Single Nucleotide Polymorphism Sites Research Articles

Genetic variation analysis of Guanling cattle based on whole-genome resequencing.

The objective of this study was to unravel the genetic traits of Guanling cattle, pinpoint genes advantageous for muscle growth, and lay a foundation for the preservation of genetic diversity and further analysis of regulation mechanism of important economic traits in local cattle breed. In this study, we sequenced the whole genome of 3 Guanling cattle in Guizhou province using the Illumina HiSeq cBo sequencing platform. And, high- multiplex polymerase chain reaction technology was employed to detect high-quality single nucleotide polymorphism (SNP) sites of other 55 Guanling cattle. Our study identified 166,411 non-synonymous SNPs (nsSNPs) and 42,423 insertions and deletions (indels). Through SNP annotation, gene function enrichment analysis, and comparing with Simmental, Angus, and Limousin cattle, we identified six genes (LEPR, AKAP9, SIX4, SPIDR, PRG4, FASN) which are potentially influential on meat quality traits, playing crucial roles in muscle growth, fat metabolism, and bodily support. We also examined polymorphisms at seven SNP sites in Guanling cattle and found that all seven were in Hardy-Weinberg equilibrium. These findings suggested that these gene sites are stable and widespread in the Guanling cattle population. Our research lays the groundwork for future genetic enhancement and variety identification of Guanling cattle.

Genomic Single Nucleotide Polymorphism (SNP) markers and mitochondrial haplotypes illuminate the origins of Crown-of-Thorns Starfish (Acanthaster solaris) outbreaks in the South China Sea.

Outbreaks of the coral predator Crown-of-Thorns Starfish (CoTS) pose a severe threat to coral reefs in the Indo-Pacific Ocean. In 2018, the South China Sea (SCS) experienced significant CoTS outbreaks, leading to extensive coral mortality across the Xisha, Zhongsha, Dongsha, and Nansha Islands, severely impacting the coral reef ecosystem. To explore the origins of these outbreaks, we conducted a comprehensive genomic analysis using data from genomic single nucleotide polymorphism sites (SNPs) and mitochondrial haplotypes. Our analysis reveals that CoTS populations in the SCS, which exhibit moderate genetic diversity and may have undergone positive selection or population expansion. There was limited genetic differentiation among CoTS populations from XS, ZS, and NS groups. Especially between the XS and ZS groups, there was almost no genetic differentiation. The populations from XS, ZS, and NS groups have strong genetic connections with populations in Vietnam and the Philippines. There was high gene flow from Vietnam to the Xisha Islands and from the Philippines to the Nansha Islands, suggesting that the CoTS populations in these regions primarily originate from these neighboring countries. The comprehensive analyses of SNP and mitochondrial genomes have provided valuable insights into the population genetics of CoTS. This research has generated significant genomic resources and facilitated important studies on the genetics of the CoTS species. By identifying potential source populations and understanding the genetic basis of their spread, managers can develop more effective conservation strategies to protect vulnerable coral reef ecosystems in the SCS.

Abstract A019: Identification and Genomic Analysis of miRNA-mRNA Binding Site SNPs in Hepatitis C Virus-Associated Hepatocellular Carcinoma

Abstract Hepatitis C virus (HCV) infection is a major risk factor for hepatocellular carcinoma (HCC), a leading cause of cancer-related deaths globally. MicroRNAs (miRNAs) play critical roles in gene regulation, and single nucleotide polymorphisms (SNPs) within miRNA binding sites can disrupt this regulation, potentially influencing HCC pathogenesis. The present study aims to identify and characterize SNPs within miRNA binding sites of genes associated with HCV- induced HCC (HCV-HCC). A comprehensive bioinformatics analysis workflow was devised. HCV-specific differentially expressed (DE) miRNAs and mRNAs were extracted from literature and databases. SNPs in the 3' UTRs of HCV-HCC genes were identified, and their impact on miRNA binding was predicted using PoymiRTS and TargetScan. RNAhybrid was used to estimate minimum folding energy (MFE) differences (ΔMFE) between mutant and wild types. Functional enrichment analysis was conducted to elucidate the biological processes affected by these SNPs. Our analysis revealed several significant alterations in miRNA-mRNA binding pairs due to SNPs within genes associated with HCV-HCC. For instance, among the downregulated genes in HCV-HCC, the SNP in CTSB (rs3088245; 193T>C) predicted to increase the stability of the miR-27b - CTSB duplex (ΔMFE: 1.4), suggesting enhanced binding and potentially improved repression of CTSB. Conversely, the SNP in EGR2 (rs61865883; 287 T>A) decreased the stability of the miR-140-5p - EGR2 mRNA interaction (ΔMFE: -1.9), suggesting a weaker binding affinity and potentially reduced repression of EGR2 expression. Considering the tumor suppressor role of EGR2, presence of this SNP is likely to reflect good prognosis. For the upregulated gene oncogene, XIAP, the SNP (rs6648556; 5892 T>C) is predicted to enhance the binding of miR- 200c (ΔMFE: 1.5), likely resulting in increased suppression of XIAP expression in patients carrying this SNP and improved prognosis. Our study emphasizes the importance of considering genetic variations in miRNA binding sites in the context of HCV-HCC. These SNPs may serve as genetic modifiers influencing disease susceptibility and progression. Further experimental studies are warranted to explore their potential as therapeutic agents and predictors of prognosis in HCV-HCC patients. Citation Format: Azeem M Butt, Anum Qamar, Shafiqa Siddique. Identification and Genomic Analysis of miRNA-mRNA Binding Site SNPs in Hepatitis C Virus-Associated Hepatocellular Carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A019.

Molecular characterization and B-cell epitope analysis of the TSP11 gene in Echinococcus infection strains from Yunnan Province.

This study investigates the molecular intricacies of the transmembrane protein TSP11 gene in Echinococcus strains isolated from livestock and patients in Yunnan Province afflicted with Echinococcus granulosus (E. granulosus) between 2016 and 2020. Gene typing analysis of the ND1 gene revealed the presence of the G1 type, G5 type and untyped strains, constituting 52.4, 38.1 and 9.5%, respectively. The analysis of 42 DNA sequences has revealed 24 novel single nucleotide polymorphic sites, delineating 11 haplotypes, all of which were of the mutant type. Importantly, there were no variations observed in mutation sites or haplotypes in any of the hosts. The total length of the TSP11 gene's 4 exons is 762 bp, encoding 254 amino acids. Our analysis posits the existence of 6 potential B-cell antigenic epitopes within TSP11, specifically at positions 49-KSN-51, 139-GKRG-142, 162-DNG-164, 169-NGS-171, 185-DS-186 and 231-PPRFTN-236. Notably, these epitopes exhibit consistent presence among various intermediate hosts and haplotypes. However, further validation is imperative to ascertain their viability as diagnostic antigens for E. granulosus in the Yunnan Province.

Comparative analysis of chloroplast genome of Lonicera japonica cv. Damaohua.

Lonicera japonica is a well-known medicinal plant, and the Damaohua cultivar is one of the oldest known honeysuckle cultivars in China. The 155,151 bp chloroplast genome of this cultivar was obtained through Illumina sequencing. The genome includes a pair of inverted repeats (IRa and IRb; 23,789 bp each), a large single-copy region (88,924 bp), and a small single-copy (SSC) region (18,649 bp). In total, 127 unique genes were identified: 80 protein-coding, 39 tRNA, and 8 rRNA genes. Only ycf3 contained two introns. Eighty-nine large repetitive sequences and 54 simple sequence repeats were detected. Fifty potential RNA editing sites were predicted. Adaptive evolution analysis revealed that infA, matK, petB, petD, rbcL, rpl16, rpl2, rps3, ycf1, and ycf2 were positively selected, possibly reflecting the specific environmental adaptations of this cultivar. Sequence alignment and analysis revealed several candidate fragments for Lonicera species identification, such as the intergenic regions rpoB-petN, rbcL-accD, and psaA-ycf3. The IR region boundary and phylogenetic analysis revealed that the L. japonica cv. Damaohua chloroplast genome was most closely related to the L. japonica genome, but there were five distinct differences between the two. There are four sites with high variability between L. japonica and L. japonica cv. Damaohua with nucleotide variability (Pi) greater than 0.002, including rps2-rpoC2, atpB-rbcL, ycf1, and ycf1-trnN GUU. The differences between L. japonica and L. japonica cv. Damaohua were further confirmed by the single nucleotide polymorphism sites between these two species. Therefore, this study revealed that the chloroplast genome can serve as a universal super barcode for plant identification, which can identify differences and help distinguish Lonicera japonica from related species. An understanding of Lonicera japonica cv. Damaohua chloroplast genomics and a comparative analysis of Lonicera species will provide a scientific basis for breeding, species identification, systematic evolution analysis, and chloroplast genetic engineering research on medicinal honeysuckle plants.

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

Population Genetic Characteristics of the Cultivated Coffea arabica with Whole-Genome Resequencing

Coffea arabica L. (C. arabica) is an economically important agricultural crop and the most popular beverage worldwide. To analyze genetic diversity and provide genetic resources for the selection and breeding of superior varieties of C. arabica, 61 cultivated Arabica coffee accessions were analyzed in the study, including 12 resequencing accessions from previous research and 49 accessions that were resequenced in this study. Single nucleotide polymorphisms (SNPs) and insertion–deletions (InDels) were statistically analyzed. Based on SNP variations, a genetic structure analysis, phylogenetic tree construction, and principal component analysis were performed for the 61 coffee accessions. The results showed that a total of 805.46 Gb of raw whole-genome resequencing data was obtained from the 61 coffee accessions, with 781.29 Gb of high-quality sequencing data after filtering. In total, 7,013,820 SNP sites and 1,074,329 InDel sites were detected. The average sequencing depth ranged from 6.69× to 19.35×, and the coverage ranged from 85.49% to 96.43%. The population genetic structure and phylogenetic analysis of the 61 coffee accessions revealed four lineages, suggesting that they had at least four ancestral genetic components. Catimor exhibited the highest genetic diversity, while Geisha had the lowest genetic diversity. The selective sweep analysis indicated that among the selected genes in Catimor, disease-resistance genes were significantly more numerous than in other coffee varieties. The genome resequencing data and genetic markers identified from the 61 cultivated Arabica coffee materials provided insights into the genetic variation in Arabica coffee germplasm and facilitated extensive genetic research.

Mitochondrial genome insights into the spatio-temporal distribution and genetic diversity of Dendrobium hancockii Rolfe (Orchidaceae).

With its distinctive evolutionary rate and inheritance patterns separate from the nuclear genome, mitochondrial genome analysis has become a prominent focus of current research. Dendrobium hancockii Rolfe, a species of orchid with both medicinal and horticultural value, will benefit from the application of the fully assembled and annotated mitochondrial genome. This will aid in elucidating its phylogenetic relationships, comparative genomics, and population genetic diversity. Based on sequencing results from Illumina combined with PacBio and Nanopore, the mitochondrial genome map of D. hancockii was constructed. Comparative analysis was conducted from the perspectives of phylogeny across multiple species, selection pressure on protein-coding genes, and homologous segments. The population diversity of D. hancockii was analyzed using single nucleotide polymorphism (SNP) data from the mitochondrial genome and single-copy nuclear genes. This research constructed a circular mitochondrial map for D. hancockii, spanning 523,952 bp, containing 40 unique protein-coding genes, 37 transfer RNA genes, and 4 ribosomal RNA genes. Comparative analysis of mitochondrial genes from 26 land plants revealed a conserved gene cluster, "rpl16-ccmFn-rps3-rps19," particularly within the Dendrobium genus. The mitochondrial genome of D. hancockii exhibits a lower point mutation rate but significant structural variation. Analysis of 103 resequencing samples identified 19,101 SNP sites, dividing D. hancockii into two major groups with limited gene flow between them, as supported by population diversity, genetic structure analysis, principal component analysis, and phylogenetic trees. The geographical distribution and genetic differentiation of D. hancockii into two major groups suggest a clear phytogeographical division, likely driven by ancient geological or climatic events. The close alignment of mitochondrial data with nuclear gene data highlights the potential of the mitochondrial genome for future studies on genetic evolution in this species.

Development of a Spot Test (ST) Based on the Nucleic Acid Amplification Test (NAAT) for Ginseng Species Authentication

It is very challenging to distinguish between Panax ginseng (P. ginseng) and Panax quinquefolius (P. quinquefolius) based on their physical appearances. Therefore, a molecular test that can be performed in commercial settings is needed to address this challenge. We have selected a locus containing a single nucleotide polymorphism (SNP) site in the Dammarendiol-II Synthase (DS) gene to differentiate between P. ginseng and P. quinquefolius. An isothermal nucleic acid amplification test (NAAT) developed previously was adapted and used on a spot test (ST) format consisting of a sample pad, a nitrocellulose membrane, and an adsorbent pad. An ST with a colorimetric detection method has been developed with visual detection based on 20-nm gold nanoparticles. The presence of the target DNA will generate a red color signal, which is visible to the naked eye. The assay comprises the capture probes, constructed based on the DS gene of ginseng, and the ends of the probe sequences sit on the SNP site for the P. ginseng and P. quinquefolius sequences; the detection probe employed in the assay is tagged with gold. The NAAT amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT technique was used to authenticate two ginseng root samples, showing greater specificity ratios than our previous work.

Development and validation of a 5K low-density SNP chip for Hainan cattle

BackgroundThis study aimed to design and develop a 5K low-density liquid chip for Hainan cattle utilizing targeted capture sequencing technology. The chip incorporates a substantial number of functional single nucleotide polymorphism (SNP) loci derived from public literature, including SNP loci significantly associated with immunity, heat stress, meat quality, reproduction, and other traits. Additionally, SNPs located in the coding regions of immune-related genes from the Bovine Genome Variation Database (BGVD) and Hainan cattle-specific SNP loci were included.ResultsA total of 5,293 SNPs were selected, resulting in 9,837 DNA probes with a coverage rate of 85.69%, thereby creating a Hainan cattle-specific 5K Genotyping by Target Sequencing (GBTS) liquid chip. Evaluation with 152 cattle samples demonstrated excellent clustering performance and a detection rate ranging from 96.60 to 99.07%, with 94.5% of SNP sites exhibiting polymorphism. The chip achieved 100% gender coverage and displayed a heterozygosity rate between 14.20% and 29.65%, with a repeatability rate of 99.65–99.85%. Analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed the potential regulatory roles of exonic SNPs in immune response pathways.ConclusionThe development and validation of the 5K GBTS liquid chip for Hainan cattle represent a valuable tool for genome analysis and genetic diversity assessment. Furthermore, it facilitates breed identification, gender determination, and kinship analysis, providing a foundation for the efficient utilization and development of local cattle genetic resources.

Employing genome-wide association studies to investigate acid adaptation mechanisms in Listeria monocytogenes

Listeria monocytogenes is a critical foodborne pathogen known to develop adaptation traits in mildly acidic food processing environments. This study analyzed the genomic data of 49 strains derived from clinical and food sources, utilizing genome-wide association studies (GWAS) to explore the correlation between the genotypic and phenotypic traits of L. monocytogenes, thereby identifying the genetic determinants of its acid adaptation capability. The findings revealed no significant association between the collected acid adaptation genes and the bacterial growth parameters. The GWAS results indicated that numerous single nucleotide polymorphism (SNP) sites were significantly correlated with the growth parameters of L. monocytogenes in a pH = 5.0 acidic environment, whereas the associations diminished as the pH approached neutrality at pH = 6.7. Analysis and annotation of synonymous mutation loci revealed that non-synonymous mutations primarily impact function. The phenotypes pH = 5.0, ΔpH (5.0–5.5), SNPλ, and SNPμmax show the strongest associations with non-synonymous mutation loci. The genes lmo0017, lmo1173, lmo0794, and lmo2783 are significant non-synonymous mutation loci influencing acid adaptation. These genes play critical roles in intracellular pH regulation, cell wall synthesis and environmental response control, directly or indirectly affecting bacterial acid tolerance. Future research could leverage existing data combined with machine learning and causal inference methods to further dissect the genotype-phenotype causal relationships, identifying causative genetic factors that govern the acid adaptation in L. monocytogenes, providing insights for risk assessment and management strategies in food safety.

ALLELE-SPECIFIC QPCR METHOD FOR SNP AND INDEL GENOTYPING BASED ON FRET

The proposed Allele-specific q-PCR (ASQ) method for genotyping of single nucleotide polymorphism (SNP) and insertion-deletion polymorphism (InDel), based on Fluorescence resonance energy transfer (FRET). Initially named by its developers as Kompetitive Allele Specific PCR (KASP), this method is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer’s 3′ end base is positioned at the SNP site. The tool allows the design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available (http://primerdigital.com/tools/). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, translating into a greater likelihood of research success.

Fine genetic mapping and transcriptomic analysis revealed major gene modulating the clear stripe margin pattern of watermelon peel.

The peel stripe margin pattern is one of the most important quality traits of watermelon. In this study, two contrasted watermelon lines [slb line (P1) with a clear peel stripe margin pattern and GWAS-38 line (P2) with a blurred peel stripe margin pattern] were crossed, and biparental F2 mapping populations were developed. Genetic segregation analysis revealed that a single recessive gene is modulating the main-effect genetic locus (Clcsm) of the clear stripe margin pattern of peel. Bulked segregant analysis-based sequencing (BSA-Seq) and fine genetic mapping exposed the delimited Clcsm locus to a 19.686-kb interval on chromosome 6, and the Cla97C06G126680 gene encoding the MYB transcription factor family was identified. The gene mutation analysis showed that two non-synonymous single-nucleotide polymorphism (nsSNP) sites [Chr6:28438793 (A-T) and Chr6:28438845 (A-C)] contribute to the clear peel stripe margin pattern, and quantitative real-time polymerase chain reaction (qRT-PCR) also showed a higher expression trend in the slb line than in the GWAS-38 line. Further, comparative transcriptomic analysis identified major differentially expressed genes (DEGs) in three developmental periods [4, 12, and 20 days after pollination (DAP)] of both parental lines. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses indicated highly enriched DEGs involved in metabolic processes and catalytic activity. A total of 44 transcription factor families and candidate genes belonging to the ARR-B transcription factor family are believed to regulate the clear stripe margin trait of watermelon peel. The gene structure, sequence polymorphism, and expression trends depicted significant differences in the peel stripe margin pattern of both parental lines. The ClMYB36 gene showed a higher expression trend for regulating the clear peel stripe margin of the slb line, and the ClAPRR5 gene depicted a higher expression for modulating the blurred peel stripe margin in the GWAS-38 line. Overall, our fine genetic mapping and transcriptomic analysis revealed candidate genes differentiating the clear and blurred peel stripe patterns of watermelon fruit.

GCphase: an SNP phasing method using a graph partition and error correction algorithm

BackgroundThe utilization of long reads for single nucleotide polymorphism (SNP) phasing has become popular, providing substantial support for research on human diseases and genetic studies in animals and plants. However, due to the complexity of the linkage relationships between SNP loci and sequencing errors in the reads, the recent methods still cannot yield satisfactory results.ResultsIn this study, we present a graph-based algorithm, GCphase, which utilizes the minimum cut algorithm to perform phasing. First, based on alignment between long reads and the reference genome, GCphase filters out ambiguous SNP sites and useless read information. Second, GCphase constructs a graph in which a vertex represents alleles of an SNP locus and each edge represents the presence of read support; moreover, GCphase adopts a graph minimum-cut algorithm to phase the SNPs. Next, GCpahse uses two error correction steps to refine the phasing results obtained from the previous step, effectively reducing the error rate. Finally, GCphase obtains the phase block. GCphase was compared to three other methods, WhatsHap, HapCUT2, and LongPhase, on the Nanopore and PacBio long-read datasets. The code is available from https://github.com/baimawjy/GCphase.ConclusionsExperimental results show that GCphase under different sequencing depths of different data has the least number of switch errors and the highest accuracy compared with other methods.

Polycyclic aromatic hydrocarbon and its adducts in peripheral blood: Gene and environment interaction among Chinese population

BackgroundBenzo(a)pyrene (B[a]P) is the most widely concerned polycyclic aromatic hydrocarbons (PAHs), which metabolizes benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) in vivo to produce carcinogenic effect on the body. Currently, there is limited research on the role of the variation of metabolic enzymes in this process. MethodsWe carried out a study including 752 participants, measured the concentrations of 16 kinds PAHs in both particle and gaseous phases, urinary PAHs metabolites, leukocyte BPDE-DNA adduct and serum BPDE- Albumin (BPDE-Alb) adduct, and calculated daily intake dose (DID) to assess the cumulative exposure of PAHs. We conducted single nucleotide polymorphism sites (SNPs) of metabolic enzymes, explored the exposure–response relationship between the levels of exposure and BPDE adducts using multiple linear regression models. ResultOur results indicated that an interquartile range (IQR) increase in B[a]P, PAHs, BaPeq, 1-hydroxypyrene (1-OHP), 1-hydroxynaphthalene (1-OHNap) and 2-hydroxynaphthalene (2-OHNap) were associated with 26.53 %, 24.24 %, 28.15 %, 39.15 %, 12.85 % and 14.09 % increase in leukocyte BPDE-DNA adduct (all P < 0.05). However, there was no significant correlation between exposure with serum BPDE-Alb adduct (P > 0.05). Besides, we also found the polymorphism of CYP1A1(Gly45Asp), CYP2C9 (Ile359Leu), and UGT1A1(downstream) may affect BPDE adducts level. ConclusionOur results indicated that leukocyte BPDE-DNA adduct could better reflect the exposure to PAHs. Furthermore, the polymorphism of CYP1A1, CYP2C9 and UGT1A1affected the content of BPDE adducts.

The mutagenic effect of cold plasma on Medicago sativa L.

To explore the feasibility of using cold plasma as a mutagenesis breeding technology for forage crops, in this study we used the Medicago sativa L. cultivar, Zhongcao No. 3, as the experimental material. The effects of plasma treatments on Medicago sativa L. were analyzed through the use of plasma and activated water. Treatments with plasma and activated water inhibited plant height but promoted root growth. By creating a closed environment, adding a dielectric barrier plate, and combining these two treatment methods, the greatest impact can be had on the growth of Medicago sativa L. seeds. After treatment, the plant heights were approximately half those of the control group, and the root lengths were approximately 1.6 times those of the control group. Through emission spectroscopy, it was found that active particles such as O, NO2, and N2* were present and could be considered to have produced plasma-activated water through contact with the water surface, thus affecting the survival and growth of the seeds. Whole-genome resequencing (WGRS) was performed on the wild-type and selected mutants after treatment, with an average sequencing depth of 115.93×, an average genome alignment rate of 91.72 %, and an average genome coverage rate of 91.85 %. Various types of mutations were detected and annotated. After filtering, 7,822,324 SNP (single nucleotide polymorphisms) sites, 2,161,917 indel sites, 200,544 SV sites, 238 CNV (copy number variation) sites. The SNPs, indels (insertions/deletions), and SVs (structural variations) were mainly heterozygous, with heterozygosity rates of 87.13 %, 92.16 %, and 83.49 %, respectively. The CNVs were dominated by low copy numbers, accounting for 53.77 %. These results indicate that plasma treatment has significant effects on plant growth and genome of Medicago sativa L.

The addition of Psathyrostachys Huashanica Keng 6Ns large segment chromosomes has positive impact on stripe rust resistance and plant spikelet number of common wheat

BackgroundDeveloping novel germplasm by using wheat wild related species is an effective way to rebuild the wheat resource bank. The Psathyrostachys huashanica Keng (P. huashanica, 2n = 2x = 14, NsNs) is regarded as a superior species to improve wheat breeding because of its multi-resistance, early maturation and numerous tiller traits. Introducing genetic components of P. huashanica into the common wheat background is the most important step in achieving the effective use. Therefore, the cytogenetic characterization and influence of the introgressed P. huashanica large segment chromosomes in the wheat background is necessary to be explored.ResultsIn this study, we characterized a novel derived line, named D88-2a, a progeny of the former characterized wheat-P. huashanica partial amphiploid line H8911 (2n = 7x = 49, AABBDDNs). Cytological identification showed that the chromosomal composition of D88-2a was 2n = 44 = 22II, indicating the addition of exogenous chromosomes. Genomic in situ hybridization demonstrated that the supernumerary chromosomes were a pair of homologues from the P. huashanica and could be stably inherited in the common wheat background. Molecular markers and 15 K SNP array indicated that the additional chromosomes were derived from the sixth homoeologous group (i.e., 6Ns) of P. huashanica. Based on the distribution of the heterozygous single-nucleotide polymorphism sites and fluorescence in situ hybridization karyotype of each chromosome, this pair of additional chromosomes was confirmed as P. huashanica 6Ns large segment chromosomes, which contained the entire short arm and the proximal centromere portion of the long arm. In terms of the agronomic traits, the addition line D88-2a exhibited enhanced stripe rust resistance, improved spike characteristics and increased protein content than its wheat parent line 7182.ConclusionsThe new wheat germplasm D88-2a is a novel cytogenetically stable wheat-P. huashanica 6Ns large segment addition line, and the introgressed large segment alien chromosome has positive impact on plant spikelet number and stripe rust resistance. Thus, this germplasm can be used for genetic improvement of cultivated wheat and the study of functional alien chromosome segment.

Whole-Genome Resequencing Analysis of the Camelus bactrianus (Bactrian Camel) Genome Identifies Mutations and Genes Affecting Milk Production Traits.

Milk production is an important trait that influences the economic value of camels. However, the genetic regulatory mechanisms underlying milk production in camels have not yet been elucidated. We aimed to identify candidate molecular markers that affect camel milk production. We classified Junggar Bactrian camels (9-10-year-old) as low-yield (<1.96 kg/d) or high-yield (>2.75 kg/d) based on milk production performance. Milk fat (5.16 ± 0.51 g/100 g) and milk protein (3.59 ± 0.22 g/100 g) concentrations were significantly lower in high-yielding camels than those in low-yielding camels (6.21 ± 0.59 g/100 g, and 3.93 ± 0.27 g/100 g, respectively) (p < 0.01). There were no apparent differences in gland tissue morphology between the low- and high-production groups. Whole-genome resequencing of 12 low- and 12 high-yield camels was performed. The results of selection mapping methods, performed using two methods (FST and θπ), showed that 264 single nucleotide polymorphism sites (SNPs) overlapped between the two methods, identifying 181 genes. These genes were mainly associated with the regulation of oxytocin, estrogen, ErbB, Wnt, mTOR, PI3K-Akt, growth hormone synthesis/secretion/action, and MAPK signaling pathways. A total of 123 SNPs were selected, based on significantly associated genomic regions and important pathways for SNP genotyping, for verification in 521 additional Bactrian camels. This analysis showed that 13 SNPs were significantly associated with camel milk production yield and 18 SNPs were significantly associated with camel milk composition percentages. Most of these SNPs were located in coding regions of the genome. However, five and two important mutation sites were found in the introns of CSN2 (β-casein) and CSN3 (κ-casein), respectively. Among the candidate genes, NR4A1, ADCY8, PPARG, CSN2, and CSN3 have previously been well studied in dairy livestock. These observations provide a basis for understanding the molecular mechanisms underlying milk production in camels as well as genetic markers for breeding programs aimed at improving milk production.

Development and application of a 1K functional liquid chip for lactation performance in Bactrian camels.

The advancement of high-throughput, high-quality, flexible, and cost-effective genotyping platforms is crucial for the progress of dairy breeding in Bactrian camels. This study focuses on developing and evaluating a 1K functional liquid single nucleotide polymorphism (SNP) array specifically designed for milk performance in Bactrian camels. We utilized RNA sequencing data from 125 lactating camels to identify and select 1,002 loci associated with milk production traits for inclusion in the SNP array. The array's performance was then assessed using 24 randomly selected camels. Additionally, the array was employed to genotype 398 individuals, which allowed for population validation to assess the polymorphism of SNP sites. The SNP array demonstrated high overall SNP call rates (> 99%) and a remarkable 100% consistency in genotyping. Population validation results indicate that camels from six breeding areas in Northwest China share a similar genetic background regarding lactation functionality. This study highlights the potential of the SNP array to accelerate the breeding process of lactating Bactrian camels and provides a robust technical foundation for improving lactation performance.

Hairpin-Empowered Invasive Reaction Combined with Catalytic Hairpin Assembly Cascade Amplification for the Specific Detection of Single-Nucleotide Polymorphisms.

Single-nucleotide polymorphism (SNP) is widely used in the study of disease-related genes and in the genetic study of animal and plant strains. Therefore, SNP detection is crucial for biomedical diagnosis and treatment as well as for molecular design breeding of animals and plants. In this regard, this article describes a novel technique for detecting SNP using flap endonuclease 1 (FEN 1) as a specific recognition element and catalytic hairpin assembly (CHA) cascade reaction as a signal amplification strategy. The mutant target (MT) was hybridized with a biotin-modified upstream probe and hairpin-type downstream probe (DP) to form a specific three-base overlapping structure. Then, FEN 1 was employed for three-base overlapping structure-specific recognition, namely, the precise SNP site identification and the 5' flap of DP dissociation. After dissociation, the hybridized probes were magnetically separated by a streptavidin-biotin complex. Especially, the ability to establish such a hairpin-type DP provided a powerful tool that could be used to hide the cut sequence (CS) and avoid false-positive signals. The cleaved CS initiated the CHA reaction and allowed superior fluorescence signal generation. Owing to the high specificity of FEN 1 for single base recognition, only the MT could be distinguished from the wild-type target and mismatched DNA. Owing to the dual signal amplification, as low as 0.36 fM MT and 1% mutation abundance from the mixtures could be detected, respectively. Furthermore, it could accurately identify SNPs from human cancer cells, as well as soybean leaf genome extracts. This strategy paves the way for the development of more precise and sensitive tools for diagnosing early onset diseases as well as molecular design breeding tools.