAC clones containing all of the ScBx genes were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of these genes, including their promoters, introns and 3’UTRs. Comparative analysis showed that the ScBx genes are similar to those of other Poaceae species, especially to the TaBx genes. The polymorphisms present both in the coding sequences and non-coding regions of ScBx in relation to other Bx genes are predicted to have an impact on the expression, structure and properties of the encoded proteins.

The specificity and genetic background of the rye (Secale cereale L.) tissue culture response

Rye is one of the most important crops in Eastern and Northern Europe. Despite the numerous beneficial features of rye, its annual production decreases successively which correlates with the lack of progress in its breeding compared with other cereals. Biotechnological methods could effectively improve the breeding of rye. However, their application is highly limited by the absence of an efficient procedure for plant regeneration in vitro, since rye is one of the most recalcitrant cereals with regard to the tissue culture response (TCR), and successful regeneration is highly dependent on genotype. Efforts to understand the genetic mechanisms controlling TCR of rye have elucidated some basic aspects, and several genes and genome regions controlling this trait have been identified. The aim of this review is to summarize the limited current knowledge of this topic.

Expression analysis of somatic embryogenesis-related SERK, LEC1, VP1 and NiR ortologues in rye (Secale cereale L.).

The genetic basis of the regeneration process in cultured immature embryos of rye (Secale cereale L.) was analyzed. The experiments were designed to reveal differences between the in vitro culture responses of two inbred lines: L318 (a high regeneration ability) and L9 (a low potential for regeneration). The rye ortologues of plant genes previously recognized as crucial for somatic embryogenesis and morphogenesis in vitro were identified. Using oligonucleotide primers designed to conserved regions of the genes Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon 1 (LEC1), Viviparous 1 (VP1) and NiR (encoding ferredoxin-nitrite reductase), it was possible to amplify specific homologous sequences from rye RNA by RT-PCR. The transcript levels of these genes were then measured during the in vitro culture of zygotic embryos, and the sites of expression localized. The expression profiles of these genes indicate that their function is likely to be correlated with the in vitro response of rye. In line L9, increased expression of the rye SERK ortologue was observed at most stages during the culture of immature embryos. The suppression of ScSERK expression appears to start after the induction of somatic embryogenesis and lasts up to plant regeneration. The rye ortologues of the LEC1 and VP1 genes may function in a complimentary manner and have a negative effect on the production of the embryogenic callus. The expression of the rye NiR ortologue during in vitro culture reveals its importance in the process of plant regeneration.