A System for Distinguishing Octoploid Strawberry Cultivars Using High-Throughput SNP Genotyping

Cultivated strawberry (Fragaria × ananassa) is an important commercial berry crop grown throughout the world. Improved strawberry cultivars are developed to meet the needs of consumers and breeders. Strawberries are usually propagated through runners, which sometimes lead to mislabeling or misinterpretation of cultivars. However, perfect identification of strawberry cultivars is essential for germplasm maintenance and for breeding programs. Molecular marker technology has been widely used to distinguish cultivars of other crops, but marker development in octoploid strawberries is complicated. Therefore, SNP marker with high-density and even distribution in the genome has been used currently as efficient DNA markers. In this report, previously published high-quality poly high resolution (PHR) SNPs from the 90 K Axiom® SNP array were utilized to develop a Fluidigm 24 SNPs genotyping system. Hundred nine (109) octoploid strawberry cultivars were screened using this 24 SNPs chip set. In addition, 24 SNPs were mapped to six chromosomes of diploid strawberry (Fragaria vesca). Our developed SNPs fluidigm genotyping is automatable, easy and reliable for processing and interpretation of data. Thus, this high-throughput SNP genotyping system will be a useful tool for distinguishing strawberry cultivars and find out parent-offspring relationship.     

Analysis of gene-derived SNP marker polymorphism in US wheat (Triticum aestivum L.) cultivars

In this study, we developed 359 detection primers for single nucleotide polymorphisms (SNPs) previously discovered within intron sequences of wheat genes and used them to evaluate SNP polymorphism in common wheat (Triticum aestivum L.). These SNPs showed an average polymorphism information content (PIC) of 0.18 among 20 US elite wheat cultivars, representing seven market classes. This value increased to 0.23 when SNPs were pre-selected for polymorphisms among a diverse set of 13 hexaploid wheat accessions (excluding synthetic wheats) used in the wheat SNP discovery project (). PIC values for SNP markers in the D genome were approximately half of those for the A and B genomes. D genome SNPs also showed a larger PIC reduction relative to the other genomes (P < 0.05) when US cultivars were compared with the more diverse set of 13 wheat accessions. Within those accessions, D genome SNPs show a higher proportion of alleles with low minor allele frequencies (<0.125) than found in the other two genomes. These data suggest that the reduction of PIC values in the D genome was caused by differential loss of low frequency alleles during the population size bottleneck that accompanied the development of modern commercial cultivars. Additional SNP discovery efforts targeted to the D genome in elite wheat germplasm will likely be required to offset the lower diversity of this genome. With increasing SNP discovery projects and the development of high-throughput SNP assay technologies, it is anticipated that SNP markers will play an increasingly important role in wheat genetics and breeding applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.     

Effects of ascertainment bias and marker number on estimations of barley diversity from high-throughput SNP genotype data

The capability of molecular markers to provide information of genetic structure is influenced by their number and the way they are chosen. This study evaluates the effects of single nucleotide polymorphism (SNP) number and selection strategy on estimates of germplasm diversity and population structure for different types of barley germplasm, namely cultivar and landrace. One hundred and sixty-nine barley landraces from Syria and Jordan and 171 European barley cultivars were genotyped with 1536 SNPs. Different subsets of 384 and 96 SNPs were selected from the 1536 set, based on their ability to detect diversity in landraces or cultivated barley in addition to corresponding randomly chosen subsets. All SNP sets except the landrace-optimised subsets underestimated the diversity present in the landrace germplasm, and all subsets of SNP gave similar estimates for cultivar germplasm. All marker subsets gave qualitatively similar estimates of the population structure in both germplasm sets, but the 96 SNP sets showed much lower data resolution values than the larger SNP sets. From these data we deduce that pre-selecting markers for their diversity in a germplasm set is very worthwhile in terms of the quality of data obtained. Second, we suggest that a properly chosen 384 SNP subset gives a good combination of power and economy for germplasm characterization, whereas the rather modest gain from using 1536 SNPs does not justify the increased cost and 96 markers give unacceptably low performance. Lastly, we propose a specific 384 SNP subset as a standard genotyping tool for middle-eastern landrace barley.     

Population structure, genetic diversity and linkage disequilibrium in elite winter wheat assessed with SNP and SSR markers

Modern genomics approaches rely on the availability of high-throughput and high-density genotyping platforms. A major breakthrough in wheat genotyping was the development of an SNP array. In this study, we used a diverse panel of 172 elite European winter wheat lines to evaluate the utility of the SNP array for genomic analyses in wheat germplasm derived from breeding programs. We investigated population structure and genetic relatedness and found that the results obtained with SNP and SSR markers differ. This suggests that additional research is required to determine the optimum approach for the investigation of population structure and kinship. Our analysis of linkage disequilibrium (LD) showed that LD decays within approximately 5–10 cM. Moreover, we found that LD is variable along chromosomes. Our results suggest that the number of SNPs needs to be increased further to obtain a higher coverage of the chromosomes. Taken together, SNPs can be a valuable tool for genomics approaches and for a knowledge-based improvement of wheat.     

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44?K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.     

High-Throughput RAD-SNP Genotyping for Characterization of Sugar Beet Genotypes

High-throughput single-nucleotide polymorphism (SNP) genotyping provides a rapid way of developing resourceful sets of markers for delineating genetic structure and for understanding the basis of the taxonomic discrimination. In this paper, we present a panel of 192 SNPs for effective genotyping in sugar beet using a high-throughput marker array technology, QuantStudio 12K Flex system, coupled with Taqman OpenArray technology. The selected SNPs were evaluated for genetic diversity among a set of 150 individuals representing 15 genotypes (10 individuals each) from five cytoplasmic male steriles (CMSs), five pollinators, and five commercial varieties. We demonstrated that the proposed panel of 192 SNPs effectively differentiated the studied genotypes. A higher degree of polymorphism was observed among the CMSs as compared to pollinators and commercial varieties. PCoA and STRUCTURE analysis revealed that CMSs, pollinators, and varieties clustered into three distinct subpopulations. Our results demonstrate the utility of the identified panel of 192 SNPs coupled with TaqMan OpenArray technology as a wide set of markers for high-throughput SNP genotyping in sugar beet.     

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

水稻单核苷酸多态性及其应用现状

单核苷酸多态性（single nucleotide polymorphisms, SNPs）在水稻中数量多,分布密度高,遗传稳定性高。水稻SNPs的发现方法主要有对样本DNA的PCR产物直接测序、从SSR区段检测SNPs和从基因组序列直接搜索等。目前已有多种基因分型技术运用到了水稻SNPs检测,SNPs检测的高度自动化使水稻SNPs基因分型非常方便。单核苷酸多态性在水稻遗传图谱的构建、基因克隆和功能基因组学研究、标记辅助选择育种、遗传资源分类及物种进化等方面的应用具有巨大潜力。     

A whole‐genome SNP array (RICE6K) for genomic breeding in rice

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics‐based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole‐genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion–deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty‐five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.     

LSGermOPA,a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) germplasm fingerprinting

To deploy a high-throughput genotyping platform in germplasm management, we designed and tested a custom OPA (Oligo Pool All), LSGermOPA, for assessing the genetic diversity and population structure of the USDA cultivated lettuce (Lactuca sativa L.) germplasm collection using Illumina’s GoldenGate assay. This OPA contains 384 EST (expressed sequence tag)-derived SNP (single nucleotide polymorphism) markers selected from a large set of SNP markers experimentally validated and mapped by the Compositae Genome Project. Used for genotyping were DNA samples prepared from bulked leaves of five randomly-selected seedlings from each of 380 lettuce accessions. High-quality genotype data were obtained from 354 of the 384 SNPs. The reproducibility of automatic genotype calls was 99.8% as calculated from the four pairs of duplicated DNA samples in the assay. An unexpectedly high percentage of heterozygous genotypes at the polymorphic loci for most accessions indicated a high level of heterogeneity within accessions. Only 148 homogenous accessions, collectively comprising all five horticultural types, were used in subsequent analyses to demonstrate the usefulness of LSGermOPA. The results of phylogenetic relationship, population structure and genetic differentiation analyses were consistent with previous reports using other marker systems. This suggests that LSGermOPA is capable of revealing sufficient levels of polymorphism among lettuce cultivars and is appropriate for rapid assessment of genetic diversity and population structure in the lettuce germplasm collection. Challenges and strategies for effective genotyping and managing lettuce germplasm are discussed.     

Discovery and development of exome‐based,co‐dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.)

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)‐based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP‐based wheat markers have been made available via the use of next‐generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial‐scale breeding programmes. To identify exome‐based co‐dominant SNP‐based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re‐sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co‐dominant. Validation of a subset of these putative co‐dominant markers confirmed that 96% were true polymorphisms and 65% were co‐dominant SNP assays. The new co‐dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web‐based database to facilitate their use on genotyping programmes worldwide.     

High-throughput SNP discovery and genotyping in durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.)

We describe the application of complexity reduction of polymorphic sequences (CRoPS^®) technology for the discovery of SNP markers in tetraploid durum wheat (Triticum durum Desf.). A next-generation sequencing experiment was carried out on reduced representation libraries obtained from four durum cultivars. SNP validation and minor allele frequency (MAF) estimate were carried out on a panel of 12 cultivars, and the feasibility of genotyping these SNPs in segregating populations was tested using the Illumina Golden Gate (GG) technology. A total of 2,659 SNPs were identified on 1,206 consensus sequences. Among the 768 SNPs that were chosen irrespective of their genomic repetitiveness level and assayed on the Illumina BeadExpress genotyping system, 275 (35.8%) SNPs matched the expected genotypes observed in the SNP discovery phase. MAF data indicated that the overall SNP informativeness was high: a total of 196 (71.3%) SNPs had MAF >0.2, of which 76 (27.6%) showed MAF >0.4. Of these SNPs, 157 were mapped in one of two mapping populations (Meridiano × Claudio and Colosseo × Lloyd) and integrated into a common genetic map. Despite the relatively low genotyping efficiency of the GG assay, the validated CRoPS-derived SNPs showed valuable features for genomics and breeding applications such as a uniform distribution across the wheat genome, a prevailing single-locus codominant nature and a high polymorphism. Here, we report a new set of 275 highly robust genome-wide Triticum SNPs that are readily available for breeding purposes.     

Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR.

Single nucleotide polymorphisms (SNPs) are the most abundant form of DNA polymorphism. These polymorphisms can be used in plants as simple genetic markers for many breeding applications, for population studies, and for germplasm fingerprinting. The great increase in the available DNA sequences in the databases has made it possible to identify SNPs by "database mining", and the single most important factor preventing their widespread use appears to be the genotyping cost. Many genotyping platforms rely on the use of sophisticated, automated equipment coupled to costly chemistry and detection systems. A simple and economical method involving a single PCR is reported here for barley SNP genotyping. Using the tetra-primer ARMS-PCR procedure, we have been able to assay unambiguously five SNPs in a set of 132 varieties of cultivated barley. The results show the reliability of this technique and its potential for use in low- to moderate-throughput situations; the association of agronomically important traits is discussed.     

Characterization of genetic diversity and population structure in wheat using array based SNP markers

Genetic diversity is crucial for successful adaptation and sustained improvement in crops. India is bestowed with diverse agro-climatic conditions which makes it rich in wheat germplasm adapted to various niches. Germplasm repository consists of local landraces, trait specific genetic stocks including introgressions from wild relatives, exotic collections, released varieties, and improved germplasm. Characterization of genetic diversity is done using morpho-physiological characters as well as by analyzing variations at DNA level. However, there are not many reports on array based high throughput SNP markers having characteristics of genome wide coverage employed in Indian spring wheat germplasm. Amongst wheat SNP arrays, 35K Axiom Wheat Breeder’s Array has the highest SNP polymorphism efficiency suitable for genetic mapping and genetic diversity characterization. Therefore, genotyping was done using 35K in 483 wheat genotypes resulting in 14,650 quality filtered SNPs, that were distributed across the B (~?50%), A (~?39%), and D (~?10%) genomes. The total genetic distance coverage was 4477.85 cM with 3.27 SNP/cM and 0.49 cM/SNP as average marker density and average inter-marker distance, respectively. The PIC ranged from 0.09 to 0.38 with an average of 0.29 across genomes. Population structure and Principal Coordinate Analysis resulted in two subpopulations (SP1 and SP2). The analysis of molecular variance revealed the genetic variation of 2% among and 98% within subpopulations indicating high gene flow between SP1 and SP2. The subpopulation SP2 showed high level of genetic diversity based on genetic diversity indices viz. Shannon’s information index (I)?=?0.648, expected heterozygosity (He)?=?0.456 and unbiased expected heterozygosity (uHe)?=?0.456. To the best of our knowledge, this study is the first to include the largest set of Indian wheat genotypes studied exclusively for genetic diversity. These findings may serve as a potential source for the identification of uncharacterized QTL/gene using genome wide association studies and marker assisted selection in wheat breeding programs.

     

Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.)

Food security is a global concern and substantial yield increases in cereal crops are required to feed the growing world population. Wheat is one of the three most important crops for human and livestock feed. However, the complexity of the genome coupled with a decline in genetic diversity within modern elite cultivars has hindered the application of marker‐assisted selection (MAS) in breeding programmes. A crucial step in the successful application of MAS in breeding programmes is the development of cheap and easy to use molecular markers, such as single‐nucleotide polymorphisms. To mine selected elite wheat germplasm for intervarietal single‐nucleotide polymorphisms, we have used expressed sequence tags derived from public sequencing programmes and next‐generation sequencing of normalized wheat complementary DNA libraries, in combination with a novel sequence alignment and assembly approach. Here, we describe the development and validation of a panel of 1114 single‐nucleotide polymorphisms in hexaploid bread wheat using competitive allele‐specific polymerase chain reaction genotyping technology. We report the genotyping results of these markers on 23 wheat varieties, selected to represent a broad cross‐section of wheat germplasm including a number of elite UK varieties. Finally, we show that, using relatively simple technology, it is possible to rapidly generate a linkage map containing several hundred single‐nucleotide polymorphism markers in the doubled haploid mapping population of Avalon × Cadenza.     

The Wheat 660K SNP array demonstrates great potential for marker‐assisted selection in polyploid wheat

The rapid development and application of molecular marker assays have facilitated genomic selection and genome‐wide linkage and association studies in wheat breeding. Although PCR‐based markers (e.g. simple sequence repeats and functional markers) and genotyping by sequencing have contributed greatly to gene discovery and marker‐assisted selection, the release of a more accurate and complete bread wheat reference genome has resulted in the design of single‐nucleotide polymorphism (SNP) arrays based on different densities or application targets. Here, we evaluated seven types of wheat SNP arrays in terms of their SNP number, distribution, density, associated genes, heterozygosity and application. The results suggested that the Wheat 660K SNP array contained the highest percentage (99.05%) of genome‐specific SNPs with reliable physical positions. SNP density analysis indicated that the SNPs were almost evenly distributed across the whole genome. In addition, 229 266 SNPs in the Wheat 660K SNP array were located in 66 834 annotated gene or promoter intervals. The annotated genes revealed by the Wheat 660K SNP array almost covered all genes revealed by the Wheat 35K (97.44%), 55K (99.73%), 90K (86.9%) and 820K (85.3%) SNP arrays. Therefore, the Wheat 660K SNP array could act as a substitute for other 6 arrays and shows promise for a wide range of possible applications. In summary, the Wheat 660K SNP array is reliable and cost‐effective and may be the best choice for targeted genotyping and marker‐assisted selection in wheat genetic improvement.     

High-throughput single nucleotide polymorphism (SNP) identification and mapping in the sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) genome with genotyping by sequencing (GBS) analysis

Sesame (Sesamum indicum L. syn. Sesamum orientale L.) is considered to be the first oil seed crop known to man. Despite its versatile use as an oil seed and a leafy vegetable, sesame is a neglected crop and has not been a subject of molecular genetic research until the last decade. There is thus limited knowledge regarding genome-specific molecular markers that are indispensible for germplasm enhancement, gene identification, and marker-assisted breeding in sesame. In this study, we employed a genotyping by sequencing (GBS) approach to a sesame recombinant inbred line (RIL) population for high-throughput single nucleotide polymorphism (SNP) identification and genotyping. A total of 15,521 SNPs were identified with 14,786 SNPs (95.26 %) located along sesame genome assembly pseudomolecules. By incorporating sesame-specific simple sequence repeat (SSR) markers developed in our previous work, 230.73 megabases (99 %) of sequence from the genome assembly were saturated with markers. This large number of markers will be available for sesame geneticists as a resource for candidate polymorphisms located along the physical chromosomes of sesame. Defining SNP loci in genome assembly sequences provides the flexibility to utilize any genotyping strategy to survey any sesame population. SNPs selected through a high stringency filtering protocol (770 SNPs) for improved map accuracy were used in conjunction with SSR markers (50 SSRs) in linkage analysis, resulting in 13 linkage groups that encompass a total genetic distance of 914 cM with 432 markers (420 SNPs, 12 SSRs). The genetic linkage map constitutes the basis for future work that will involve quantitative trait locus (QTL) analyses of metabolic and agronomic traits in the segregating RIL population.