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.

Genome-Wide Association Analysis for Hybrid Breeding in Wheat.

Disclosure of markers that are significantly associated with plant traits can help develop new varieties with desirable properties. This study determined the genome-wide associations based on DArTseq markers for six agronomic traits assessed in eight environments for wheat. Moreover, the association study for heterosis and analysis of the effects of markers grouped by linkage disequilibrium were performed based on mean values over all experiments. All results were validated using data from post-registration trials. GWAS revealed 1273 single nucleotide polymorphisms with biologically significant effects. Most polymorphisms were predicted to be modifiers of protein translation, with only two having a more pronounced effect. Markers significantly associated with the considered set of features were clustered within chromosomes based on linkage disequilibrium in 327 LD blocks. A GWAS for heterosis revealed 1261 markers with significant effects.

Evaluation of genetic structure in European wheat cultivars and advanced breeding lines using high-density genotyping-by-sequencing approach.

BACKGROUND: The genetic diversity and gene pool characteristics must be clarified for efficient genome-wide association studies, genomic selection, and hybrid breeding. The aim of this study was to evaluate the genetic structure of 509 wheat accessions representing registered varieties and advanced breeding lines via the high-density genotyping-by-sequencing approach. RESULTS: More than 30% of 13,499 SNP markers representing 2162 clusters were mapped to genes, whereas 22.50% of 26,369 silicoDArT markers overlapped with coding sequences and were linked in 3527 blocks. Regarding hexaploidy, perfect sequence matches following BLAST searches were not sufficient for the unequivocal mapping to unique loci. Moreover, allelic variations in homeologous loci interfered with heterozygosity calculations for some markers. Analyses of the major genetic changes over the last 27 years revealed the selection pressure on orthologs of the gibberellin biosynthesis-related GA2 gene and the senescence-associated SAG12 gene. A core collection representing the wheat population was generated for preserving germplasm and optimizing breeding programs. CONCLUSIONS: Our results confirmed considerable differences among wheat subgenomes A, B and D, with D characterized by the lowest diversity but the highest LD. They revealed genomic regions that have been targeted by breeding.

How Machine Learning Methods Helped Find Putative Rye Wax Genes Among GBS Data.

The standard approach to genetic mapping was supplemented by machine learning (ML) to establish the location of the rye gene associated with epicuticular wax formation (glaucous phenotype). Over 180 plants of the biparental F2 population were genotyped with the DArTseq (sequencing-based diversity array technology). A maximum likelihood (MLH) algorithm (JoinMap 5.0) and three ML algorithms: logistic regression (LR), random forest and extreme gradient boosted trees (XGBoost), were used to select markers closely linked to the gene encoding wax layer. The allele conditioning the nonglaucous appearance of plants, derived from the cultivar Karlikovaja Zelenostebelnaja, was mapped at the chromosome 2R, which is the first report on this localization. The DNA sequence of DArT-Silico 3585843, closely linked to wax segregation detected by using ML methods, was indicated as one of the candidates controlling the studied trait. The putative gene encodes the ABCG11 transporter.

Putative candidate genes responsible for leaf rolling in rye (Secale cereale L.).

BACKGROUND: Rolling of leaves (RL) is a phenomenon commonly found in grasses. Morphology of the leaf is an important agronomic trait in field crops especially in rice; therefore, majority of the rice breeders are interested in RL. There are only few studies with respect to RL of wheat and barley; however, the information regarding the genetic base of RL with respect to the shape of leaf in rye is lacking. To the best of our knowledge, this is the first study on the localization of loci controlling RL on high density consensus genetic map of rye. RESULTS: Genotypic analysis led to the identification of 43 quantitative trait loci (QTLs) for RL, grouped into 28 intervals, which confirms the multigenic base of the trait stated for wheat and rice. Four stable QTLs were located on chromosomes 3R, 5R, and 7R. Co-localization of QTL for RL and for different morphological, biochemical and physiological traits may suggests pleiotropic effects of some QTLs. QTLs for RL were associated with QTLs for such morphological traits as: grain number and weight, spike number per plant, compactness of spike, and plant height. Two QTLs for RL were found to coincide with QTLs for drought tolerance (4R, 7R), two with QTLs for heading earliness (2R, 7R), one with α-amylase activity QTL (7R) and three for pre-harvest sprouting QTL (1R, 4R, 7R). The set of molecular markers strongly linked to RL was selected, and the putative candidate genes controlling the process of RL were identified. Twelve QTLs are considered as linked to candidate genes on the base of DArT sequences alignment, which is a new information for rye. CONCLUSIONS: Our results expand the knowledge about the network of QTLs for different morphological, biochemical and physiological traits and can be a starting point to studies on particular genes controlling RL and other important agronomic traits (yield, earliness, pre-harvest sprouting, reaction to water deficit) and to appoint markers useful in marker assisted selection (MAS). A better knowledge of the rye genome and genes could both facilitate rye improvement itself and increase the efficiency of utilizing rye genes in wheat breeding.

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.

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.

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 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.

The importance of chromosomes from the sixth homeologic group in the restoration of male fertility in winter triticale with Triticum timopheevii cytoplasm.

The sterilising cytoplasm from Triticum timopheevii is presently considered to be the most promising as regards to the seed production of triticale hybrid cultivars. This study was aimed at the utilisation of Diversity Arrays Technology (DArT) for the preliminary identification of genomic regions with loci controlling male sterility/fertility in the field-grown F2 generation of the interline hybrid between male sterile line CMS-Salvo 15/1 and restorer line Stan I. The fertility of plants was examined by visual scoring as well as by the assessment of seed setting within bagged spikes. For DNA analyses, 92 individuals representing opposite phenotypes (male sterile vs. fully male fertile) were chosen from the whole F2 population, which consisted of 414 plants. The constructed genetic map consists of 759 DArT markers distributed in 24 linkage groups that cover a distance of 974.4 cM. Application of the interval mapping method and the Kruskal-Wallis test enabled the identification of six genomic regions engaged in the restoration of male fertility within the mapping population. The most effective restorer genes were found on chromosomes of the sixth homeologic group, i.e. on 6R (the most efficient), 6A and 6B. Additionally, linkage groups assigned to chromosomes 1BS, 3A and 3A/3B were important for the determination of male fertility.

Detection of the quantitative trait loci for α-amylase activity on a high-density genetic map of rye and comparison of their localization to loci controlling preharvest sprouting and earliness.

The objectives of the research were to determine the position of quantitative trait loci (QTL) for α-amylase activity on the genetic map of a rye recombinant inbred line population-S120 × S76-and to compare them to known QTL for preharvest sprouting and heading earliness. Fourteen QTL for α-amylase activity on all seven chromosomes were identified. The detected QTL were responsible for 6.09-23.32% of α-amylase activity variation. The lowest LOD value (2.22) was achieved by locus QAa4R-M3 and the highest (7.79) by locus QAa7R-M1. Some QTL intervals for features of interest overlapped partially or completely. There were six overlapping QTL for α-amylase activity and preharvest sprouting (on 1R, 3R, 4R, 6R, 7R) and the same number for preharvest sprouting and heading earliness (on 1R, 2R, 6R, 7R). Furthermore, there was one interval partially common to all three traits, mapped on the long arm of chromosome 1R. Testing of lines originating from hybrid breeding programs, such as S120 and S76, may provide important information about the most significant genes and markers for selection in commercial breeding. Among the statistically significant markers selected in the Kruskal-Wallis test (P < 0.005), there were 55 common ones for preharvest sprouting and heading earliness (1R, 2R, 6R), 30 markers coinciding between α-amylase activity and preharvest sprouting (5R, 7R) and one marker for α-amylase activity and heading earliness (6R). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9627-1) contains supplementary material, which is available to authorized users.

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.

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.

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.

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.

New genetic map of rye composed of PCR-based molecular markers and its alignment with the reference map of the DS2 x RXL10 intercross.

A new genetic map of rye, developed by using the 541 x Ot1-3 F2 intercross, consists of 148 marker loci, including 99 RAPDs, 18 SSRs, 14 STSs, 9 SCARs and 7 ISSRs, and spans the distance of 1401.4 cM. To the 7 rye chromosomes, 8 linkage groups were assigned and compared with the reference map of the DS2 x RXL10 F2 intercross by using 24 common markers. The 2 combined maps contain altogether 611 marker loci (70-109 per chromosome) and constitute a substantial source of information useful for further genomic studies in rye. From 21 to 37 RAPD marker loci are distributed randomly along each chromosome length and their total number for all 7 rye chromosomes is 177. This abundance of RAPD marker loci in the rye genetic map can be exploited for development of SCARs in regions containing important genes or QTL.

Identification of sterility-inducing cytoplasms in rye using the plasmotype-genotype interaction test and newly developed SCAR markers.

A series of 25 rye (Secale cereale L.) inbred lines was tested with respect to three mitochondrial sequence-characterized amplified region (SCAR) polymorphisms. The analysis revealed a close association between the marker-determined mitotypes and plasmotypes (cytoplasm types known from breeding data) represented by the inbreds. The mitochondrial markers also confirmed normal (N-) cytoplasmic character of three wild rye species: Secale montanum, S. vavilovii and S. kuprijanovii. For 186 plants from open-pollinated cultivars of Turkish and South American origin, cytoplasm identification was performed with the use of crossing with double non-restoring tester. In 77 plants the normal (N) cytoplasm was detected, and for 63 of these the PCR analysis was performed producing results which were consistent with the genetic data based on testcrossing and phenotype assessment. The mitochondrial markers also confirmed a presence of sterility-inducing cytoplasm in the remaining 109 plants. Moreover, the markers allowed for differentiation between Pampa (P-) and Vavilovii (V-) cytoplasmic individuals. For 60 plants the latter results were verified using crosses with a line maintaining P-cytoplasmic sterility and acting as a restorer of the V-cytoplasm. For two of these plants contradicting results were produced with the applied methods of cytoplasm identification and the basis of this discrepancy is discussed. Regardless of the identification method, widespread occurrence of a sterility-inducing cytoplasm was revealed, especially in South American populations.

Rye SCAR markers for male fertility restoration in the P cytoplasm are also applicable to marker-assisted selection in the C cytoplasm.

The study aimed at testing the usefulness of recently developed SCAR markers on rye (Secale cereale L.) chromosome 4R in hybrid breeding based on the C source of male sterility-inducing cytoplasm. Of 10 markers studied, 4 revealed polymorphisms between 2 inbred lines (544cms-C and Ot0-20) crossed to develop F2 and BC1 mapping populations. Analyses performed on 94 F2 and 93 BC1 plants allowed to extend a formerly constructed genetic map of chromosome arm 4RL. Three SCAR markers (SCP14M55, SCP15M55 and SCP16M58) were mapped in the vicinity of gene Rfc1, which restores male fertility in the C cytoplasm. The 3 tested SCAR markers proved to be effective in marker-assisted selection (MAS) for male fertility/sterility.