Mapping Of Markers Research Articles

Advances in molecular preclinical therapy mediated by imaging.

Several advances have been made toward understanding the biology of cancer and most of them are due to robust genetic studies that led to the scientific recognition that although many patients have the same type of cancer their tumors may have harbored different molecular alterations. Personalized therapy and the development of advanced techniques of preclinical imaging and new murine models of disease are emerging concepts that are allowing mapping of disease markers in vivo and in some cases also receptor targeted therapy. Aim of this review is to illustrate some emerging models of disease that allow patient tumor implantation in mice for subsequent drug testing and advanced approaches for therapy mediated by preclinical imaging. In particular we discuss targeted therapy mediated by high frequency ultrasound and magnetic resonance, two emerging techniques in molecular preclinical therapy.

A physical map of important QTLs, functional markers and genes available for sesame breeding programs.

Sesame is one of the oldest oilseed crops grown mainly in Africa and Asia. Although genetic and genomic studies on sesame have started late, the past 5years have witnessed extensive progresses in these areas on this crop. Important genomic sequence resources such as functional markers, genes and QTLs linked to agronomically important traits, have been generated through linkage mapping and association analysis to assist sesame improvement programs. However, most of these data are scattered in different maps making them hard to be exploited efficiently in breeding programs. In this study, we report a comprehensive physical map gathering 151 published genomic sequence resources which highlighted some hotspot functional regions in the sesame genome. Moreover, 83,135 non-redundant SSRs have been supplied along with their physical position and motif composition. This will assist future research in fine mapping or pinpointing more functional genes based on the already published QTLs and functional markers. This physical map represents a good landmark for further non-overlapping genetic and genomic studies working towards sesame improvement.

GWAS in practical cattle breeding in Czech Republic, single step method, genetic progress

Development of genetic evaluation of animals is permanent process. It was going from estimated breeding value (EBV) calculated by CC-test, across a BLUP – animal model and RR-TDM, to the genomic enhanced breeding value (GEBV) using genetic markers. Methods of genetic evaluation become a part of marketing strategies of insemination companies. Therefore all countries and association of breeders seek to be compatible with others. Now we are in a period of massive global implementation of genomic evaluation, which combines traditional BLUP with huge quantity of genetic SNP markers. Multi-step procedures are now usual in practice, which work with deregressed proofs. Development of methods attained to the single-step procedure (ssGBLUP) which overcomes some difficulties of previous methods, improves reliabilities of evaluation and compares all animals, genotyped and ungenotyped, in entire nation-wide population. Genomic evaluation influence above all young genotyped animals. In Czech Republic single-step procedure is routinely used for national evaluation of milk, linear type traits, reproduction and longevity. GEBVs are accompanied by genomic reliabilities. Genetic trends over last 20 years are in some traits different for genomic evaluation compared to traditional BLUP evaluation, although input data and genetic parameters (heritability) are the same and genotyped animals were only small proportion from entire evaluated population. Differences in genetic trends increase mainly in new batches of animals. Reason of it could be in the changed variability of breeding values and “genomic correction” of relationship between animals, which is expanded from genotyped animals to others individuals in a population. Keywords: genomic breeding value, single-step, genomic relationship, genetic trend, SNP References Bauer, J. et al. (2014) Approximation of the reliability of single-step genomic breeding values for dairy cattle in the Czech Republic. Anim. Sci. Papers and Reports, 32, pp. 301-306. Bauer, J., Přibyl, J. and Vostrý, L. (2015) Contribution of domestic production records and Interbull EBV on approximate reliabilities of single-step genomic breeding values in dairy cattle. Czech J. Anim. Sci., 60, 263-267. Candrak, J., Kadlecik O. and Schaeffer L.R. (1997) The use of test-day model for Slovak cattle populations. In: Proc. 48th Annual Meeting of the European Association for Animal Production, Vienna, Austria, August 25–28. Christensen, O.F. and Lund, M.S. (2010) Genomic prediction when some animals are not genotyped. Genet.Sel.Evol. 42, pp. 2. Fisher, R.A. (1918) The correlation between relatives in the supposition of Mendelian inheritance. Trans. Roy. Soc. Edinb. 52, pp. 399-433. Fragomeni, B.O. et al. (2015) Hot topic: Use of genomic recursions in single-step genomic best linear unbiased predictor (BLUP) with a large number of genotypes. J. Dairy Sci., 98, pp. 4090-4094. Gao, H. et al. (2012) Comparison on genomic predictions using three GBLUP methods and two single step blending methods in the Nordic Holstein population. Genet. Sel.Evol. 44, pp. 8. Legarra A., Aguilar I. and Misztal, I. (2009) A relationship matrix including full pedigree and genomic information. J. Dairy Sci., 92, pp. 4656-4663. Masuda, Y. et al. (2016) Implementation of genomic recursions in single-step genomic best linear unbiased predictor for US Holsteins with a large number of genotyped animals. J. Dairy Sci., 99, pp. 1968-1974. Mendel, G.J. (1866) Versuche uber Pflanzen-Hybriden. Verh. Naturforsch. Ver. Brunn 4, pp. 3–47 (1901, J. R. Hortic. Soc. 26, pp. 1–32). Meuwissen, T.H.E., Hayes, B.J. and Goddard, M.E. (2001) Prediction of total genetic value using genome-wide dense marker maps. Genetics, 157, pp. 1819–1829. Misztal, I., Legarra A. and Aguilar, I. (2009) Computing procedures for genetic evaluation including phenotypic, full pedigree, and genomic information. J. Dairy Sci., 92, pp. 4648–4655. Misztal, I. et al. (2013) Methods to approximate reliabilities in single-step genomic evaluation. J. Dairy Sci., 96, pp. 647-654. Pesek, P., Přibyl, J. and Vostrý, L . (2015) Genetic variances of SNP loci for milk yield in dairy cattle. J. Appl. Genet., 56, pp. 339-347. Přibyl, J. et al. (2014) Domestic and Interbull information in the single step genomic evaluation of Holstein milk production. Czech J. Anim. Sci., 59, pp. 409-415. Přibyl, J. et al. (2015) Domestic estimated breeding values and genomic enhanced breeding values of bulls in comparison with their foreign genomic enhanced breeding values. Animal, 9, pp. 1635-1642. VanRaden, P.M. (2008) Efficient methods to compute genomic predictions. J. Dairy Sci., 91, pp. 4414–4423. VanRaden, P.M. et al. (2011) Genomic evaluations with many more genotypes. Genet. Sel.Evol. 43, pp. 10. Wright, S. (1921) Systems of mating. Genetics. 6, pp. 111-178. Zavadilova, L. et al. (2014) Single-step genomic evaluation for linear type traits of Holstein cows in Czech Republic. Anim. Sci. Papers and Reports vol. 32, pp. 201-208.

Comparison of genomic breeding values of Holstein in Czech Republic

Genomic evaluation by single-step procedure allows efficient implanting of genetic SNP markers into complicated procedure of random regression test-day-model of milk traits. Test-day records and pedigree files on nation-wide scale are combined with genomic relationship into common evaluation of both genotyped and ungenotyped animals. Due to strong import of foreign sperm into small national populations is the reliability of evaluation of young animals low. This is particularly seen in evaluation of young bulls, which frequently have both parents foreign. Genomic evaluation helps and notably improves reliability of evaluation. ssGBLUP procedure is advantageous especially for small populations. Domestic genomic evaluation of young animals has medium to high correlation with foreign Interbull values. Interbull conversion of values of bulls according MACE, which works with progeny tested bulls, is more reliable than conversion according GMACE procedure, which works with genomic evaluation of young animals. Keywords: genomic breeding value, ssGBLUP, test day model, MACE, GMACE References Bauer, J. et al. (2014) Approximation of the reliability of single-step genomic breeding values for dairy cattle in the Czech Republic. Anim. Sci. Papers and Reports, 32, pp. 301-306. Bauer, J., Přibyl, J. and Vostrý, L. (2015) Contribution of domestic production records and Interbull EBV on approximate reliabilities of single-step genomic breeding values in dairy cattle. Czech J. Anim. Sci., 60, 263-267. Christensen, O.F. and Lund, M.S. (2010) Genomic prediction when some animals are not genotyped. Genet.Sel.Evol. 42, pp. 2. Forni, S., Aguilar I. and Misztal, I. (2011) Different genomic relationship matrices for single step analysis using phenotypic, pedigree and genomic information. Genet. Sel. Evol., 43, pp. 1. Legarra A., Aguilar I. and Misztal, I. (2009) A relationship matrix including full pedigree and genomic information. J. Dairy Sci., 92, pp. 4656-4663. Meuwissen, T.H.E., Hayes, B.J. and Goddard, M.E. (2001) Prediction of total genetic value using genome-wide dense marker maps. Genetics, 157, pp. 1819–1829. Misztal, I., Legarra A. and Aguilar, I. (2009) Computing procedures for genetic evaluation including phenotypic, full pedigree, and genomic information. J. Dairy Sci., 92, pp. 4648–4655. Pesek, P., Přibyl, J. and Vostrý, L . (2015) Genetic variances of SNP loci for milk yield in dairy cattle. J. Appl. Genet., 56, pp. 339-347. Plemdat, (2015) Descriptions of Breeding values Evaluation. Retrieved on 10 th June 2015 From www.plemdat.cz. Přibyl, J. et al. (2014) Domestic and Interbull information in the single step genomic evaluation of Holstein milk production. Czech J. Anim. Sci., 59, pp. 409-415. Přibyl, J. et al. (2015) Domestic estimated breeding values and genomic enhanced breeding values of bulls in comparison with their foreign genomic enhanced breeding values. Animal, 9, pp. 1635-1642. Vitezica, Z.G. et al. (2011) Bias in genomic predictions for populations under selection. Genet. Res. (Camb), 93, pp. 357-366. Zavadilova, L., Jamrozik, J. and Schaeffer, L.R. (2005a) Genetic parameters for test- day model with random regressions for production traits of Czech Holstein cattle. Czech J. Anim. Sci., 50, pp. 142-154. Zavadilova, L., Němcova, E. and Wolf, J. (2005b) Definition of subgroups for fixed regression in the test-day animal model for milk production of Holstein cattle in the Czech Republic. Czech J. Anim. Sci., 50, pp. 7-13. Zavadilova, L. et al. (2014) Single-step genomic evaluation for linear type traits of Holstein cows in Czech Republic. Anim. Sci. Papers and Reports vol. 32, pp. 201-208.

Genetic Mapping of SSR Markers in Eight Soybean Chromosomes Based on F2 Population B3462 x B3293

<p>Genetic Mapping of SSR Markers in Eight Soybean<br />Chromosomes Based on F2 Population B3462 x B3293. I<br />Made Tasma, Ahmad Warsun, Dani Satyawan, Saptowo<br />J. Pardal, and Slamet. Aluminum toxicity is one of the main<br />contrains for cultivating soybean in acid soils. Genetic<br />Hak Cipta © 2011, BB-Biogen<br />mapping of SSR markers is one step for detecting aluminumtoxicity<br />tolerant QTLs in soybean. Another step is to<br />phenotype the same population at various aluminum-toxicity<br />environments. The objectives of this study were to analyze<br />the segregation of SSR markers in progenies of an F2<br />population and map the markers in 8 soybean chromosomes.<br />The F2 population was previously developed by<br />crossing the Al-tolerant parent B3462 and the Al-sensitive<br />parent B3293. Polymorphic SSR markers in the parents were<br />used to PCR amplify DNA of the 100 F2 progenies. PCR<br />products were separated using agarose or polyacrylamide<br />gels. A Chi-Square test was done with a null hypothesis that<br />progenies segregated in a 1 : 2 : 1 ratio. Results showed that<br />125 SSR markers were polymorphics in the parents. Out of<br />125 polymorphic markers, 122 were segregated in the<br />progenies of the F2 population. Among the segregating<br />markers, 114 were segregated in a 1 : 2 : 1 ratio. Only 8<br />markers (5.6%) did not follow the 1 : 2 : 1 ratio. One hundred<br />and nineteen SSR markers were mapped in 8 soybean<br />chromosomes. These include 18 markers in chromosome<br />A2, 10 in B1, 16 (C1), 16 (F), 10 (G), 23 (J), 16 (L), and 10 (N).<br />Total genetic maps covered was 1,194.8 cM with average<br />map distances between two adjacent markers of 10.7 cM.<br />Further SSR marker enrichment is required to fill in the gaps<br />of several chromosomal regions. Genetic maps presented in<br />this study should be useful for detection of Al-toxicity<br />tolerant QTLs in soybean.</p>

Urinary Peptide Analysis Differentiates Pancreatic Cancer From Chronic Pancreatitis.

Differentiation of pancreatic cancer (PCA) from chronic pancreatitis (CP) is challenging. We searched for peptide markers in urine to develop a diagnostic peptide marker model. Capillary electrophoresis-mass spectrometry was used to search for peptides in urine of patients with PCA (n = 39) or CP (n = 41). Statistical different peptides were included in a peptide multimarker model. Peptide markers were sequence identified and validated by immunoassay and immunohistochemistry (IHC). Applied to a validation cohort of 54 patients with PCA and 52 patients with CP, the peptide model correctly classified 47 patients with PCA and 44 patients with CP (area under the curve, 0.93; 87% sensitivity; 85% specificity). All 5 patients with PCA with concomitant CP were classified positive. Urine proteome analysis outperformed carbohydrate antigen 19-9 (area under the curve, 0.84) by a 15% increase in sensitivity at the same specificity. From 99 healthy subjects, only four were misclassified. Fetuin-A was the most prominent peptide marker source for PCA as verified by immunoassay and IHC. In silico protease mapping of the peptide markers' terminal sequences pointed to increased meprin-A activity in PCA, which in IHC was associated with neoangiogenesis. Urinary proteome analysis differentiates PCA from CP and may serve as PCA screening tool.

Genetic epidemiology of pharmacogenetic variants in South East Asian Malays using whole-genome sequences.

Expanding the scope of pharmacogenomic research by including multiple global populations is integral to building robust evidence for its clinical translation. Deep whole-genome sequencing of diverse ethnic populations provides a unique opportunity to study rare and common pharmacogenomic markers that often vary in frequency across populations. In this study, we aim to build a diverse map of pharmacogenetic variants in South East Asian (SEA) Malay population using deep whole-genome sequences of 100 healthy SEA Malay individuals. We investigated the allelic diversity of potentially deleterious pharmacogenomic variants in SEA Malay population. Our analysis revealed 227 common and 466 rare potentially functional single nucleotide variants (SNVs) in 437 pharmacogenomic genes involved in drug metabolism, transport and target genes, including 74 novel variants. This study has created one of the most comprehensive maps of pharmacogenetic markers in any population from whole genomes and will hugely benefit pharmacogenomic investigations and drug dosage recommendations in SEA Malays.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-016-0347-4) contains supplementary material, which is available to authorized users.

Advances in Quantitative Trait Loci, Mapping and Importance of Markers Assisted Selection in Plant Breeding Research

Advances in Quantitative Trait Loci, Mapping and Importance of Markers Assisted Selection in Plant Breeding Research

Mapping QTLs for morpho-agronomic traits in proso millet (Panicum miliaceum L.)

Proso millet (Panicum miliaceum L.) is the cereal crop with the low water requirement and increasingly being used for human consumption. It is the most common rotational crop within wheat-based dryland production systems in the semiarid High Plains of the USA. However, there is no published genetic map for this species, which prevents the identification of quantitative trait loci (QTL). The objectives of the present study were (1) construction of a genetic linkage map and (2) identification of DNA markers linked to QTLs for morpho-agronomic traits. A total of 93 recombinant inbred lines derived from a single F1 (“Huntsman” × “Minsum”) were genotyped with GBS-SNP markers and phenotyped for nine morpho-agronomic traits in the field during 2013 and 2014 at Scottsbluff and Sidney, NE. IciMapping v.4.0.6.0 was used for constructing a genetic linkage map and mapping QTL. The RILs exhibited significant variation for a wide range of traits, and several traits showed evidence of genotype × environment interactions. A total of 833 GBS-SNP markers formed 18 major and 84 minor linkage groups, whereas 519 markers remained ungrouped. A total of 117 GBS-SNP markers were distributed on the 18 major linkage groups spanning a genome length of 2137 cM of proso millet with an average distance of 18 cM between markers. The length and number of markers in each of the 18 major linkage groups ranged from 54.6 to 236 cM and 4 to 12, respectively. A total of 18 QTLs for eight morpho-agronomic traits were detected on 14 linkage groups, each of which explained 13.2–34.7 % phenotypic variance. DNA markers flanking the QTLs were identified, which will aid in marker-assisted selection of these traits. To our knowledge, this is the first genetic linkage map and QTL mapping in proso millet, which will support further genetic analysis and genomics-assisted genetic improvement of this crop.

New insights into the tectonic inversion of North Canterbury and the regional structural context of the 2010–2011 Canterbury earthquake sequence, New Zealand

AbstractThe 2010–2011 Canterbury earthquake sequence highlighted the existence of previously unknown active faults beneath the North Canterbury plains and Pegasus Bay, South Island, New Zealand. We provide new insights into the geometry and kinematics of ongoing deformation by analyzing marine seismic data to produce new maps of regional faults and cross‐sectional reconstructions of deformation history. Active faulting and folding extends up to 30 km offshore, and involves reactivation of sets of Late Cretaceous‐Paleogene normal faults under NW‐SE tectonic compression. The active faults consist predominantly of NE‐SW striking, SE‐dipping reverse faults, and less commonly E‐W to NW‐SE faults suitably oriented for strike‐slip reactivation. Additionally, newly developing reverse faults obliquely segment and overprint the inherited basement fabric and impose geometric and kinematic complexities revealed by mapping and reverse displacement profiles of markers. The Quaternary reverse slip rates decrease from 0.1–0.3 mm/yr beneath northern Pegasus Bay to <0.05 mm/yr approaching Banks Peninsula. Fault growth modeling involving trishear fault‐propagation folding mechanisms successfully restores an evolutionary sequence of progressive fault inversion, revealing a history of reactivated individual faults. Tectonic inversion and overprinting processes beneath Pegasus Bay are immature and <1.2 ± 0.4 Ma old, with no evidence of systematic spatial migration of deformation. Our marine data analyses give insights into the structural context of the 2010–2011 Canterbury earthquake sequence, while the combined onshore to offshore data provide an excellent illustration of fault growth associated with immature inversion tectonics, in which selective fault reactivation results from compressive stress imposed across a complex network of inherited faults.

A highly recombined, high-density, eight-founder wheat MAGIC map reveals extensive segregation distortion and genomic locations of introgression segments.

SummaryMultiparent Advanced Generation Intercross (MAGIC) mapping populations offer unique opportunities and challenges for marker and QTL mapping in crop species. We have constructed the first eight‐parent MAGIC genetic map for wheat, comprising 18 601 SNP markers. We validated the accuracy of our map against the wheat genome sequence and found an improvement in accuracy compared to published genetic maps. Our map shows a notable increase in precision resulting from the three generations of intercrossing required to create the population. This is most pronounced in the pericentromeric regions of the chromosomes. Sixteen percent of mapped markers exhibited segregation distortion (SD) with many occurring in long (>20 cM) blocks. Some of the longest and most distorted blocks were collinear with noncentromeric high‐marker‐density regions of the genome, suggesting they were candidates for introgression fragments introduced into the bread wheat gene pool from other grass species. We investigated two of these linkage blocks in detail and found strong evidence that one on chromosome 4AL, showing SD against the founder Robigus, is an interspecific introgression fragment. The completed map is available from http://www.niab.com/pages/id/326/Resources.

Gametocidal System for Dissecting Wheat Chromosomes.

Gametocidal (Gc) system is a novel method of inducing chromosome mutations in wheat by using Gc genes of wheat-related species. Many deletion lines were efficiently produced by this system and used for physical mapping of genes and DNA markers. A large stocks with chromosome deletions in homozygous state as well as the lines with Gc genes are available in the gene bank of Japan (KOMUGI). In this chapter, I describe the method of inducing breakage in a target chromosome using lines with the Gc gene on chromosome 2C of Aegilops cylindrica (a wheat-related wild species) and nullisomic-tetrasomics.

<b>Mapping and validation of molecular markers of genes <i>Dt1</i> and <i>Dt2</i> to determine the type of stem growth in soybean

The type of stem growth in soybean is a distinguishing feature of cultivars. The genes Dt1 and Dt2 affect the termination of the stem, and the types of growth are classified as determinate, semi- determinate and indeterminate. Phenotypic characterization of the type of growth is complex and is occasionally erroneously described. The objective of this study was to map and validate molecular markers to classify the type of soybean growth to facilitate the description of cultivars and genotypic selection. Two populations were used for mapping and validation of molecular markers F2:3: T 117 (semi-determinate growth type) x Igra RA 518 RR (indeterminate growth type) and CD 235RR (determinate growth type) x Igra RA 518 RR. The study demonstrates that the association of the molecular marker with the gene GmTFL1b was efficient in the classification of soybean growth types. The marker sat_064 is connected to the Dt2 gene, which is located in the Liaison Group G of the consensus map of soybeans with a recombination frequency of 19.4%. Molecular markers for Dt1 and Dt2 genes efficiently described the genotypes of soybean stem growth and selection.

Selection of promising apple genotypes for columnar growth habit and scab resistance using diagnostic DNA markers

Monogenic scab resistance and columnar growth habit are important breeding traits of apple. The use of molecular markers very accurately determines the presence of necessary genes in the genome early during ontogenesis and reduces the time of selection. The purpose of this study was molecular genetic testing of initial forms of apple and hybrid seedlings of apple to identify carriers of the target alleles of genes for monogenic scab resistance (Rvi6) and columnar growth habit (Co) and the clarification of the pattern of inheritance of the Co and Rvi6 genes in hybrid progeny populations. This paper presents the results of molecular genetic analysis of varieties Valuta, Uspenskoe, Belarusskoe sladkoe and seedlings of the Valuta×Uspenskoe and Valuta×Belarusskoe sladkoe hybrid families for genes controlling columnar growth habit (Co) and scab resistance (Rvi6). The presence of the dominant allele of the Co gene was diagnosed with primers 29f1 and JWI1r flanking the 5’-end of the insert at the Co locus controlling columnar genotypes. Allelic status of the Rvi6 gene was determined with the AL07-SCAR marker mapping at about 0.2 cM from the Rvi6 locus. The correlation frequency of inheritance of the allelic states of these genes has been determined. Valuta×Uspenskoe crosse yielded 48.1 % columnar genotypes and 77.8 % scab-immune genotypes; Valuta×Belarusskoe sladkoe crosses, 46.8 % and 68.0 %, respectively. The observed segregation conforms to the expected Mendelian ratios: 1:1 for columnar habit and 3:1 for scab resistance. The joint inheritance of columnar growth habit and monogenic resistance to scab has been analyzed. The hybrid seedlings that had the dominant Co allele together with the Rvi6 gene in the homozygous dominant state (Rvi6 Rvi6) in their genome have been identified, which can significantly intensify the selection process and run it into 100 % of hybrid seedlings with monogenic scab resistance and up to 50 % of genotypes with columnar growth habit.

High-resolution tyramide-FISH mapping of markers tightly linked to the male-fertility restoration (Ms) locus of onion

Tyramide FISH was used to locate relatively small genomic amplicons from molecular markers linked to Ms locus onto onion chromosome 2 near the centromere, a region of relatively low recombination. Fluorescence in situ hybridization (FISH) has not been readily exploited for physical mapping of molecular markers in plants due to the technical challenge of visualizing small single-copy probes. Signal amplification using tyramide (tyr) FISH can increase sensitivity up to 100-fold. We used tyr-FISH to physically locate molecular markers tightly linked to the nuclear male-fertility (Ms) restoration locus of onion onto mitotic metaphase, pachytene, and super-stretched pachytene chromosomes. Relatively short genomic amplicons (846-2251 bp) and a cDNA clone (666 bp) were visualized in 9-42 % of observed cells. The markers were assigned to proximal locations close to the centromere on the long arm of chromosome 2, a region of lower recombination, revealing that tightly linked markers may be physically distant from Ms. This result explains why several labs have identified molecular markers tightly linked to the Ms locus after screening relatively few DNA clones or primers and segregating progenies. Although these markers are still useful for marker-aided selection, our results indicate that map-based cloning of Ms will likely be difficult due to reduced recombination near this gene.

A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass.

The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic self-incompatibility (SI) mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. Here, we report a fine-mapping approach to identify the male component of the S-locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins. Our results represent a major step forward toward understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S-locus.

Application of Population Sequencing (POPSEQ) for Ordering and Imputing Genotyping-by-Sequencing Markers in Hexaploid Wheat

The advancement of next-generation sequencing technologies in conjunction with new bioinformatics tools enabled fine-tuning of sequence-based, high-resolution mapping strategies for complex genomes. Although genotyping-by-sequencing (GBS) provides a large number of markers, its application for association mapping and genomics-assisted breeding is limited by a large proportion of missing data per marker. For species with a reference genomic sequence, markers can be ordered on the physical map. However, in the absence of reference marker order, the use and imputation of GBS markers is challenging. Here, we demonstrate how the population sequencing (POPSEQ) approach can be used to provide marker context for GBS in wheat. The utility of a POPSEQ-based genetic map as a reference map to create genetically ordered markers on a chromosome for hexaploid wheat was validated by constructing an independent de novo linkage map of GBS markers from a Synthetic W7984 × Opata M85 recombinant inbred line (SynOpRIL) population. The results indicated that there is strong agreement between the independent de novo linkage map and the POPSEQ mapping approach in mapping and ordering GBS markers for hexaploid wheat. After ordering, a large number of GBS markers were imputed, thus providing a high-quality reference map that can be used for QTL mapping for different traits. The POPSEQ-based reference map and whole-genome sequence assemblies are valuable resources that can be used to order GBS markers and enable the application of highly accurate imputation methods to leverage the application GBS markers in wheat.

Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Transcriptome-enabled marker discovery and mapping of plastochron-related genes in Petunia spp.

BackgroundPetunia (Petunia × hybrida), derived from a hybrid between P. axillaris and P. integrifolia, is one of the most economically important bedding plant crops and Petunia spp. serve as model systems for investigating the mechanisms underlying diverse mating systems and pollination syndromes. In addition, we have previously described genetic variation and quantitative trait loci (QTL) related to petunia development rate and morphology, which represent important breeding targets for the floriculture industry to improve crop production and performance. Despite the importance of petunia as a crop, the floriculture industry has been slow to adopt marker assisted selection to facilitate breeding strategies and there remains a limited availability of sequences and molecular markers from the genus compared to other economically important members of the Solanaceae family such as tomato, potato and pepper.ResultsHere we report the de novo assembly, annotation and characterization of transcriptomes from P. axillaris, P. exserta and P. integrifolia. Each transcriptome assembly was derived from five tissue libraries (callus, 3-week old seedlings, shoot apices, flowers of mixed developmental stages, and trichomes). A total of 74,573, 54,913, and 104,739 assembled transcripts were recovered from P. axillaris, P. exserta and P. integrifolia, respectively and following removal of multiple isoforms, 32,994 P. axillaris, 30,225 P. exserta, and 33,540 P. integrifolia high quality representative transcripts were extracted for annotation and expression analysis. The transcriptome data was mined for single nucleotide polymorphisms (SNP) and simple sequence repeat (SSR) markers, yielding 89,007 high quality SNPs and 2949 SSRs, respectively. 15,701 SNPs were computationally converted into user-friendly cleaved amplified polymorphic sequence (CAPS) markers and a subset of SNP and CAPS markers were experimentally verified. CAPS markers developed from plastochron-related homologous transcripts from P. axillaris were mapped in an interspecific Petunia population and evaluated for co-localization with QTL for development rate.ConclusionsThe high quality of the three Petunia spp. transcriptomes coupled with the utility of the SNP data will serve as a resource for further exploration of genetic diversity within the genus and will facilitate efforts to develop genetic and physical maps to aid the identification of QTL associated with traits of interest.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1931-4) contains supplementary material, which is available to authorized users.