QTL Mapping Using RIL Population.

Genetic maps are an excellent tool for the analysis of important traits, the development of which is the result of the combined expression of several genes, enabling the genomic localization of the factors determining them. Such features, characterized by a normal distribution of values, are referred to as quantitative or polygenic. The analysis of their genetic background using a chromosome map is called the mapping of quantitative traits loci (QTL). QTL analysis is a statistical method of determining the genetic association of phenotypic data (trait measurements) with genotypic data (DNA markers assigned to linkage groups).There are numerous tools developed for QTL mapping. This chapter introduces Windows QTL Cartographer with Composite Interval Mapping (CIM) method, which estimates the QTL position by combining interval mapping with multiple regression. The genotypic and phenotypic data used in the exemplary QTL mapping procedure were obtained for the recombinant inbred line (RIL) population of rye. Plant height, assessed in three seasons, was the exemplary trait under study.

Genetic Map Construction Using F2 and RIL/DH Mapping Population.

Genetic mapping is the determination of the position and relative genetic distance between genes or molecular markers in the chromosomes of a particular species. The construction of genetic maps uses data from the genotyping of the mapping population. Among the different mapping populations used, two are relatively common: the F2 and recombinant inbred lines (RILs) obtained as a result of the controlled crossing of genetically diverse parental forms (e.g., inbred lines). Also, the dihaploid (DH) population is often used in plants, but obtaining DHs in different crops, including rye, is very difficult or even impossible. Any molecular marker system can be used for genotyping. Polymorphic markers are used for linkage analysis, differentiating parental forms with segregation in the mapping population, consistent with the appropriate single-gene model. A genetic map is a great source of information on a species and can be an exquisite tool for analyzing important quantitative traits (QT).This chapter presents the procedure of genetic map construction with two different algorithms using the JoinMap5.0 program. First, the Materials section briefly informs about the mapping program, showing how to obtain a mapping population and prepare data for mapping. Finally, the Methods section describes the protocol for the mapping procedure itself.

Three new species of arbuscular mycorrhizal fungi (Glomeromycota) and Acaulospora gedanensis revised.

Studies of the morphology and the 45S nuc rDNA phylogeny of three potentially undescribed arbuscular mycorrhizal fungi (phylum Glomeromycota) grown in cultures showed that one of these fungi is a new species of the genus Diversispora in the family Diversisporaceae; the other two fungi are new Scutellospora species in Scutellosporaceae. Diversispora vistulana sp. nov. came from maritime sand dunes of the Vistula Spit in northern Poland, and S. graeca sp. nov. and S. intraundulata sp. nov. originally inhabited the Mediterranean dunes of the Peloponnese Peninsula, Greece. In addition, the morphological description of spores of Acaulospora gedanensis, originally described in 1988, was emended based on newly found specimens, and the so far unknown phylogeny of this species was determined. The phylogenetic analyses of 45S sequences placed this species among Acaulospora species with atypical phenotypic and histochemical features of components of the two inner germinal walls.

In search of the relationship between the rye polyamine oxidase (PAO) gene and resistance to powdery mildew (PM).

Powdery mildew (PM), a common cereal disease in cultivated areas, including Europe and other temperate regions, is caused by the fungus Blumeria graminis. While PM is one of the most important wheat leaf diseases globally, rye is highly tolerant to PM. It has been reported that in barley infected with PM, polyamine oxidase (PAO) activity related to the production of hydrogen peroxide (H2O2) has increased, which may promote defense against biotrophic or hemibiotrophic pathogens. The current study aimed to assess the relationship between the segregation of the polymorphic marker for rye PAO (ScPAO) and the level of PM infection in plants. The genetic mapping in two interline populations shows that ScPAO is located on chromosome 7R. Further analysis comparing ScPAO location to mapped wheat (Triticum aestivum L.) PAO duplicates suggests the ScPAO homology with TaPAO6 or TaPAO7. A possible association of ScPAO from 7R with PM resistance is demonstrated in the recombinant inbred lines (RIL)-L population phenotyped for PM infection. Finally, three novel QTLs for PM resistance on the 7R chromosome of rye are detected.

Fingerprinting, structure, and genetic relationships among selected accessions of blue honeysuckle (Lonicera caerulea L.) from European collections.

Due to its value and economic importance, the genome of Lonicera caerulea L. has been widely studied in various fields of science. In this study the genetic structure and relationships between 24 accessions of L. caerulea of different origins were assessed. A total of 692, 814, and 258 loci were amplified using 43 RAPD (random amplified polymorphic DNA), 40 ISSR (intersimple sequence repeat), and 20 R-ISSR (RAPD+ISSR) primers, respectively. Among the amplified loci, 66-78% were polymorphic and 12-20% were private. Selected R-ISSR sequences were detected in Lonicera japonica transcripts. Cluster and STRUCTURE analyses performed for each of the techniques revealed the existing differences and unknown similarities between the genotypes. The r-factor values calculated in the Mantel test indicated highly significant positive correlations between the Nei distance matrices, similar to the F ST values (F ST_RAPD = 0.223, F ST_ISSR = 0.279, F ST_R-ISSR = 0.363) determined in the analysis of molecular variance. It was found that 78%, 72%, and 64% of the genetic variations were related to the differences observed within the populations, which suggest that the variations are mainly reflected in the differences among the genotypes. The principal coordinate analysis showed greater differences between the mean distances of the Lonicera genotype pair and the actual distances of the same pairs on the Nei matrix compared to multidimensional scaling. These differences were 45%, 56%, and 42% higher for RAPD, ISSR and R-ISSR, respectively.

Three new species of arbuscular mycorrhizal fungi of the genus Diversispora from maritime dunes of Poland.

Three new species of arbuscular mycorrhizal fungi of the genus Diversispora (phylum Glomeromycota) were described based on their morphology and molecular phylogeny. The phylogeny was inferred from the analyses of the partial 45S rDNA sequences (18S-ITS-28S) and the largest subunit of RNA polymerase II (rpb1) gene. These species were associated in the field with plants colonizing maritime sand dunes of the Baltic Sea in Poland and formed mycorrhiza in single-species cultures.

A new order, Entrophosporales, and three new Entrophospora species in Glomeromycota.

As a result of phylogenomic, phylogenetic, and morphological analyses of members of the genus Claroideoglomus, four potential new glomoid spore-producing species and Entrophospora infrequens, a new order, Entrophosporales, with one family, Entrophosporaceae (=Claroideoglomeraceae), was erected in the phylum Glomeromycota. The phylogenomic analyses recovered the Entrophosporales as sister to a clade formed by Diversisporales and Glomeraceae. The strongly conserved entrophosporoid morph of E. infrequens, provided with a newly designated epitype, was shown to represent a group of cryptic species with the potential to produce different glomoid morphs. Of the four potential new species, three enriched the Entrophosporales as new Entrophospora species, E. argentinensis, E. glacialis, and E. furrazolae, which originated from Argentina, Sweden, Oman, and Poland. The fourth fungus appeared to be a glomoid morph of the E. infrequens epitype. The physical association of the E. infrequens entrophosporoid and glomoid morphs was reported and illustrated here for the first time. The phylogenetic analyses, using nuc rDNA and rpb1 concatenated sequences, confirmed the previous conclusion that the genus Albahypha in the family Entrophosporaceae sensu Oehl et al. is an unsupported taxon. Finally, the descriptions of the Glomerales, Entrophosporaceae, and Entrophospora were emended and new nomenclatural combinations were introduced.

New Glomeromycotan Taxa, Dominikia glomerocarpica sp. nov. and Epigeocarpum crypticum gen. nov. et sp. nov. From Brazil, and Silvaspora gen. nov. From New Caledonia.

Examination of fungal specimens collected in the Atlantic rain forest ecosystems of Northeast Brazil revealed many potentially new epigeous and semihypogeous glomerocarp-producing species of the phylum Glomeromycota. Among them were two fungi that formed unorganized epigeous glomerocarps with glomoid spores of almost identical morphology. The sole structure that distinguished the two fungi was the laminate layer 2 of their three-layered spore wall, which in spores of the second fungus crushed in PVLG-based mountants contracted and, consequently, transferred into a crown-like structure. Surprisingly, phylogenetic analyses of sequences of the 18S-ITS-28S nuc rDNA and the rpb1 gene indicated that these glomerocarps represent two strongly divergent undescribed species in the family Glomeraceae. The analyses placed the first in the genus Dominikia, and the second in a sister clade to the monospecific generic clade Kamienskia with Kamienskia bistrata. The first species was described here as Dominikia glomerocarpica sp. nov. Because D. glomerocarpica is the first glomerocarp-forming species in Dominikia, the generic description of this genus was emended. The very large phylogenetic distance and the fundamental morphological differences between the second species and K. bistrata suggested us to introduce a new genus, here named as Epigeocarpum gen. nov., and name the new species Epigeocarpum crypticum sp. nov. In addition, our analyses also focused on an arbuscular mycorrhizal fungus originally described as Rhizophagus neocaledonicus, later transferred to the genus Rhizoglomus. The analyses indicated that this species does not belong to any of these two genera but represents a new clade at the rank of genus in the Glomeraceae, here described as Silvaspora gen. nov.

The GAMYB gene in rye: sequence, polymorphisms, map location, allele-specific markers, and relationship with α-amylase activity.

BACKGROUND: Transcription factor (TF) GAMYB, belonging to MYB family (named after the gene of the avian myeloblastosis virus) is a master gibberellin (GA)-induced regulatory protein that is crucial for development and germination of cereal grain and involved in anther formation. It activates many genes including high-molecular-weight glutenin and α-amylase gene families. This study presents the first attempt to characterize the rye gene encoding GAMYB in relation to its sequence, polymorphisms, and phenotypic effects. RESULTS: ScGAMYB was mapped on rye chromosome 3R using high-density Diversity Arrays Technology (DArT)/DArTseq-based maps developed in three mapping populations. The ScGAMYB sequences were identified in RNA-seq libraries of four rye inbred lines. The transcriptome used for the search contained almost 151,000 transcripts with a median contig length of 500 nt. The average amount of total base raw data was approximately 9 GB. Comparative analysis of the ScGAMYB sequence revealed its high level of homology to wheat and barley orthologues. Single nucleotide polymorphisms (SNPs) detected among rye inbred lines allowed the development of allele specific-PCR (AS-PCR) markers for ScGAMYB that might be used to detect this gene in wide genetic stocks of rye and triticale. Segregation of the ScGAMYB alleles showed significant relationship with α-amylase activity (AMY). CONCLUSIONS: The research showed the strong similarity of rye GAMYB sequence to its orthologues in other Graminae and confirmed the position in the genome consistent with the collinearity rule of cereal genomes. Concurrently, the ScGAMYB coding sequence (cds) showed stronger variability (24 SNPs) compared to the analogous region of wheat (5 SNPs) and barley (7 SNPs). The moderate regulatory effect of ScGAMYB on AMY was confirmed, therefore, ScGAMYB was identified as a candidate gene for partial control of α-amylase production in rye grain. The predicted structural protein change in the aa region 362-372, caused by a single SNP (C/G) at the 1100 position in ScGAMYB cds and single aa sequence change (S/C) at the 367 position, is the likely cause of the differences in the effectiveness of ScGAMYB regulatory function associated with AMY. The development of sequence-based, allele-specific (AS) PCR markers could be useful in research and application.

A complex network of QTL for thousand-kernel weight in the rye genome.

Here, QTL mapping for thousand-kernel weight carried out within a 541 × Ot1-3 population of recombinant inbred lines using high-density DArT-based map and three methods (single-marker analysis with F parametric test, marker analysis with the Kruskal-Wallis K* nonparametric test, and the recently developed analysis named genes interaction assorting by divergent selection with χ2 test) revealed 28 QTL distributed over all seven rye chromosomes. The first two methods showed a high level of consistency in QTL detection. Each of 13 QTL revealed in the course of gene interaction assorting by divergent selection analysis coincided with those detected by the two other methods, confirming the reliability of the new approach to QTL mapping. Its unique feature of discriminating QTL classes might help in selecting positively acting QTL and alleles for marker-assisted selection. Also, interaction among seven QTL for thousand-kernel weight was analyzed using gene interaction assorting by the divergent selection method. Pairs of QTL showed a predominantly additive relationship, but epistatic and complementary types of two-loci interactions were also revealed.

Identification and mapping of a new recessive dwarfing gene dw9 on the 6RL rye chromosome and its phenotypic effects.

The introduction of high-yielding semi-dwarf varieties of wheat into cultivation has led to a "green revolution." This has required intensive research into various sources of dwarfism in wheat. However, there has been very little advancement in research on dwarfing genes in rye in comparison to wheat or barley. So far, three dominant dwarfing genes (Ddw1, Ddw3, and Ddw4) and three recessive genes (ct1, ct2, and np) have been characterized and precisely mapped in rye. There is no complete catalog of dwarfing genes available in rye. This paper presents an identification of the source of dwarfism and preliminary characterization of the new recessive gene dw9 from the BK-1 line. The gene was mapped on the long arm of the 6R chromosome and belongs to the GA-insensitive group. The initial characterization of the influence of this gene on morphological traits shows that it significantly affects the decrease of yielding trait parameters. A full evaluation can be performed after detailed breeding studies.

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.

Identification of a novel, dominant dwarfing gene (Ddw4) and its effect on morphological traits of rye.

Shortening rye stems to improve lodging resistance is among the major tasks awaiting breeders of this cereal. The most straightforward way to achieve this goal is the implementation of a dominant dwarfing gene into high yielding cultivars. The choice of dominant dwarfing genes in rye is limited to Ddw1 and Ddw3 loci, which are well characterized with respect to map position and tightly linked molecular markers on the long arms of chromosomes 5RL and 1RL, respectively. This paper reports on the identification and preliminary characterization of a novel dominant dwarfing gene, Ddw4, from line S44. This was mapped within the centromeric region of chromosome 3R. The Ddw4 gene is sensitive to exogenous gibberellin. Its introduction into the rye populational cultivar Dankowskie Amber decreased plant height by c. 54% without any negative effects on spike length and number of kernels per spike. Further genetic studies are needed to determine the perspectives for application of the newly detected dwarfing gene into breeding programs for short-stem rye.

QTL mapping for benzoxazinoid content, preharvest sprouting, α-amylase activity, and leaf rust resistance in rye (Secale cereale L.).

Mapping population of recombinant inbred lines (RILs) representing 541 × Ot1-3 cross exhibited wide variations of benzoxazinoid (BX) content in leaves and roots, brown rust resistance, α-amylase activity in the grain, and resistance to preharvest sprouting. QTL mapping of major BX species using a DArT-based map revealed a complex genetic architecture underlying the production of these main secondary metabolites engaged in stress and allelopathy responses. The synthesis of BX in leaves and roots was found to be regulated by different QTL. The QTL for the BX content, rust resistance, α-amylase activity, and preharvest sprouting partially overlapped; this points to their common genetic regulation by a definite subset of genes. Only one QTL for BX located on chromosome 7R coincided with the loci of the ScBx genes, which were mapped as two clusters on chromosomes 5RS (Bx3-Bx5) and 7R (Bx1-Bx2). The QTL common for several BX species, rust resistance, preharvest sprouting, and α-amylase activity are interesting objects for further exploration aimed at developing common markers for these important agronomic traits.

Identification of Single Nucleotide Polymorphisms Associated with Brown Rust Resistance, α-Amylase Activity and Pre-harvest Sprouting in Rye (Secale cereale L.).

Rye is a crop with relatively high resistance to biotic and abiotic stresses. However, the resistance to brown rust (Puccinia recondita f. sp. secalis) and pre-harvest sprouting are still not satisfactory. High α-amylase activity is also among the main disadvantages of this species. Therefore, effective tools, e.g. molecular markers, allowing precise and environmentally independent selection of favourable alleles are desirable. In the present study, two kinds of association mapping-genome-wide association mapping (GWAM) based on sequences of DArTSeq markers and candidate gene association mapping (CGAM) based on sequences of ScBx genes-were chosen for development of molecular markers fulfilling these criteria. The analysed population consisted of 149 diverse inbred lines (DILs). Altogether, 67 and 11 single nucleotide polymorphisms (SNPs) identified in, respectively, GWAM and CGAM, were significantly associated with the investigated traits: 2 SNPs with resistance to brown rust, 71 SNPs with resistance to pre-harvest sprouting and 5 SNPs with α-amylase activity in the grain. Fifteen SNPs were stable across all environments. The highest number (13) of environmentally stable SNPs was associated with pre-harvest sprouting resistance. The test employing the Kompetitive Allele Specific PCR method proved the versatility of four markers identified in both GWAM and CGAM.

The application of GBS markers for extending the dense genetic map of rye (Secale cereale L.) and the localization of the Rfc1 gene restoring male fertility in plants with the C source of sterility-inducing cytoplasm.

Genotyping by sequencing (GBS) is an efficient method of genotyping in numerous plant species. One of the crucial steps toward the application of GBS markers in crop improvement is anchoring them on particular chromosomes. In rye (Secale cereale L.), chromosomal localization of GBS markers has not yet been reported. In this paper, the application of GBS markers generated by the DArTseq platform for extending the high-density map of rye is presented. Additionally, their application is used for the localization of the Rfc1 gene that restores male fertility in plants with the C source of sterility-inducing cytoplasm. The total number of markers anchored on the current version of the map is 19,081, of which 18,132 were obtained from the DArTseq platform. Numerous markers co-segregated within the studied mapping population, so, finally, only 3397 unique positions were located on the map of all seven rye chromosomes. The total length of the map is 1593 cM and the average distance between markers is 0.47 cM. In spite of the resolution of the map being not very high, it should be a useful tool for further studies of the Secale cereale genome because of the presence on this map of numerous GBS markers anchored for the first time on rye chromosomes. The Rfc1 gene was located on high-density maps of the long arm of the 4R chromosome obtained for two mapping populations. Genetic maps were composed of DArT, DArTseq, and PCR-based markers. Consistent mapping results were obtained and DArTs tightly linked to the Rfc1 gene were successfully applied for the development of six new PCR-based markers useful in marker-assisted selection.

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.

DArT markers tightly linked with the Rfc1 gene controlling restoration of male fertility in the CMS-C system in cultivated rye (Secale cereale L.).

The Rfc1 gene controls restoration of male fertility in rye (Secale cereale L.) with sterility-inducing cytoplasm CMS-C. Two populations of recombinant inbred lines (RIL) were used in this study to identify DArT markers located on the 4RL chromosome, in the close vicinity of the Rfc1 gene. In the population developed from the 541×2020LM intercross, numerous markers tightly linked with the restorer gene were identified. This group contained 91 DArT markers and three SCARs additionally analyzed in the study. All these markers were mapped in the distance not exceeding 6 cM from the gene of interest. In the second mapping population (541×Ot1-3 intercross), only 9 DArT markers located closely to the Rfc1 gene were identified. Five of these DArT markers were polymorphic in both populations.

Genomic architecture of alpha-amylase activity in mature rye grain relative to that of preharvest sprouting.

Bi-directional selective genotyping (BSG) carried out on two opposite groups of F(9)(541 × Ot1-3) recombinant inbred lines (RILs) with extremely low and extremely high alpha-amylase activities in mature (dry) grain of rye, followed by molecular mapping, revealed a complex system of selection-responsive loci. Three classes of loci controlling alpha-amylase activity were discerned, including four major AAD loci on chromosomes 3R (three loci) and 6RL (one locus) responding to both directions of the disruptive selection, 20 AAR loci on chromosomes 2RL (three loci), 3R (three loci), 4RS (two loci), 5RL (three loci), 6R (two loci) and 7R (seven loci) responding to selection for low alpha-amylase activity and 17 AAE loci on chromosomes 1RL (seven loci), 2RS (two loci), 3R (two loci), 5R (two loci) and 6RL (four loci) affected by selection for high alpha-amylase activity. The majority of the discerned AA loci also showed responsiveness to selection for preharvest sprouting (PHS). Two AAD loci on chromosome arm 3RL coincided with PHSD loci. The AAD locus on chromosome arm 3RS was independent from PHS, whereas that on chromosome 6RL belonged to the PHSR class. AAR-PHSR loci were found on chromosomes 4RS (one locus) and 5R (two loci) and AAE-PHSE loci were identified on chromosomes 1RL (one locus) and 5RL (one locus). Some PHSD loci represented the AAE (chromosomes 1RL, 3RS and 3RL) or AAR classes (chromosome 5RL). AAR and AAE loci not related to PHS were found on chromosomes 1RL, 2R, 3RS, 4R, 6RL and 7RL. On the other hand, several PHS loci (1RL, 3RS, 5RL, 6RS and 7RS) had no effect on alpha-amylase activity. Allele originating from the parental line 541 mapped in six AA loci on chromosomes 2R (two loci), 5R (three loci) and 7R (one locus) exerted opposite effects on PHS and alpha-amylase activity. Differences between the AA and PHS systems of loci may explain the weak correlation between these two traits observed among recombinant inbred lines. Strategies for the breeding of sprouting-resistant varieties with low alpha-amylase and high PHS resistance are discussed.

A consensus map of chromosome 6R in rye (Secale cereale L.).

Four F(2) mapping populations derived from crosses between rye inbred lines DS2 x RXL10, 541 x Ot1-3, S120 x S76 and 544 x 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.