Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Identification and Fine-Mapping of Clubroot (Plasmodiophora brassicae) Resistant QTL in Brassica rapa

European fodder turnips (Brassica rapa ssp. rapifera) were identified as sources of clubroot resistance (CR) and have been widely used in Brassica resistance breeding. An F2 population derived from a cross between a resistant turnip and a susceptible Chinese cabbage was used to determine the inheritance and locating the resistance Quantitative Trait Loci (QTLs). The parents showed to be very resistant/susceptible to the field isolates (pathotype 4) of clubroot from Henan in China. After inoculation, 27 very resistant or susceptible individuals were selected to construct bulks, respectively. Next-generation-sequencing-based Bulk Segregant Analysis Sequencing (BSA-Seq) was used and located resistance QTL on chromosome A03 (3.3–7.5 Mb) and A08 (0.01–6.5 Mb), named Bcr1 and Bcr2, respectively. Furthermore, an F3 population including 180 families derived from F2 individuals was phenotyped and used to verify and narrow candidate regions. Ten and seven Kompetitive Allele-Specific PCR (KASP) markers narrowed the target regions to 4.3–4.78 Mb (A03) and 0.02–0.79 Mb (A08), respectively. The phenotypic variation explained (PVE) of the two QTLs were 33.3% and 13.3% respectively. The two candidate regions contained 99 and 109 genes. In the A03 candidate region, there were three candidate R genes, namely Bra006630, Bra006631 and Bra006632. In the A08 candidate region, there were two candidate R genes, namely Bra030815 and Bra030846.

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

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.

Modeling the technology of identification of wine materials and wines by PCR analysis of microsatellite loci of grapevine chloroplast DNA

Использование полиморфных микросателлитных локусов ДНК является одним из подходов к аутентификации виноматериалов и вин. При этом SSR-маркеры хлоропластной ДНК имеют большую копийность мишени на клетку и менее подвержены деградации из-за содержания в органеллах с двойной мембраной. Целью настоящей работы являлось моделирование технологии идентификации виноматериалов и вин ПЦР-анализом микросателлитных локусов хлоропластной ДНК винограда. Подобраны условия экстракции нуклеиновых кислот, постановки ПЦР с соответствующими наборами праймеров и электрофоретической детекции, направленные на практическое воспроизведение генетического тестирования пробоподготовленного биоматериала из осаждаемого винного дебриса. Представлены наглядные результаты выравнивания частичных нуклеотидных последовательностей аллелей микросателлитных локусов хлоропластной ДНК Vitis vinifera L. Проанализирована разделяющая способность метода горизонтального электрофореза в геле «Spreadex EL 300» in silico моделированием генерируемых аллельспецифичных фрагментов, позволяющая идентифицировать известные хлоротипы винограда даже при постановке ПЦР с ограниченными наборами праймеров, нацеленных на локусы cpSSR3, cpSSR5, cpSSR10, NTCP12 и ccSSR9. The use of polymorphic microsatellite DNA loci is one of the approaches to the authentication of wine materials and wines. At the same time, SSR markers of chloroplast DNA have a large target copy number per cell and are less susceptible to degradation due to their content in organelles with a double membrane. The aim of this work was to simulate the technology of identification of wine materials and wines by PCR analysis of microsatellite loci of grapevine chloroplast DNA. The conditions for the extraction of nucleic acids, PCR with the corresponding sets of primers and electrophoretic detection were selected, aimed at the practical reproduction of genetic testing of the sample prepared biomaterial from the precipitated wine debris. Illustrative results of the alignment of partial nucleotide sequences of alleles of microsatellite loci of Vitis vinifera L. chloroplast DNA are presented. The separating ability of the method of horizontal electrophoresis in «Spreadex EL 300» gel by in silico modeling the generated allele-specific fragments, which makes it possible to identify the known chlorotypes of grapevine with a limited set of primers targeting loci (cpSSR3, cpSSR5, cpSSR10, NTCP12 and ccSSR9) has been analyzed.

A High-Density Genetic Map Enables Genome Synteny and QTL Mapping of Vegetative Growth and Leaf Traits in Gardenia

The gardenia is a traditional medicinal horticultural plant in China, but its molecular genetic research has been largely hysteretic. Here, we constructed an F1 population with 200 true hybrid individuals. Using the genotyping-by-sequencing method, a high-density sex-average genetic map was generated that contained 4,249 SNPs with a total length of 1956.28 cM and an average genetic distance of 0.46 cM. We developed 17 SNP-based Kompetitive Allele-Specific PCR markers and found that 15 SNPs were successfully genotyped, of which 13 single-nucleotide polymorphism genotypings of 96 F1 individuals showed genotypes consistent with GBS-mined genotypes. A genomic collinearity analysis between gardenia and the Rubiaceae species Coffea arabica, Coffea canephora and Ophiorrhiza pumila showed the relativity strong conservation of LG11 with NC_039,919.1, HG974438.1 and Bliw01000011.1, respectively. Lastly, a quantitative trait loci analysis at three phenotyping time points (2019, 2020, and 2021) yielded 18 QTLs for growth-related traits and 31 QTLs for leaf-related traits, of which qBSBN7-1, qCD8 and qLNP2-1 could be repeatably detected. Five QTL regions (qCD8 and qSBD8, qBSBN7 and qSI7, qCD4-1 and qLLLS4, qLNP10 and qSLWS10-2, qSBD10 and qLLLS10) with potential pleiotropic effects were also observed. This study provides novel insight into molecular genetic research and could be helpful for further gene cloning and marker-assisted selection for early growth and development traits in the gardenia.

Schizophrenia-associated somatic copy number variants from 12,834 cases reveal contribution to risk and recurrent, isoform-specific NRXN1 disruptions

While inherited and de novo copy number variants (CNV) have been implicated in the genetic architecture of schizophrenia (SCZ), the contribution of somatic CNVs (sCNVs), present in some but not all cells of the body, remains unknown. Here we explore the role of sCNVs in SCZ by analyzing blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls. sCNVs were more common in cases (0.91%) than in controls (0.51%, p = 2.68e-4). We observed recurrent somatic deletions of exons 1-5 of the NRXN1 gene in 5 SCZ cases. Allele-specific Hi-C maps revealed ectopic, allele-specific loops forming between a potential novel cryptic promoter and non-coding cis regulatory elements upon deletions in the 5' region of NRXN1. We also observed recurrent intragenic deletions of ABCB11, a gene associated with anti-psychotic response, in 5 treatment-resistant SCZ cases. Taken together our results indicate an important role of sCNVs to SCZ risk and treatment-responsiveness.

Identification of Accession-Specific Variants and Development of KASP Markers for Assessing the Genetic Makeup of Crop Seeds

Background Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed testing for purity was done with a grow-out test (GOT) in the field, but these tests are time consuming and costly. Seed testing with molecular markers was introduced as a replacement for GOT early in the last decade. Recently, Kompetitive allele specific PCR (KASP) markers are promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and could be used with only a small number of accessions or varieties due to the limited genetic information and reference genomes available. Results Here, we identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. Furthermore, the total 2,925 SNPs were selected as accession-specific SNPs, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by NGS analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguish individuals from the mixed population including 50 target accessions from B. rapa core collection and outgroup. Conclusions This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).