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Abstract Background Improving the quality and shelf life of groundnut oil is one of the foremost objectives of groundnut breeding programmes. This can be achieved by marker-assisted introgression, a technique that efficiently and precisely enables breeders to develop plants with enhanced qualities. This study focused on improving the oleic acid content of an elite groundnut variety, TMV 7, by introgressing a recessive mutation responsible for the increase in oleic acid from ICG 15419. Hybridization was performed between the donor and recurrent parents to develop the F1, BC1F1, BC2F1 and BC2F2 populations. Introgressed lines with increased oleic acid in the genetic background of TMV 7 were identified using allele-specific marker, F435-F, F435SUB-R and a set of SSR markers were employed to recover the genome of the recurrent parent. Results With two backcrosses, a total of ten homozygous plants in the BC2F2 population were identified with oleic acid content ranging from 54.23 to 57.72% causing an increase of 36% over the recurrent parent. Among the ten lines, the line IL-23 exhibited the highest level of recurrent parent genome recovery of 91.12%. Conclusions The phenotypic evaluation of 10 homozygous introgressed lines indicated fewer differences for all other traits under study compared to the recurrent parent, except for oleic acid and linoleic acid content confirming the genetic background of the recurrent parent. The identified lines will be subjected to multilocation trials before their commercial release.

Background: Improving the quality and shelf life of groundnut oil is one of the foremost objectives of groundnut breeding programmes. This can be achieved by marker-assisted introgression, a technique that efficiently and precisely enables breeders to develop plants with enhanced qualities. This study focused on improving the oleic acid content of an elite groundnut variety, TMV 7, by introgressing a recessive mutation responsible for the increase in oleic acid from ICG 15419. Hybridization was performed between the donor and recurrent parents to develop the F1, BC1F1, BC2F1 and BC2F2 populations. Introgressed lines with increased oleic acid in the genetic background of TMV 7 were identified using allele-specific marker, F435-F, F435SUB-R and a set of SSR markers were employed to recover the genome of the recurrent parent. Results: With two backcrosses, a total of ten homozygous plants in the BC2F2 population were identified with oleic acid content ranging from 54.23 to 57.72% causing an increase of 36% over the recurrent parent. Among the ten lines, the line IL-23 exhibited the highest level of recurrent parent genome recovery of 91.12%. Conclusions: The phenotypic evaluation of 10 homozygous introgressed lines indicated fewer differences for all other traits under study compared to the recurrent parent, except for oleic acid and linoleic acid content confirming the genetic background of the recurrent parent. The identified lines will be subjected to multilocation trials before their commercial release. [ABSTRACT FROM AUTHOR]

Non-invasive prenatal diagnosis for single-gene disorders (NIPD) is still in development and deserves further study. The advent of next-generation sequencing technology significantly improved the detection of multiple mutations for non-invasive prenatal diagnosis for single-gene disorder purposes. However, bespoke amplicon-based NGS assays are costly. In this study, we developed a new strategy for non-invasive prenatal screening for single-gene disorders based on a capillary electrophoresis (CE) platform using an amplification refractory mutation system (ARMS)-PCR technique. Allelespecific primers for several disease-correlated mutations were designed, and subsequently, sensitivity and specificity assays were conducted. Assays on simulated two-person DNA mixtures showed that three primers targeting the mutant allele could detect minor DNA components in 1:500 mixtures. All primers showed positive results at 0.01 ng of the template DNA. Cell-free fetal DNA was extracted from a pregnant woman's peripheral blood for the detection of paternally inherited mutations. Our results showed that one primer successfully amplified the mutant allele of fetalDNA inmaternal plasma, whichwas confirmed by genotyping the genomic DNA extracted from amniotic fluid. This study suggested that the ARMS-PCR technique, a fast and cost-effective method, might be a promising method used to target de novo or paternally inherited pathogenic mutations in maternal plasma. [ABSTRACT FROM AUTHOR]

Soybean, Glycine max L., is one of the most important agricultural crops grown in a wide range of latitude. In this regard, in soybean breeding, it is necessary to pay attention to the set of genes that control the transition to the flowering stage, which will make it possible to adapt genotypes to local growing conditions as accurately as possible. The possibilities of soybean breeding for this trait have now significantly expanded due to identification of the main genes (E1–E4, GmFT2a, GmFT5a) that control the processes of flowering and maturation in soybean, depending on the day length. The aim of this work was to develop a panel of markers for these genes, which could be used for a rapid and efficient genotyping of domestic soybean cultivars and selection of plant material based on sensitivity to photoperiod and the duration of vegetation. Combinations of 10 primers, both previously developed and our own, were tested to identify different alleles of the E1–E4, GmFT2a, and GmFT5a genes using 10 soybean cultivars from different maturity groups. As a result, 5 combinations of dominant and recessive alleles for the E1–E4 genes were identified: (1) e1-nl(e1-as)/ e2-ns/e3-tr(e3-fs)/e4; (2) e1-as/e2-ns/e3-tr/E4; (3) e1-as/e2-ns/E3-Ha/e4; (4) E1/e2-ns/e3-tr/E4; (5) e1-nl/e2-ns/E3-Ha/E4. The studied cultivars contained the most common alleles of the GmFT2a and GmFT5a genes, with the exception of the ‘Cassidi’ cultivar having a rare dominant allele GmFT5a-H4. The degree of earliness of cultivars positively correlated with the number of recessive genes E1–E4, which is consistent with the data of foreign authors on different sets of cultivars from Japan and North China. Thus, the developed panel of markers can

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. [ABSTRACT FROM AUTHOR]

Spring bread wheat is the most important cereal crop, cultivated under various climatic conditions and on different latitudes. Modern molecular genetic studies of wheat are aimed at investigating the crop’s genetic potential. By now, molecular markers have been developed to identify alleles of the Vrn (vernalization response) and Ppd (photoperiod response) genes. Vrn genes are responsible for crop development rate regulation and crop yield structure. Ppd genes determine the response of plants to the length of the day, that is, the timing of flowering and the beginning of heading in plants under different cultivation conditions. The use of diagnostic DNA markers made it possible to analyze the presence of allelic combinations of the Vrn and Ppd genes in local and commercial wheat varieties from Europe, Asia, North and South Americas, Africa and Australia. This review summarizes the results of studies on the distribution of alleles of Vrn and Ppd genes in wheat breeding material over different geographical areas of its cultivation. For example, the dominant Vrn-A1a allele was found in 62% of European varieties; 52% of the studied Turkish wheat varieties carried dominant Vrn-B1 alleles. A dominant Vrn-D1 was found in 61% of Pakistani wheat accessions. Vrn-D1 is present in 41.9% of the studied varieties of Chinese wheat. Higher incidence of Ppd-D1A is typical for West European varieties. A Ppd-D1a allele was found in 58.6% of varieties preserved in the Turkish wheat collection, with a 60% frequency of this allele in commercial cultivars. Among local Afghan varieties, 97% are sensitive to photoperiod (carriers of Ppd-D1b); they are distributed throughout the country without much dependence on agroecological zones. All Pakistani varieties are insensitive to photoperiod (carriers of Ppd-D1a). In China, the highest incidence of the Ppd-D1a allele was observed in zone VII (87.5% of varieties).

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.

Prunus avium species of the Rosaceae family exhibit gametophytic self-incompatibility. Determination of the self-incompatibility genotype of individuals is essential for genetic studies and the development of informed management strategies. The PCR-based detection of S-allele helps to promote and speed up traditional breeding activity and hence molecular analysis of the perspective genotypes has become more intensive in all cherry growing countries. The alleles S3, S4 and S9 from 34 accessions of Czech collections were determined using the polymerase chain reaction (PCR) method. Initially, DNA extracts were amplified with consensus primers that amplify across the first, second, or both introns of the S-ribonuclease gene which shows a considerable length polymorphism. The new allele specific primers were designed with the goal to overcome some occurring difficulties in the detection of expected alleles by previously published allele specific primers. S-alleles fragments of standard cultivars used in this study were PCR amplified, sequenced to validate the designed primers. The study demonstrates the advantage of newly designed primers application in testing of sweet cherry genotypes.

Non-invasive prenatal diagnosis for single-gene disorders (NIPD) is still in development and deserves further study. The advent of next-generation sequencing technology significantly improved the detection of multiple mutations for non-invasive prenatal diagnosis for single-gene disorder purposes. However, bespoke amplicon-based NGS assays are costly. In this study, we developed a new strategy for non-invasive prenatal screening for single-gene disorders based on a capillary electrophoresis (CE) platform using an amplification refractory mutation system (ARMS)-PCR technique. Allele-specific primers for several disease-correlated mutations were designed, and subsequently, sensitivity and specificity assays were conducted. Assays on simulated two-person DNA mixtures showed that three primers targeting the mutant allele could detect minor DNA components in 1:500 mixtures. All primers showed positive results at 0.01 ng of the template DNA. Cell-free fetal DNA was extracted from a pregnant woman's peripheral blood for the detection of paternally inherited mutations. Our results showed that one primer successfully amplified the mutant allele of fetal DNA in maternal plasma, which was confirmed by genotyping the genomic DNA extracted from amniotic fluid. This study suggested that the ARMS-PCR technique, a fast and cost-effective method, might be a promising method used to target de novo or paternally inherited pathogenic mutations in maternal plasma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The editor GH declared a shared parent affiliation with the authors. AE [GH] department: Institute of Rare Diseases, West China Hospital of Sichuan University, Sichuan University., (Copyright © 2023 Tan, Jian, Zhang, Wang, Zhou, Xiao, Liang and Wang.)

The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology. [ABSTRACT FROM AUTHOR]

Culex tritaeniorhynchus is a major Japanese encephalitis virus vector distributed in Southeast Asia and surrounding countries. The aim of the present study is to investigate insecticide resistance status among 10 Cx. tritaeniorhynchus populations of the Mediterranean region of Turkey. Bioassay results indicated that all of the populations were resistant or at least possibly resistant to 1,1′-(2,2,2-Trichloroethane-1,1-diyl) bis (4-chlorobenzene) (DDT) (4%), [(dimethoxyphosphorothioyl) sulfanyl] butanedioate, Diethyl (malathion) (5%), and 2-[(Propan-2-yl) oxy] phenyl methylcarbamate (propoxur) (0,1%). Whereas, some of the populations were still susceptible to 3-Phenoxybenzyl (1 RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate (permethrin) (0,75%) and (S)-Cyano (3-phenoxy phenyl) methyl (1 R ,3 R)-3-(2,2-dibromoethen-1-yl)-2,2-dimethylcyclopropane-1-carboxylate (deltamethrin) (0,05%). Biochemical analysis results showed altered alpha esterase, beta esterase, para-nitrophenyl acetate (PNPA), and glutathione-s-transferase (GST) levels in some populations while all of the populations had increased oxidase levels except for the Yumurtalık population. Additionally, all of the populations had sensitive acetylcholinesterase (AChE) levels similar to the control group except for the Erzin population. Correlation analysis showed a significant correlation between mortality rates for deltamethrin and alpha esterase, beta esterase, PNPA, and GST levels while mortality rates for permethrin were significantly correlated with GST levels. An allele-specific polymerase chain reaction (AS-PCR) detected high L1014F allele frequency in the populations. Overall results indicate the urgent need for monitoring and mapping of insecticide resistance in Cx. tritaeniorhynchus populations of the study area for effective vector control management. [Display omitted] • Bioassay results indicated high resistance to DDT, malathion and propoxur. • Some of the populations started to gain resistance to permethrin and deltamethrin. • Allele-specific primers detected high frequency of kdr (L1014F) mutations. • Most of the populations had sensitive acetylcholinesterase levels. • Correlation analysis showed correlation between GST levels and mortality rates to permethrin. • Correlation analysis showed correlation between esterase and GST levels and mortality rates to deltamethrin. [ABSTRACT FROM AUTHOR]

• The complete chloroplast genome of Cyathula officinalis was sequenced, assembled, and annotated for the first time. • The complete chloroplast genome of Cyathula officinalis and four related species were compared and analyzed. • Allele-specific PCR and DNA barcoding were designed for a supplementary identification method of Cyathula officinalis. Cyathula officinalis is a medicinal and edible herb, which can remove blood stasis, stimulate menstrual flow, and ease joint movement. In this study, the complete chloroplast genome of Cyathula officinalis was sequenced, assembled, and analyzed. Compared with the chloroplast genomes of Cyathula capitata , Achyranthes bidentata , Achyranthes longifolianine and Achyranthes aspera , the basic characteristics, codon usage bias, repeat sequences, simple sequence repeats, and phylogenetic tree were analyzed. In addition, according to nucleotide diversity analysis and sequence alignment, DNA barcoding and allele-specific PCR primers were designed to identify and distinguish Cyathula officinalis from its fake drugs, which has effectively practical significance for the authentication of "Chuan Niuxi" crude drug in the market. [ABSTRACT FROM AUTHOR]

The allele-specific qPCR (ASQ) method for SNP (single nucleotide polymorphism) detection is based on the FRET (fluorescence resonance energy transfer) system, a system using position-dependent fluorescent dyes and quenches. The modified ASQ method requires two separate components: (1) the allele-specific part, two AS primers targeting the SNP with identity in the penultimate positions at the 3'-end and specific tags in the 5'-end, and (2) the universal part, two universal probes (UPs) with corresponding tags and different fluorescent dyes in the 5'-end and a single common universal probe with a quencher in the 3'-ends (Uni-Q), complementary to all UP tags. There are two major variations of the ASQ method, with either short 4-bp tags (variant A) or longer 6-bp tags (variant B), both of which have been successfully used for SNP genotyping in plants. The modified ASQ method is much cheaper compared to other similar FRET-based methods because the most expensive parts, the universal probes, have a short and linear structure, where fluorophores and quenchers are located in the ends but not incorporated inside of the sequences., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polymerase chain reaction (PCR) amplification of specific alleles allowed the rapid detection of a point mutation (missense Gly528 → Asp) in exon 11 of the low density lipoprotein receptor gene which was otherwise not detectable by exon amplification and enzymatic digestion as it does not modify the normal restriction pattern. The mutant allele, designated as FH-Palermo-1 from the origin of the first carrier family identified, gave a specific PCR product of 109 bp clearly distinct from the product of 168 bp obtained from other alleles with a nonspecific couple of primers. This method allowed us to distinguish one positive sample mixed with up to 11 parts of normal DNA. Furthermore, the specific amplification product was characterized by a Bsm I restriction site not present in nonspecific products.—Cantafora, A., I. Blotta, E. Mercuri, S. Calandra, and S. Bertolini. Simple detection of a point mutation in LDL receptor gene causing familial hypercholesterolemia in southern Italy by allele-specific polymerase chain reaction. J. Lipid Res. 1998. 39: 1101–1105.

Rosa rugosa, a vigorous ornamental shrub introduced from Asia in the 19th century, is now naturalized in coastal northeastern North America, where it occasionally grows in sympatry with the native R. blanda. To document hybridization between these species, evaluate its extent across the area of sympatry, and examine the use of morphology as a field monitoring tool, we sampled 179 individuals of parental species and putative hybrids in 13 pure and 11 mixed populations. We developed allele-specific primers to assay single nucleotide polymorphisms (SNP5) markers from one chloroplast region and four low-copy nuclear introns. Our results revealed frequent bidirectional hybridization and infrequent introgression in sympatric populations of these species. The recurrent presence of F1 hybrids in mixed populations indicated the weakness of early-acting reproductive barriers. Morphological data were concordant with molecular data and provided additional evidence for the presence of a few backcrosses. Morphological analyses yielded diagnostic characters for identifying hybrids and monitoring the hybrid zone. Such hybridization could ultimately lead to the genetic assimilation of R. blanda in mixed populations and to the formation of invasive hybrid genotypes, a phenomenon that is of economic and ecological concern because of the increasing number of exotic species worldwide. [ABSTRACT FROM AUTHOR]

The article reports on the identification of S-allele in Lithuanian sweet cherries using the polymerase chain reaction (PCR) method. The process and results of DNA extracts amplification is discussed. It notes that in all S alleles' accessions studied its combinations were identified which include S1S13 and S6S16. These combinations can be regarded as unique genotypes and therefore as universal pollinators.

Background Basal cell carcinoma (BCC) is one of the most common neoplasms in the world. Development of BCC is associated with environmental factors (especially sun exposure) as well as heritable factors. Objectives To analyse three single nucleotide polymorphisms (SNPs) in the promoter regions of interleukin (IL) genes in genomic DNA from 527 cases of BCC and 530 matched controls and to examine if DNA pooling is a useful method on which to base decisions regarding further SNP analysis. Methods The SNPs analysed were IL6–597, IL10–1082 and IL1B–511. The SNPs were first analysed from pooled DNA and afterwards from individual samples. The DNA pools resulted from a division of the samples into cases and controls, female and male, and three age groups. In these pools the allele frequencies were estimated by two methods, real-time polymerase chain reaction with allele-specific primers, and quantitative sequencing. Results No significant association was found when the allele frequencies in cases and controls were compared. However, by analysis of the individual genotypes we found SNP IL6–597 G/A to be significantly associated with BCC risk ( P =0·007). Hereby the heterozygous genotype ‘GA’ had a protective effect (odds ratio 0·64, 95% confidence interval 0·49–0·84). No significant association was found for IL10–1082 and IL1B–511. Conclusions The association of SNP IL6–597 with BCC could be found only by individual genotyping, but would have been missed if only data from the pooling analysis had been known. [ABSTRACT FROM AUTHOR]

The self-incompatibility (S) genotypes of a collection of 65 Belgian accessions of wild cherry, selected within two populations and planted in a seed orchard, were determined using polymerase chain reaction (PCR) methods. Initially, DNA extracts were amplified with consensus primers that amplify across the second intron of theS-ribonuclease gene which shows considerable length polymorphism. The provisional genotypes deduced were checked with the appropriate allele-specific primers for the known allelesS 1 toS 16. Putative new alleles were subjected to PCR with consensus primers amplifying across the first intron. Six new alleles,S 17 toS 22, were thus indicated on the basis of the estimated lengths of the first and second intron PCR products. Examples of these alleles were partially sequenced and were indeed mutually distinct and different from the known alleles. The incompatibility genotypes of all 65 accessions were determined and one triploid individual was found. Seventeen alleles were detected in all. Allele frequencies differed between samples and the expected total number of alleles in the underlying populations was estimated. The wild cherry populations differed significantly with respect to allelic frequencies from sweet cherry cultivars; allelesS 4 andS 5, which are moderately frequent in sweet cherry, were absent from the wild cherry accessions. The knowledge of theSgenotypes will be useful for studying the gene flow within the seed orchard and these approaches should also be informative in wild populations. [ABSTRACT FROM AUTHOR]

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Soybean, Glycine max L., is one of the most important agricultural crops grown in a wide range of latitude. In this regard, in soybean breeding, it is necessary to pay attention to the set of genes that control the transition to the f lowering stage, which will make it possible to adapt genotypes to local growing conditions as accurately as possible. The possibilities of soybean breeding for this trait have now signif icantly expanded due to identif ication of the main genes (E1-E4, GmFT2a, GmFT5a) that control the processes of f lowering and maturation in soybean, depending on the day length. The aim of this work was to develop a panel of markers for these genes, which could be used for a rapid and eff icient genotyping of domestic soybean cultivars and selection of plant material based on sensitivity to photoperiod and the duration of vegetation. Combinations of 10 primers, both previously developed and our own, were tested to identify different alleles of the E1-E4, GmFT2a, and GmFT5a genes using 10 soybean cultivars from different maturity groups. As a result, 5 combinations of dominant and recessive alleles for the E1-E4 genes were identif ied: (1) e1-nl(e1-as)/ e2-ns/e3-tr(e3-fs)/e4; (2) e1-as/e2-ns/e3-tr/E4; (3) e1-as/e2-ns/E3-Ha/e4; (4) E1/e2-ns/e3-tr/E4; (5) e1-nl/e2-ns/E3-Ha/E4. The studied cultivars contained the most common alleles of the GmFT2a and GmFT5a genes, with the exception of the 'Cassidi' cultivar having a rare dominant allele GmFT5a-H4. The degree of earliness of cultivars positively correlated with the number of recessive genes E1-E4, which is consistent with the data of foreign authors on different sets of cultivars from Japan and North China. Thus, the developed panel of markers can be successfully used in the selection of soybean for earliness and sensitivity to photoperiod., (Copyright © AUTHORS.)