Stefan Stojałowski

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.

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid Secale cereale × Dasypyrum villosum hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.

The application of high-density genetic maps of rye for the detection of QTLs controlling morphological traits

The development of genetic maps is, nowadays, one of the most intensive research activities of plant geneticists. One of the major goals of genome mapping is the localisation of quantitative trait loci (QTLs). This study was aimed at the identification of QTLs controlling morphological traits of rye and comparison of their localisation on genetic maps constructed with the use of genetically different germplasms. For QTL analyses, two high-density consensus maps of two populations (RIL-S and RIL-M) of recombinant inbred lines (RIL) were applied. Plant height (Ph), length of spikes (Sl) and the number of spikelets per spike (Sps) were studied in both populations. Additionally, the number of kernels per spike under isolation (Kps), the weight of kernels per spike (Kw) and thousand kernel weight (Tkw) were assessed in the RIL-M population. Except for Tkw, the majority of the traits were correlated to each other. The non-parametric Kruskal–Wallis (K-W) test and composite interval mapping (CIM) revealed 18/48 and 24/18 regions of rye chromosomes engaged in the determination of Ph, Sl and Sps in the RIL-S and RIL-M populations, respectively. An additional 18/15 QTLs controlling Kps, Kw and Tkw were detected on a map of the RIL-M population. A numerous group of QTLs detected via CIM remained in agreement with the genomic regions found when the K-W test was applied. Frequently, the intervals indicated by CIM were narrower.

Mapping of sequence-specific markers and loci controlling preharvest sprouting and alpha-amylase activity in rye (Secale cereale L.) on the genetic map of an F2 (S120 × S76) population.

Location of the loci that control preharvest sprouting and alpha-amylase activity in rye was studied based on intercross S120×S76, consisting of 110 genotypes of F2 and F3 progenies. The genetic map currently consists of 141 loci distributed in 11 linkage groups, covering a distance of 506.4 cM, and was enriched during this study with 24 sequence-specific markers (7 SCARs, 7 SSRs, and 10 STSs). The extended map was applied for composite interval mapping of the loci controlling preharvest sprouting and α-amylase activity, revealing 3 significant QTLs for preharvest sprouting, located on chromosomes 3R, 5R and 6R (in 1999), and one QTL for α-amylase activity found on chromosome 2R (in 2000).

A consensus map of chromosome 6R in rye [Secale cereale L.]

Four F2 mapping populations derived from crosses between rye inbred lines DS2×RXL10, 541×Ot1-3, S120×S76 and 544×Ot0-20 were used to develop a consensus map of chromosome 6R. Thirteen marker loci that were polymorphic in more than one mapping population constituted the basis for the alignment of the four maps using the JoinMap v. 3.0 software package. The consensus map consists of 104 molecular marker loci including RFLPs, RAPDs, AFLPs, SSRs, ISSRs, SCARs, STSs and isozymes. The average distance between the marker loci is 1.3 cM, and the total map length is 135.5 cM. This consensus map may be used as a source of molecular markers for the rapid development of new maps of chromosome 6R in any mapping population.

Determining the plasmotypic structure of rye populations by SCAR markers

In rye (Secale cereale L.), 2 types of cytoplasmic male sterility are known: Pampa type (CMS-P) and Vavilovii type (CMS-V). As an alternative method to the conventional plasmotype-genotype interaction test, for identification of the cytoplasm type, the use of sequence-characterised amplified region (SCAR) markers was validated in this study. In over 2600 individual rye plants, representing 26 populations originating from Poland (18 cultivars), Iran (5 populations of primitive rye), and South America (3 populations), the cytoplasm type was determined by using a set of 3 SCAR markers. For about 10% of these individuals, the plasmotype-genotype interaction test was performed in parallel. The results of both tests were fully consistent. In the majority of the Polish populations, CMS-V was present, and only 4 populations contained CMS-P. Primitive Iranian populations contained predominantly normal cytoplasm, and only occasionally CMS-P was identified in them. South American populations displayed a mixture of normal cytoplasm, CMS-P and CMS-V. This work validates the use of SCAR markers as a reliable and quick method to determine the plasmotypic diversity of rye populations on a large scale.

Phenotypic effect and chromosomal localization of Ddw3, the dominant dwarfing gene in rye (Secale cereale L.)

AbstractDwarfing genes play a major role in development of semi-dwarf cultivars of various cereals, but in rye this type of cultivar is still not common. Several recessive and dominant dwarfing genes in rye have been reported. Among the known dominant dwarfing genes in rye, two are well characterised: Ddw1 from the 5RL chromosome and Ddw2 located on 7R. This study was aimed at characterisation of the K11 source of dominantly inherited dwarfism found in plant materials grown in the Plant Breeding and Acclimatization Institute (Radzików, Poland). Mapping analyses in this study indicate that the dominant dwarf gene under investigation is located on the 1RL chromosome and is independent from previously known genes. The gene was named Ddw3. Phenotypic effect of the Ddw3 was tested on two pairs of near-isogenic lines. Six morphological traits were analysed in two or three growing seasons: plant height, length of the second internode from the base, number of internodes, tillering, spike length, and number of spikelets per spike. No negative or weak influences on yield-related traits were observed. The examination of the dwarf plants’ reaction to seedling treatment by gibberellic acid solution revealed sensitivity of Ddw3 to this growth regulator. This report is the first evidence of the dominant Ddw3 gene in rye and its presence on the 1R chromosome.

Bidirectional selective genotyping approach for the identification of quantitative trait loci controlling earliness per se in winter rye (Secale cereale L.)

The genes controlling earliness of plants include genes responsible for vernalisation (Vrn) and photoperiod (Ppd), and those that are not entirely associated with a response to temperature or light. The last group of loci is known as earliness per se (Eps). Eps genes have been most commonly reported in the scientific literature as quantitative trait loci (QTL). The objective of this study was to use a bidirectional selective genotyping (BSG) method for the identification of loci controlling Eps, assessed at the heading stage in a newly developed rye population of recombinant inbred lines. We identified four linkage groups representing fragments of chromosomes, 1R, 5R, 6R and 7R, and containing, in total, 155 DArT markers significantly associated with earliness in rye. The results are discussed in the context of previously published QTL analyses.

The Development of Hybrid Triticale

Triticale possesses a number of attributes that make it amenable for development into a hybrid cereal breeding system. It possesses a tendency for outcrossing, a large percentage of open flowers, longer duration of flowering, a high percentage of anther extrusion including high amount of pollen shed, and a longer pollen lifetime in comparison with the wheat. Furthermore, it possess the rye genome that can be exploited for heterosis, and it is self-pollinating meaning the development of inbred lines is straightforward. Finally, cytoplasmic male sterility (CMS) systems are available making the production of hybrid seed potentially cost-effective. Major challenges must be tackled before hybrid triticale becomes a reality, including the sorting of triticale germplasm into hybrid pools and the refining of CMS systems. This chapter outlines previous efforts to examine the biology and suitability of triticale to be developed into a hybrid cereal and current efforts and strategies to sort triticale germplasm into heterotic pools and develop CMS systems using Triticum timopheevi and Secale cereale cytoplasm. Preliminary work on hybrid seed production using the T. timopheevi CMS system has also been completed giving a glimpse of how seed production might occur on a commercial scale. Finally, areas for future hybrid triticale research are laid out, including the importance of defining heterotic pools, refining the CMS systems and improving the hybrid seed production system, as well as considerations on biomass production and potential effects of heterosis on triticale targeted at this production system.

Effective BAC clone anchoring with genotyping-by-sequencing and Diversity Arrays Technology in a large genome cereal rye

Identification of bacterial artificial chromosome (BAC) clones containing specific sequences is a prerequisite for many applications, such as physical map anchoring or gene cloning. Existing BAC library screening strategies are either low-throughput or require a considerable initial input of resources for platform establishment. We describe a high-throughput, reliable, and cost-effective BAC library screening approach deploying genotyping platforms which are independent from the availability of sequence information: a genotyping-by-sequencing (GBS) method DArTSeq and the microarray-based Diversity Arrays Technology (DArT). The performance of these methods was tested in a very large and complex rye genome. The DArTseq approach delivered superior results: a several fold higher efficiency of addressing genetic markers to BAC clones and anchoring of BAC clones to genetic map and also a higher reliability. Considering the sequence independence of the platform, the DArTseq-based library screening can be proposed as an attractive method to speed up genomics research in resource poor species.