Developing KASP Markers for Identification of Basmati Rice Varieties

Basmati is considered a unique varietal group of rice (Oryza sativa L.) because of its aroma and superior grain quality. Previous genetic analyses of rice showed that most of the Basmati varieties are classified into the aromatic group. Despite various efforts, genomic relationship of Basmati rice with other varietal groups and genomic variation in Basmati rice are yet to be understood. In the present study, we resequenced the whole genome of three traditional Basmati varieties at a coverage of more than 25X using Illumina HiSeq2500 and mapped the obtained sequences to the reference genome sequences of Nipponbare (japonica rice), Kasalath (aus rice), and Zhenshan 97 (indica rice). Comparison of these sequences revealed common single nucleotide polymorphisms (SNPs) in the genic regions of three Basmati varieties. Analysis of these SNPs revealed that Basmati varieties showed fewer sequence variations compared with the aus group than with the japonica and indica groups. Gene ontology (GO) enrichment analysis indicated that SNPs were present in genes with various biological, molecular, and cellular functions. Additionally, functional annotation of the Basmati mutated gene cluster shared by Nipponbare, Kasalath, and Zhenshan 97 was found to be associated with the metabolic process involved in the cellular aromatic compound, suggesting that aroma is an important specific genomic feature of Basmati varieties. Furthermore, 30 traditional Basmati varieties were classified into three different groups, aromatic (22 varieties), aus (four varieties), and indica (four varieties), based on genome-wide SNPs. All 22 aromatic Basmati varieties harbored the fragrant-inducing Badh2 allele. We also performed comparative analysis of 13 key agronomic and grain quality traits of Basmati rice and other rice varieties. Three traits including length-to-width ratio of grain (L/W ratio), panicle length (PL), and amylose content (AC) showed significant (P < 0.05 and P < 0.01) differences between the aromatic and indica/aus groups. Comparative analysis of genome structure, based on genome sequence variation and GO analysis, revealed that the Basmati genome was derived mostly from the aus and japonica groups. Overall, whole-genome sequence data and genetic diversity information obtained in this study will serve as an important resource for molecular breeding and genetic analysis of Basmati varieties.

Single nucleotide polymorphism (SNP) data can be obtained using one of the numerous uniplex or multiplex SNP genotyping platforms that combine a variety of chemistries, detection methods, and reaction formats. Kompetitive Allele Specific PCR (KASP) is one of the uniplex SNP genotyping platforms, and has evolved to be a global benchmark technology. However, there are no publications relating either to the technology itself or to its application in crop improvement programs. In this review, we provide an overview of the different aspects of the KASP genotyping platform, discuss its application in crop improvement, and compare it with the chip-based Illumina GoldenGate platform. The International Maize and Wheat Improvement Center routinely uses KASP, generating in excess of a million data points annually for crop improvement purposes. We found that (1) 81 % of the SNPs used in a custom-designed GoldenGate assay were transferable to KASP; (2) using KASP, negative controls (no template) consistently clustered together and rarely produced signals exceeding the threshold values for allele calling, in contrast to the situation observed using GoldenGate assays; (3) KASP’s average genotyping error in positive control DNA samples was 0.7–1.6 %, which is lower than that observed using GoldenGate (2.0–2.4 %); (4) KASP genotyping costs for marker-assisted recurrent selection were 7.9–46.1 % cheaper than those of the BeadXpress and GoldenGate platforms; and (5) KASP offers cost-effective and scalable flexibility in applications that require small to moderate numbers of markers, such as quality control analysis, quantitative trait loci (QTL) mapping in bi-parental populations, marker-assisted recurrent selection, marker-assisted backcrossing, and QTL fine mapping.

Greenbug [Schizaphis graminum (Rondani)] is a serious insect pest that not only damages cereal crops, but also transmits several destructive viruses. The emergence of new greenbug biotypes in the field makes it urgent to identify novel greenbug resistance genes in wheat. CWI 76364 (PI 703397), a synthetic hexaploid wheat (SHW) line, exhibits greenbug resistance. Evaluation of an F2:3 population from cross OK 14319 × CWI 76364 indicated that a dominant gene, designated Gb9, conditions greenbug resistance in CWI 76364. Selective genotyping of a subset of F2 plants with contrasting phenotypes by genotyping-by-sequencing identified 25 SNPs closely linked to Gb9 on chromosome arm 7DL. Ten of these SNPs were converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for genotyping the entire F2 population. Genetic analysis delimited Gb9 to a 0.6-Mb interval flanked by KASP markers located at 599,835,668bp (Stars-KASP872) and 600,471,081bp (Stars-KASP881) on 7DL. Gb9 was 0.5cM distal to Stars-KASP872 and 0.5cM proximal to Stars-KASP881. Allelism tests indicated that Gb9 is a new greenbug resistance gene which confers resistance to greenbug biotypes C, E, H, I, and TX1. TX1 is one of the most widely virulent biotypes and has overcome most known wheat greenbug resistance genes. The introgression of Gb9 into locally adapted wheat cultivars is of economic importance, and the KASP markers developed in this study can be used to tag Gb9 in cultivar development.

Oryza glaberrima accessions harbor genes for tolerance to abiotic stresses such as mineral deficiency in problem soils. This genetic potential could be exploited in interspecific crosses with Oryza sativa, as in the case of the ‘New Rice for Africa’ (NERICA) varieties; however, to attain this goal it would be desirable to develop a high-throughput marker system to specifically detect O. glaberrima introgressions in an O. sativa background. Therefore, a single nucleotide polymorphism (SNP) genotyping analysis of an O. glaberrima accession (CG14) with two O. sativa lines (WAB56-104 and WAB181-18) was performed on a genome-wide basis. Comparison of CG14 and the WAB lines resulted in a set of 9,523 polymorphic SNPs which would be suitable to detect O. glaberrima introgressions in upland NERICAs. In addition, a set of 1,540 polymorphic SNPs between O. glaberrima versus O. sativa was identified. A subset of SNPs which were evenly distributed in the genome was then used to design a flexible and cost-effective SNP genotyping panel using the Competitive Allele-Specific PCR technology (KASP). This SNP genotyping panel consists of 2,015 SNPs successfully converted into KASP markers, providing 745 polymorphic SNPs for the parents O. glaberrima CG14/O. sativa WAB56-104 (upland NERICA), and 752 for O. glaberrima TOG5681/O. sativa IR64 (lowland NERICA). KASP markers were successfully validated by mapping O. glaberrima introgressions in NERICA-derived breeding lines. This new SNP genotyping panel will be useful in modern breeding applications such as QTL mapping and/or marker-assisted selection.

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

There is an increasing demand for horse milk, but the current domestic performance of the horse milk has not yet conducted a systematic in-depth study. We carried out a pilot study on 13 single nucleotide polymorphisms (SNPs) located within five genes in Kazakh horse using Kompetitive Allele Specific PCR (KASPTM). An association study with average daily milk production of 60 Kazakh mares was performed for 6/13 polymorphic SNPs. The results showed that only two SNPs rs114262445 (NUMB), rs1136347938 (CA8) out of the 6 polymorphic markers were significantly associated with milk production in Kazakh horses. These results indicate the potential of using SNPs for the NUMB and CA8 genes as candidate genes for selection and farm production outputs. This study is relevant for future horse genomic studies.

ABSTRACTThe waxy protein primarily controls the synthesis of amylose, which is a key determinant of rice (Oryza sativa L.) cooking and processing qualities. Knowledge of the diversity of waxy gene in Chinese rice is essential to validate molecular markers for marker‐assisted selection and to trace the origin of Chinese glutinous rice. The waxy (Wx) gene in the wild rice has rarely been studied, and the origin of Chinese glutinous rice is not well understood. The objectives of our investigation were (i) to identify the diversity of Wx and molecular markers for marker‐assisted breeding and (ii) trace the origin of Chinese glutinous rice. We examined the sequence variations for the waxy gene of 98 accessions of cultivated rice and 134 accessions of wild rice from a previously established microcore collection of Chinese rice germplasm. A total of 51 and 226 single nucleotide polymorphisms (SNPs) or insertions or deletions were found in the cultivated rice and the wild rice, respectively. Wild rice accessions had much higher diversity than cultivated rice and nonglutinous rice had much higher diversity than glutinous rice whereas the genetic diversity of indica rice was similar to that of japonica rice. Polymorphisms of CTn microsatellite, G/T SNP, and 23‐bp insertion in the waxy gene and their relationship to amylose content were explored using cultivated rice. The G/T allele and 23‐bp insertion were better associated with amylose content than with the CTn alleles, and they were validated as molecular markers for marker‐assisted selection. All the wild rice accessions with 23‐bp insertion or T allele came from South China and the glutinous rice originated from wild rice of South China parallel with indica and japonica differentiation.

Using a novel and fast genotyping method called Kompetitive Allele Specific PCR (KASP™), we carried out a pilot study on 48 single nucleotide polymorphisms (SNPs) belonging to 40 genes in French Alpine (n = 24) and Saanen (n = 25) goats reared in Romania. Furthermore, the associations of the 13 polymorphic genetic variants with milk production and composition were investigated. Thirty-five SNPs did not show polymorphism in the studied populations. Polymorphic SNPs were detected in the following genes: CAST, CLEC4E, DES, GHRHR, HSP90AA1, IL15RA, IL1RN, IL8, MITF, PPRC1, SOCS3, TNF and TNFSF13. The studied Alpine population was in Hardy-Weinberg disequilibrium at the g.62894878A>G locus (rs671391101) (P < 0.05). The results showed that four SNPs rs671391101 (GHRHR), rs640582069 (IL1RN) rs635583012 (SOCS3) and rs635969404 (IL15RA) out of the 13 polymorphic markers were significantly associated with milk production, protein, fat and lactose content in the Alpine breed. However, no significant effect was recorded in the Saanen population regarding milk yield or milk chemical composition. The current results provide new insights for the development of SNP marker-assisted selection technology in the goat industry and confirm the potential of using SNPs for the GHRHR, IL1RN, SOCS3, and IL15RA genes as candidate genes for selection, highlighting the direct implications of such genes for farm production outputs. The results from this study are relevant for future goat genomic studies and the inclusion of the associated traits into up-to-date selection schemes.

Cloning of the candidate gene <italic>BnCRY2</italic>, associated with the early-flowering locus <italic>BnaC08cqDTF</italic>, identified an A/G single-nucleotide polymorphism (SNP) within the third exon of the corresponding alleles. Based on this SNP, a KASP marker named BN900449 was developed. This marker, along with four additional markers—multiplex PCR markers PB05 and PB06, and KASP markers A015348 and K000272—tightly linked to the early-flowering loci <italic>qFT.A02-1</italic> and <italic>qFTA10</italic>, was used to genotype a natural population of 497 <italic>Brassica napus</italic> L. accessions. Genotypic analysis revealed that each of the three loci effectively divided the population into two groups with significantly different flowering times. Notably, accessions carrying two of the early-flowering loci flowered, on average, earlier than those carrying only one. Three representative accessions, each harboring a different single-locus allele (<italic>BnaC08cqDTF</italic>, <italic>qFTA10</italic>, and <italic>qFT.A02-1</italic>), were selected for diallel crosses aimed at pyramiding early-flowering alleles. Marker-assisted selection subsequently yielded 41 extremely early-flowering lines, which exhibited flowering times 3-4 days earlier than their respective parents. Among these, 195 lines were identified that combined extreme earliness with double-low seed quality. Multi-location yield trials conducted at five high-altitude sites over two consecutive years showed that the average yield of selected lines exceeded that of <italic>Brassica rapa</italic> L. cultivar Haoyou 11—the predominant spring-sown variety in high-altitude regions—by 23.07%. This study not only expands the genetic framework for developing extremely early- flowering <italic>B. napus</italic> spring-type germplasm but also provides elite parental resources for breeding early-maturing, high-yielding, and high-quality rapeseed cultivars.

BackgroundCompletely sequenced plant genomes provide scope for designing a large number of microsatellite markers, which are useful in various aspects of crop breeding and genetic analysis. With the objective of developing genic but non-coding microsatellite (GNMS) markers for the rice (Oryza sativa L.) genome, we characterized the frequency and relative distribution of microsatellite repeat-motifs in 18,935 predicted protein coding genes including 14,308 putative promoter sequences.ResultsWe identified 19,555 perfect GNMS repeats with densities ranging from 306.7/Mb in chromosome 1 to 450/Mb in chromosome 12 with an average of 357.5 GNMS per Mb. The average microsatellite density was maximum in the 5' untranslated regions (UTRs) followed by those in introns, promoters, 3'UTRs and minimum in the coding sequences (CDS). Primers were designed for 17,966 (92%) GNMS repeats, including 4,288 (94%) hypervariable class I types, which were bin-mapped on the rice genome. The GNMS markers were most polymorphic in the intronic region (73.3%) followed by markers in the promoter region (53.3%) and least in the CDS (26.6%). The robust polymerase chain reaction (PCR) amplification efficiency and high polymorphic potential of GNMS markers over genic coding and random genomic microsatellite markers suggest their immediate use in efficient genotyping applications in rice. A set of these markers could assess genetic diversity and establish phylogenetic relationships among domesticated rice cultivar groups. We also demonstrated the usefulness of orthologous and paralogous conserved non-coding microsatellite (CNMS) markers, identified in the putative rice promoter sequences, for comparative physical mapping and understanding of evolutionary and gene regulatory complexities among rice and other members of the grass family. The divergence between long-grained aromatics and subspecies japonica was estimated to be more recent (0.004 Mya) compared to short-grained aromatics from japonica (0.006 Mya) and long-grained aromatics from subspecies indica (0.014 Mya).ConclusionOur analyses showed that GNMS markers with their high polymorphic potential would be preferred candidate functional markers in various marker-based applications in rice genetics, genomics and breeding. The CNMS markers provided encouraging implications for their use in comparative genome mapping and understanding of evolutionary complexities in rice and other members of grass family.

Maize streak virus (MSV) is responsible for streak disease of maize and poses a serious threat to maize production in sub-Saharan Africa. Polygenic resistance to MSV has become an essential requirement in modern maize cultivars to mitigate yield losses. Many single nucleotide polymorphism (SNP) markers linked to putative MSV resistance loci have been identified for use in forward breeding. This study aimed to validate, using the high-throughput kompetitive allele specific PCR (KASP) assay, the diagnostic ability of the three SNP markers linked to the loci for the Msv1 resistance trait in 151 early generations inbred lines with diverse genetic backgrounds, together with nine MSV-resistant elite lines and a susceptible check (cv. Pool-16). The phenotypic responses were determined by MSV inoculation using viruliferous leafhoppers (Cicadulina triangular) under screenhouse conditions. Based on an established MSV disease rating system, the maize lines were categorized into resistant, moderately resistant, susceptible, and highly susceptible. The three SNPs associated with MSV resistance were detected in 133 lines, which were categorized as resistant (54), moderately resistant (76), and susceptible (1). The 18 early generation lines without these SNPs were classified as moderately resistant (10), susceptible (5), and highly susceptible (3). This study confirms the strong association of SNPs with MSV resistance and their usefulness for forward breeding in maize while emphasizing the need to identify additional markers to screen lines for MSV resistance without any ambiguity.

Optimization of a breeding program for increased genetic gain requires quality assurance (QA) and quality control (QC) at key phases of the breeding process. One vital phase in a breeding program that requires QC and QA is the choice of parents and successful hybridizations to combine parental attributes and create variations. The objective of this study was to determine parental diversity and confirm hybridity of cowpea F1 progenies using KASP (Kompetitive Allele-Specific PCR)-based single nucleotide polymorphism (SNP) markers. A total of 1,436 F1 plants were derived from crossing 220 cowpea breeding lines and landraces to 2 elite sister lines IT99K-573-1-1 and IT99K-573-2-1 as male parents, constituting 225 cross combinations. The progenies and the parents were genotyped with 17 QC SNP markers via high-throughput KASP genotyping assay. The QC markers differentiated the parents with mean efficiency of 37.90% and a range of 3.4–82.8%, revealing unique fingerprints of the parents. Neighbor-Joining cladogram divided the 222 parents into 3 clusters. Genetic distances between parents ranged from 0 to 3.74 with a mean of 2.41. Principal component analysis (PCA) depicted a considerable overlap between parents and F1 progenies with more scatters among parents than the F1s. The differentiation among parents and F1s was best contributed to by 82% of the markers. As expected, parents and F1s showed a significant contrast in proportion of heterozygous individuals, with mean values of 0.02 and 0.32, respectively. KASP markers detected true hybridity with 100% success rate in 72% of the populations. Overall, 79% of the putative F1 plants were true hybrids, 14% were selfed plants, and 7% were undetermined due to missing data and lack of marker polymorphism between parents. The study demonstrated an effective application of KASP-based SNP assay in fingerprinting, confirmation of hybridity, and early detection of false F1 plants. The results further uncovered the need to deploy markers as a QC step in a breeding program.

Breeding for resistance to stripe rust (caused by Puccinia striiformis f. tritici) is essential for reducing losses in yield and quality in wheat. To identify genes for use in breeding, a biparental population of 186 recombinant inbred lines (RILs) from a cross of the Chinese landrace Mingxian 169 and CIMMYT-derived line P9936 was evaluated in field nurseries either artificially or naturally inoculated in two crop seasons. Each of the RILs and parents was genotyped with the wheat 55K single-nucleotide polymorphism (SNP) 'Breeders' array and a genetic linkage map with 8,225 polymorphic SNP markers spanning 3,593.37 centimorgans was constructed. Two major quantitative trait loci (QTL) and two minor QTL were identified. The major QTL QYr.nwafu-3BS.2 and QYr.nwafu-7BL on chromosomes arms 3BS and 7BL were detected in all field locations and explained an average 20.4 and 38.9% of phenotypic variation stripe rust severity, respectively. QYr.nwafu-3BS.2 likely corresponds to the locus Yr30/Sr2 and QYr.nwafu-7BL may be a resistance allele identified previously in CIMMYT germplasm. The other minor QTL had limited individual effects but increased resistance when in combinations with other QTL. Markers linked to QYr.nwafu-7BL were converted to kompetitive allele-specific polymerase chain reaction markers and validated in a panel of wheat accessions. Wheat accessions carrying the same haplotype as P9936 at the identified SNP loci had lower average stripe rust severity than the average severity of all other haplotypes.

BackgroundPre-harvest sprouting (PHS) of wheat grain leads to a reduction in grain yield and quality. The availability of markers for marker-assisted selection (MAS) of PHS resistance will serve to enhance breeding selection and advancement of lines for cultivar development. The aim of this study was to identify candidate regions and develop molecular markers for PHS resistance in wheat. This was achieved via high density mapping of single nucleotide polymorphism (SNP) markers from an Illumina 90 K Infinium Custom Beadchip in a doubled haploid (DH) population derived from a RL4452/‘AC Domain’ cross and subsequent detection of quantitative trait loci (QTL) for PHS related traits (falling number [FN], germination index [GI] and sprouting index [SI]). SNP marker sequences flanking QTL were used to locate colinear regions in Brachypodium and rice, and identify genic markers associated with PHS resistance that can be utilized for MAS in wheat.ResultsA linkage map spanning 2569.4 cM was constructed with a total of 12,201 SNP, simple sequence repeat (SSR), diversity arrays technology (DArT) and expressed sequence tag (EST) markers. QTL analyses using Multiple Interval Mapping (MIM) identified four QTL for PHS resistance traits on chromosomes 3B, 4A, 7B and 7D. Sequences of SNPs flanking these QTL were subject to a BLASTN search on the International Wheat Genome Sequencing Consortium (IWGSC) database (http://wheat-urgi.versailles.inra.fr/Seq-Repository). Best survey sequence hits were subject to a BLASTN search on Gramene (www.gramene.org) against both Brachypodium and rice databases, and candidate genes and regions for PHS resistance were identified. A total of 18 SNP flanking sequences on chromosomes 3B, 4A, 7B and 7D were converted to KASP markers and validated with matching genotype calls of Infinium SNP data.ConclusionsOur study identified candidate genes involved in abscissic acid (ABA) and gibberellin (GA) metabolism, and flowering time in four genomic regions of Brachypodium and rice respectively, in addition to 18 KASP markers for PHS resistance in wheat. These markers can be deployed in future genetic studies of PHS resistance and might also be useful in the evaluation of PHS in germplasm and breeding material.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0340-1) contains supplementary material, which is available to authorized users.

With the continued emergence of Genome-Wide Association study-identified risk loci for complex diseases as well as a sharp increase in the number of variants of uncertain significance (VUS) identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Our lab has previously published a novel variant modeling pipeline which combines CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer variants in ~1 month. In this study, we utilize our pipeline to model risk Single Nucleotide Polymorphism (SNP) rs6983267 in human colorectal cancer (CRC) cells and present the first functional characterization for this risk SNP at the most precise level possible with current technology, isogenic clones of the same cell lines that only differ by a single base. Rs6983267 is located within a known Mycenhancer and has been shown to exert risk-allele specific increase in enhancer activity. However, we do not observe risk-allele specific upregulation of Mycexpression in our isogenic models. Instead, we find that presence of the risk allele confers significant effects within the cell locally, modulating activity of adjacent binding site for WNT transcription factor, TCF7L2, and a subset of genes associated with enhancer function. Through 3D interaction analysis, we identify difference in interactions incisacross the three genotypes including the loss of interaction with Mycin the heterozygous and homozygous reference lines, indicating genotype-specific changes of enhancer gene targets in CRC cells. Together, these variations lead to global effects, affecting transcriptional expression of genes involved in WNT signaling, cell-to-cell adhesion and PI3K-Akt signaling pathways. These results suggest the loss of a single risk allele in CRC cells confers a transcriptional change associated with overall decreased cancer activity despite expression of the putative target gene of the enhancer, Myc, not being significantly affected. Interestingly, engineered loss of both risk alleles in CRC cells does not further the overall effect, indicating the activation of a compensatory mechanism, possibly due to redundancy of regulatory pathways affected. Activation of such compensatory mechanisms could demonstrate an important limitation to the use of established cancer cell lines in the functional modeling of risk-associated variants, which are the current standard models in the field. Our study provides an important contribution to the current efforts of functional characterization of novel VUSs and non-coding SNPs associated with cancer risk. It will become important to consider validation standards and functional thresholds for inclusion of lower penetrance susceptibility alleles in clinical decision-making as the field continues to advance towards the ultimate goal of precision medicine in cancer. Citation Format: Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. Comprehensive functional modeling of pan-cancer risk SNP rs6983267 in human CRC cells via scarless CRISPR/Cas9 genome editing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2470.