CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 . However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid  but not type I , while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field‐applicable markers to better track resistance Africa‐wide.Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi‐omics approach, followed‐up with functional validation to show that a directionally selected haplotype of a cytochrome P450,  Together, these interventions are credited with a greater than 70% decrease in malaria burdens since their introduction  and all target‐site resistance genes sequences from An. funestus. We also included the entire genomic regions of the major quantitative trait locus (QTLs) associated with pyrethroid resistance which are the 120 kb BAC clone of the rp1 containing the major CYP6 P450 cluster on chromosome 2 (right arm), as well as the 113 kb BAC clone sequence for the rp2 chromosome 2 (left arm). A total of 1302 target sequences were included (with redundancy). Baits were designed using the SureSelect DNA Advanced Design Wizard in the eArray program of Agilent. The bait size was 120 bp for paired‐end sequencing using the “centred” option with a bait tiling frequency (indicating the amount of bait overlap) of “x3”.The sequence capture array was designed prior to the release of the 2.2An. funestus laboratory colonies (the FANG and FUMOZ) and field mosquitoes from Cameroon, Malawi and Uganda were utilised in this study. The FANG colony is a fully insecticide susceptible colony derived from Angola  and resistant  mosquitoes. The variation of exposure time was associated with the level of resistance in the population.Two For the PoolSeq experiment, there were sufficient individuals for two likely “susceptible” and three “resistant” replicates of 40 individuals each from Malawi and one “susceptible” and one “resistant” replicate also of 40 individuals from Cameroon. Genomic DNA was extracted per individual using the DNeasy Blood and Tissue kit (Qiagen) and individuals were species ID molecularly . For FST analyses, variant calling was carried out using varscan (2.4.4)  . Additionally, average FST for nonoverlapping sliding windows of 1000 SNPs was calculated in poolfstat for (i) all Malawi replicates combined and (ii) Cameroon versus Malawi replicates.The PoolSeq data was analysed in the R package rp1 locus according to prior research  and separate comparisons of “Malawi Dead (2×) versus Alive (3×) replicates” and “Cameroon versus Malawi” input to the Cochran‐Mantel‐Haenszel (CMH) test script “CMH‐test.pl”. The “Cameroon versus Malawi” comparison served as a positive control for this method as we expected to see a strong peak around the Wickham,  for pool2.3.2strandngs 3.4 (Strand Life Sciences). Alignment and mapping were performed using the “DNA alignment” option against the whole genome (version AfunF1) which was constructed into three chromosomes using synteny from An. gambiae  chromosomes using nucmer, from mummer v3.0. Nucmer alignment placed 46% (644 of 1392), totalling 217,255,185 bp or 96%  of the AfunF1 assembly (96%). For reference, the AfunF3 chromosomal assembly is slightly shorter in length at 210,975,222 bp as of VectorBase release 56. The 2L to 3R transposition between An. gambiae and An. funestus was accounted for by renaming of the ordered contigs and unplaced scaffolds were placed at the end of the ordered contigs. Aligned and mapped reads were used to create a DNA variant analysis experiment. Before variant detection, SNP preprocessing was performed to reduce false positive calls: (i) split read realignment of partially aligned split reads and full‐length reads with gaps introduced by initial alignment, (ii) local realignment to reduce alignment artefacts around indels, and (iii) base quality score recalibration to reduce errors and systematic bias.Initial processing and quality assessment of the sequenced data was performed as for the PoolSeq data and analysed using strandngs 3.4 and default parameters. A SNP multi sample report was generated for each sample. For each variant, its effect was predicted using the transcript annotation (version AfunF1.4). To identify SNPs significantly associated with permethrin resistance, two approaches were used. First, we used a differential allele frequency‐based approach where a variant was significant in relation to permethrin resistance if the supporting read of the SNP is found in 35%–100% of the alive mosquitoes (R) after permethrin exposure but present at low frequency in dead mosquitoes (1%–35%) (C) (R‐C comparison). Both sets of mosquitoes were also compared to the fully susceptible laboratory colony, FANG (S), with significant SNPs having frequency >35% but <35% in FANG (S) in R‐S and C‐S comparisons. This assumes that SNPs associated with resistance will be present at higher frequency in alive mosquitoes (>35%) but lower in dead (<35%). Additionally, a minimum threshold of five out of 10 individuals of the same category was required to include a SNP. The second approach assessed the significant association between each variant and permethrin resistance by estimating the unpaired t‐test unpaired of each variant between each comparison  and a Manhattan plot of –Log10 of p‐value created. A SNP frequency cutoff of three or more samples was applied for this approach.All variant types  were detected by comparing against the FUMOZ AfunF1 genome using the MAQ independent model implemented in CYP9K1 gene was analysed across Africa using the sureselect data. CYP9K1 polymorphisms were retrieved from the SNP Multisample report file generated through Strand NGS 3.4 for each population. Bioedit  (primers sequences: Table 2), 85.7 µM dNTP mixes, 0.34 µM each of forward and reverse primers, 0.015 U of Phusion HotStart II DNA polymerase (Fermentas) and 10.71 µl of dH20, 1 µl cDNA to a total volume of 14 µl. Amplification was carried out using the following conditions: one cycle at 98°C for 1 min; 35 cycles each of 98°C for 20 s (denaturation), 60°C for 30 s , and extension at 72°C for 2 min; and one cycle at 72°C for 5 min . PCR products were cleaned individually with QIAquick PCR Purification Kit (Qiagen) and cloned into pJET1.2/blunt cloning vector using the CloneJET PCR Cloning Kit (Fermentas). These were used to transform cloned E. coli DH5α, plasmids miniprepped with the QIAprep Spin Miniprep Kit (Qiagen) and sequenced on both strands using the pJET1.2F and R primers provided in the cloning kit.RNA was extracted using the PicoPure RNA isolation Kit  from three pools each of 10 permethrin‐resistant females from Tororo in Uganda. The RNA was used to synthesize cDNA using SuperScript III (Invitrogen) with oligo‐dT20 and RNAse H (New England Biolabs). Full length coding sequences of 2.4.2CYP9K1 was used to prepare the P450 for expression by fusing it to a bacterial ompA+2 leader sequence allowing translocation to the membrane following previously established protocols  into E. coli JM109. Membrane expression and preparation was performed as for , and the reaction started by vortexing at 1200 rpm and 30°C for 90 min. Reactions were quenched with 0.1 ml ice‐cold methanol and incubated for 5 min to precipitate protein. Tubes were centrifuged at 16,000 rpm and 4°C for 15 min, and 100 µl of supernatant and transferred into HPLC vials for analysis. All reactions were carried out in triplicate with experimental samples (+NADPH) and negative controls (−NADPH). Per sample volumes of 100 µl were loaded onto isocratic mobile phase (90:10 v/v methanol to water) with a flow rate of 1 ml/min, a wavelength of 226 nm and peaks separated with a 250 mm C18 column  on an Agilent 1260 Infinity at 23°C. For DDT, a solubilizing agent sodium cholate (1 mM) was added as described in Mitchell et al. (t‐test used to assess significance.Metabolism assays were conducted with permethrin (a type I pyrethroid insecticide), deltamethrin (a type II) and the organochlorine DDT. Assay protocols have been described previously  and aligned to the An. funestus F3 FUMOZ reference genome , Cochran‐Mantel‐Haenszel tests of association and divergence (FST) estimates were applied to all biallelic variant sites. These estimates were plotted as ‐log10 p‐values Manhattan plots for 1000 SNP sliding‐window global FSTs estimated in the R package poolfstat  but does include six cuticular genes and one cytochrome P450 (CYP301A1). The average FST in this region is 0.018 versus a background of FST of 0.0005 for chromosome 3. Although a substantial 32‐fold difference in FST averages, the absolute FST of this region was low. Furthermore, on inspection of the pairwise stF plots  and CYP9M2 (AFUN016005). There were also four cellular retinaldehyde binding proteins, three CRAL‐TRIO domain‐containing proteins and the remaining 31 genes lacked annotation. The peak around 97.7 Mb did not overlap any gene but is downstream of the 3′ end of gene AFUN003294 which encodes an ETS family transcriptional repressor. The only other visually concordant region of FST and −log10p‐values was observed towards the end of the X chromosome from positions 14.4 to 14.7 mb overlapping four genes including a homologue of “single‐minded” (AFUN005600) and an unannotated gene (AFUN020237) with homology to “stasimon”. Although the SNP with the highest CMH ‐log10p‐value is outside of this region at position 14,172,028.Sequence data obtained for each Fme Table   on chromosome 3, rp2  on chromosome 2, as well as rp3 (CYP9J11) on chromosome 3. Further evidence of the association of polymorphisms at rp1 with the resistance phenotype was the presence of the carboxylesterase gene (AFUN015787) located within this same genomic region. Two cuticle protein genes also presented abundant significant SNPs (AFUN009934 and AFUN009937) for all three comparisons. Two genes showed common amino acid changes for all three comparisons, the P450 CYP6AK1 (AFUN000518) and the UDP‐glucuronosyl transferase (AFUN004976). For the resistant versus field‐susceptible (R‐C), four significantly different nonsynonymous sites were identified in the immune response gene APL1C followed by three in carboxylesterase (AFUN015787) , 73 between resistant and FANG strain (R‐S), and 64 between Cameroon susceptible and FANG Figure . Most of7) Table . Further3.2.2rp1 QTL (CYP6P9a and the pseudo‐P450 AFUN008357) and rp2 (CYP6M1c and CYP6S2). As for Cameroon, three cuticle protein genes had the most significant SNPs between the three comparisons. For nonsynonymous substitutions, two genes showed common amino acid changes in all three comparisons, the P450 CYP6AK1 (AFUN000518) and a cuticle protein (AFUN009934). As for Cameroon, APL1C had the most nonsynonymous substitutions in the R‐C comparison  were found to be significant between Ugandan permethrin resistant and field‐susceptible mosquitoes (R‐C), with 85 between resistant and the FANG (R‐S) and 92 between Ugandan susceptible and FANG  and rp2 (CYP6M1b and CYP6N2) QTLs. The gene with the most significant SNPs is the P450 CYP6M1b. The gene with most nonsynonymous substitutions in the R‐C comparison alone was the P450 CYP6AK1 with three sites followed by the cuticle protein (AFUN009936) and cytochrome P450 CYP4H19 (AFUN001746) with two such sites each  Table , with 52ch Table . As in Ct‐test in each country which provided the following results.A second approach consisted of detecting significant SNPs by 3.2.4CYP304B1 on chromosome 2 (p = 1.7 × 10−5). Analysis of the 29 SNPs with Bonferroni‐corrected p < .001 revealed seven SNPs that were also detected with the frequency‐based filtering approach above (Table CYP6M1c (AFUN010919) on the rp2 QTL. Some of these 29 SNPs also belong to genes known to be significantly overexpressed in resistant mosquitoes such the P450 CYP315A1 and the glutathione S‐transferase GSTe3 , the chymotrypsin‐like protease (AFUN015111) (D476G) and the decarboxylase, AFUN007527 (V169L). A comparison of the 10 resistant Cameroon mosquitoes to FANG detected a lowest p‐value of 7.8 x 10−48 in a cuticular protein gene (AFUN004689). Regions with most SNPs between Cameroon and FANG were found in the rp1 QTL, a Zinc finger protein (AFUN015873) and a cluster of ABC transporter genes around ABCG4  on the X chromosome (p = 2.9 × 10−6) (Table CYP6AG1 (K262Q), or to immune response genes such as the transmembrane protease serine 13 (AFUN003078) (H61Y), serine protease 14 (AFUN000319) (N18H), chymotrypsin‐like elastase (AFUN015884) (T40K) and the C‐type lectin AFUN002085 (L63R). A comparison of the resistant mosquitoes from Uganda to FANG determined an intergenic substitution between the P450 gene CYP6P9a and a carboxylesterase gene (AFUN015793) in the rp1 QTL region as most significant p‐value of 2.28 × 10−50  Table , which i6) Table . Six non50 Table . Overall4 Figure . Interes3.2.6p‐value of 3.0 × 10−8)  Malawi mosquitoes the top significant was a synonymous substitution in the ABC transporter gene (ABCG4/AFUN007162) on chromosome 3 (8) Table . Nonsynots Table . Anotherts Table .3.33.3.1CYP9K1 was coexpressed with cytochrome p450 reductase (CPR) in E. coli, as expected from a good‐quality functional enzyme with a predominant expression at 450 nm and low P420 content  ‐difference spectrum was obtained when t Figure . Recombi3.3.2CYP9K1 exhibited contrasting activity towards permethrin (type I) and deltamethrin (type II). While no metabolic activity was observed with permethrin (0.47% depletion), CYP9K1 depleted 64%  of deltamethrin in 90 min (as determined by the disappearance of substrate [20 µM] after 90 min) compared to controls (with no NADPH)  Figure . For DDT3.43.4.1CYP9K1 gene  was analysed between 10 permethrin‐resistant and 10 susceptible mosquitoes from each of the three countries and from FANG. Analysis of these 70 mosquitoes revealed 137 substitutions and 72 haplotypes of the 2.7 kb gene‐body of CYP9K1 across the continent. When mosquitoes were analysed by country, however, a stark contrast was observed between Uganda and other samples. This was evident for most parameters assessed, notably the lower number of substitution sites in Uganda  versus Cameroon (123) and Malawi (42). A similar paucity of haplotypes was observed, with just five haplotypes in Uganda versus 38 and 29 in Cameroon and Malawi, respectively. Not surprisingly therefore, haplotype diversity in Uganda was also very low (0.19) in contrast to Cameroon (0.99) and Malawi (0.97) (Table A 2707 bp genomic fragment spanning the full 7) Table . Similar7) Table . Further7) Table . A simil7) Table  or the n7) Table . Analysi7) Table  reveals 3.4.2CYP9K1 sequences supported the high genetic diversity of this gene across the continent with several haplotypes clustering, mostly by their geographical origin   haplotype tree further highlighted the high polymorphism of ) Figure a–b. The n Figure a–b or thn Figure a–b.4CYP9K1 is a major driver of pyrethroid resistance in East African populations of the major malaria vector An. funestus.As malaria prevention still relies heavily on insecticide‐based interventions, it is essential to improve our understanding of the mechanisms driving resistance in malaria vectors to prolong the effectiveness of these tools by implementing suitable resistance management strategies. The present study used a multi‐omics approach, and one of these approaches detected that the cytochrome P450 4.1Drosophila , which was observed between Malawi and Cameroon. This is probably due to the fixation of selected alleles at these two P450 genes . This highlights the potential important role played by ABC transporters in the resistance to insecticides in general as reported recently  may reflect resistance or underlying differences in population structure. Despite this potentially confounding factor, we consider the significant signal at CYP9K1 between Uganda resistant and FANG as a signature of resistance. This is because country‐specific PoolSeq results and RNAseq that show a high overexpression of this gene only in Uganda  in E. coli followed by metabolism assays revealed that CYP9K1 metabolises the type II pyrethroid, deltamethrin. Recombinant CYP9K1 had a depletion rate similar to those observed for other cytochrome P450s genes in An. funestus including CYP6P9b , shown to be conferring pyrethroid resistance in the An. coluzzii population of Bioko Island  known to metabolize carbaryl  and a cis‐regulatory element (Knirps) are both interacting with the 5′ UTR of the P450 gene CYP321A8, leading to its upregulation of expression  and haplotypes (5 vs. 38 in Cameroon) identified. Strong selection on the CYP9K1 allele in Uganda is probably driven by the scale up of pyrethroid‐based interventions, notably the mass distribution of bed nets. Scale up of bed nets has been strongly associated with the escalation of pyrethroid resistance in southern African An. funestus populations  were able to increase the catalytic efficiency of these enzymes   , PRJEB24520 (Cameroon SureSelect), PRJEB47287  and PRJEB24506 (FANG SureSelect).Figure S1‐S6Click here for additional data file.Table S1‐S16Click here for additional data file.


Litopenaeus vannamei. To identify the genes associated with ammonia nitrogen tolerance, we performed an extreme phenotype genome-wide association study method (XP-GWAS) on a population of 200 individuals. The single nucleotide polymorphism (SNP) genotyping array method was used to construct the libraries and 36,048 SNPs were genotyped. Using the MLM, FarmCPU and Blink models, six different SNPs, located on SEQ3, SEQ4, SEQ5, SEQ7 and SEQ8, were determined to be significantly associated with ammonia nitrogen tolerance. By integrating the results of the GWAS and the biological functions of the genes, seven candidate genes  were found to be associated with ammonia nitrogen tolerance in L. vannamei. These genes are involved in cell transcription, cell division, metabolism, and immunity, providing the basis for further study of the genetic mechanisms of ammonia nitrogen tolerance in L. vannamei. Further candidate gene association analysis in the offspring population revealed that the SNPs in the genes zinc finger protein OZF-like (OZF) and homeobox protein Hox-B7-like (HOX7) were significantly associated with ammonia nitrogen tolerance trait of L. vannamei. Our results provide fundamental genetic information that will be useful for further investigation of the molecular mechanisms of ammonia nitrogen tolerance. These associated SNPs may also be promising candidates for improving ammonia nitrogen tolerance in L. vannamei.Ammonia nitrogen tolerance is an economically important trait of the farmed penaeid shrimp  Litopenaeus vannamei is naturally distributed along the Pacific coast of Central and South America. It is an important farmed penaeid shrimp that provides approximately 80% of the world’s total penaeid shrimp output  and ionic ammonia (NH4+) (Ammonia nitrogen in aquaculture water has two forms: non-ionic ammonia (NHa (NH4+) . Non-iona (NH4+) , causinga (NH4+) .L. vannamei aquaculture, it is important to improve the germplasm of L. vannamei and cultivate varieties with strong tolerance to high ammonia nitrogen. At present, selection of L. vannamei is generally based on population and family selection  method, the traits were found to have low heritability (0.13 and 0.17). L. vannamei larvae and found that the heritability of ammonia nitrogen tolerance traits of larvae was low.To ensure sustainable development of election . De DonaL. vannamei, using high-throughput next-generation sequencing transcriptome data, 96,040 SNP markers of L. vannamei were successfully identified. Several studies have identified transcriptomic changes and differentially expressed genes in L. vannamei after high ammonia nitrogen stress. L. vannamei based on comparative transcriptomic and metabolomic analyses of ammonia-tolerant and ammonia-sensitive L. vannamei families. L. vannamei using marker-trait correlation analyses.Traditional breeding methods have long cycles and limited genetic progress , especiaL. vannamei and mapped a QTL associated with ammonia nitrogen tolerance. Due to limitations of marker density, QTL analysis only identified one gene, LOC113809108, annotated as the ATP synthase g subunit.Previous transcriptomic and metabolomic analyses have focused on the genetic bases of ammonia tolerance in shrimp. Quantitative trait loci (QTL) linkage mapping is an essential method for identifying relevant genes. Ictalurus punctatus) (Cyprinus carpio) (Larimichthys crocea) , found multiple significant loci and speculated that these two traits were controlled by micro-efficient polygenes. However, no study has investigated SNPs associated with ammonia nitrogen tolerance in L. vannamei using a GWAS.Genome-wide association study (GWAS) can be used to identify functional genes in a genome. Until now, genes related to economic traits of important aquatic animals have been identified by GWAS, including catfish (nctatus) , carp (C carpio) , and lar crocea) . Wang  . XP-GWASL. vannamei using an XP-GWAS. Six different analysis methods were used to identify loci significantly associated with ammonia tolerance traits in L. vannamei and successfully mapped to potential genes associated with ammonia nitrogen tolerance. Finally, we confirmed that the use of XP-GWAS is feasible in L. vannamei, and is a cost-saving approach to genotyping.In this study, we assessed ammonia nitrogen tolerance traits in All animal experiments were carried out following the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. The animal protocols were approved by the Animal Ethics Committee of Guangdong Ocean University . Shrimp were reared by the shrimp breeding company Guoxing Aquaculture Science and Technology Co., Ltd. in Zhanjiang City, Guangdong Province, P.R. China. In March 2020, we established 38 different families, and each family was cultured separately. After 1 month, 10 families were randomly selected and we moved 30 individuals from each family into the same pool for common environment breeding, each family was fluorescently labeled by visible implant elastomer. After 2 months of culture, 20 shrimps from each family were randomly selected for the high ammonia nitrogen stress experiment.A population of 200 individuals was used for the challenge experiment . An ammonia nitrogen stress concentration of 93 mg/L was used based on the results of the semi-lethal high ammonia nitrogen concentration of 96 h in preliminary experiments. For stress experiments, 20 shrimps from each family were placed in a 3 × 3 m test pool. After 7 days of suspension, the concentration of ammonia nitrogen was adjusted to 93 mg/L by adding analytical grade ammonium chloride.During the experiment, water was replaced daily, the water temperature was maintained at 26 ± 2°C, the pH was maintained at 8.1 ± 0.2, the salinity was maintained at 30 ± 1, and the dissolved oxygen was maintained at 6–8 mg/L. Deaths were observed each hour and the survival time was recorded. Shrimp were considered dead if they lay on the pool floor with no touch response. After the time of death was recorded, the muscle tissues of each shrimp were preserved individually in absolute ethanol for subsequent DNA extraction. The experiment was concluded until all shrimp had died.n = 120) with the EasyPure® Genomic DNA Kit (TRANSGEN BIOTECH) in the Nanhai Economic Shrimp Breeding and Culture Laboratory. The quality and concentration of extracted DNA was determined by agarose testing and a NanoDrop 2000 UV spectrophotometer (Thermo Fisher). DNA was stored at −20°C until further use.To reduce costs and improve efficiency, we chose extreme individuals for experiments based on Resistance to high ammonia nitrogen was measured as the number of hours to death. We used the Genobaits® Prawn 40K Panel developed by MOLBREEDING®  as the genotyping method: it integrates the relevant SNPs including WSSV resistance, ammonia nitrogen tolerance, and feed conversion efficiency, SNPs were filtered using plink v1.9 . SNPs wep-value) of each SNP across the genome was calculated to illustrate the GWAS results. The threshold p-value for genome-wide significance was calculated using Bonferroni correction based on the estimated number of independent markers. Considering the marker number and the number of genotyping populations, loci that ranked in the top 200 in the results of all GWAS model analyses were finally included as significant SNPs.We used six different models to implement GWAS, including General Linear Model (GLM), Mixed Linear Model (MLM), Compressed MLM (CMLM), Settlement of MLM Under Progressively Exclusive Relationship (SUPER), Fixed and random model Circulating Probability Unification (FarmCPU) and Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (Blink). All the GWAS analyses used the GAPIT R package . MLM canL. vannamei (NCBI assembly ID: ASM378908v1). To do this, we defined a region that was expected to contain causative genes. Because the SNPs detected in this study are on different scaffolds, we assumed they have independent effects on investigated traits, so we defined an independent region that 300 kb upstream and downstream of each SNP to search candidate genes. To annotate the genes, we used the BLAST + tool to generate a sequence alignment using the non-redundant protein database (NCBI), the reference genome used in this study is ASM378908v1.We annotated candidate genes around the significant SNPs based on the reference genome of L.vannamei, we performed High ammonia nitrogen stress on a progeny population with 150 shrimp. Based on the results of the semi-lethal high ammonia nitrogen concentration of 48 h in preliminary experiments, ammonia nitrogen stress concentration was set to 383 mg/L. SNPs of gene HOX and OZF were examined by direct sequencing of PCR products , information of primers designed for this procedure is listed in 2-test using SPSS version 24.0. Significance level for the analysis was specified at p < 0.05.To validate the association of SNPs with high ammonia nitrogen tolerance traits in 50) was 98 h. Statistical analysis revealed that the mortality data of L. vannamei were approximately normally distributed. Besides, the range of survival time in the sensitive and tolerant groups was 14.5–88 h and 110–136 h, respectively.The average body weight and body length of individual shrimp were 5.24 ± 2.07 g and 77.49 ± 10.85 mm, respectively. Mortality was observed beginning at 14.5 h after the start of stress and lasted until 136 h , the medp < 0.00001). After filtering, 36,048 quality compliant SNP loci were retained for GWAS analysis. We evaluated these eight models for false positives and false negatives based on the Q-Q plots . The six associated SNPs are scattered over SEQ5, SEQ4, SEQ3, SEQ7, and SEQ8.We assessed the SNP quality and excluded 2,845 SNPs with a call rate that lower than 90%, 5,569 SNPs with minimum allele frequency that lower than 0.01, and 945 SNPs with significant deviation from Hardy–Weinberg equilibrium  was 76.5 h. After genotyping these SNPs of two genes in the progeny population, SNPs of HOX7 and OZF were significantly associated with ammonia nitrogen tolerance  simultaneously were considered to be significantly associated with high ammonia nitrogen tolerance in this study. MLM is one kind of single SNP analysis methods, some studies have found that multilocus models performed better than single locus models (Generally, Bonferroni correction is used to avoid false positives. However, the Bonferroni-corrected  in GWAS . In the quencing . Howevers models , the resL. vannamei, triggering gene transcription, replication, and the immune response. Zinc finger protein OZF is a member of zinc finger family and function to bind DNA, RNA, protein, and lipid substrates (L. vannamei. HOX7 is a member of the HOX gene family, which encodes transcription factors that regulate cell division (The SNPs related to ammonia nitrogen tolerance found in this study mainly have functions related to cell transcription, cell division, metabolism, and immunity. Ammonia nitrogen tolerance may stimulate physiological reactions in bstrates . Zuo et division . HOX gendivision . UPF2 isdivision . VPS11, division . The TMEdivision . HoweverL. vannamei. After analysis, the SNP site had a significant effect on the growth characteristics of L. vannamei. L. vannamei, found that one SNP, g.155 A>G, belonging to the synonymous mutation Gln→Gln, was screened in the CAT sequence and significantly associated with low hemolytic oxygen tolerance traits in L. vannamei. In our study, the SNP of HOX7 is a synonymous mutation Lys→Lys, the SNP of OZF is a nonsynonymous mutation Leu→Pro. As several studies have described, nonsynonymous SNPs change their encoded amino acid sequence, thus affecting gene functions and interactions (In this study, one SNP A>C in the intron of gene HOX7 and one SNP T>C in the exon of gene OZF were screened. ractions . Synonymractions . In addiractions , so in tp-values of these SNPs did not reach the Bonferroni-corrected p-value threshold in MLM, the results were similar to those obtained by L. vannamei against Vibrio parahaemolyticus, which involved analysis of whole individuals.Compared with methods such as transcriptome analysis and QTL mapping, GWAS are costly because they require SNP genotyping across the genome of hundreds of individuals . The numL. vannamei. Thus, the implementation of MAS may not be successful. Similar to our findings, Piscirickettsia salmonis; they concluded that it was due to a micro effect polygenic trait with low p-values for SNPs associated with this trait, suggesting that genomic selection will be a more efficient approach to such traits. Molecular information from genotyped SNPs may be incorporated in breeding programs through the application of genomic selection (Quantitative traits are often influenced by multiple genes with small effects, so the benefit of conducting MAS depends on the effect of each SNP . Previouelection , where eelection . Such an


The major soy protein QTL, cqProt-003, was analysed for haplotype diversity and global distribution, and results indicate 304 bp deletion and variable tandem repeats in protein coding regions are likely causal candidates.Glyma.20G085100 are strongly correlated with the high-protein phenotype and likely represent causal variation. Structural variation has previously been found in the same gene, for which we report the global distribution of the 304 bp deletion and have identified additional nested variation present in high-protein individuals. Mapping variation at the cqProt-003 locus across demographic groups suggests that the high-protein haplotype is common in wild accessions (94.7%), rare in landraces (10.6%) and near absent in cultivated breeding pools (4.1%), suggesting its decrease in frequency primarily correlates with domestication and continued during subsequent improvement. However, the variation that has persisted in under-utilized wild and landrace populations holds high breeding potential for breeders willing to forego seed oil to maximize protein content. The results of this study include the identification of distinct haplotype structures within the high-protein population, and a broad characterization of the genomic context and linkage patterns of cqProt-003 across global populations, supporting future functional characterization and modification.Here, we present association and linkage analysis of 985 wild, landrace and cultivar soybean accessions in a pan genomic dataset to characterize the major high-protein/low-oil associated locus cqProt-003 located on chromosome 20. A significant trait-associated region within a 173 kb linkage block was identified, and variants in the region were characterized, identifying 34 high confidence SNPs, 4 insertions, 1 deletion and a larger 304 bp structural variant in the high-protein haplotype. Trinucleotide tandem repeats of variable length present in the second exon of gene The online version contains supplementary material available at 10.1007/s00122-022-04045-8. Glycine max (L.) Merr.)  and exotic landrace populations that still harbour genomic variation at loci that are of value to breeders  may be responsible for the phenotypic effect (Fliege Protein content in soybean is associated with a major quantitative trait locus (QTL); cqProt-003 on chromosome 20 that underwent a genetic bottleneck during domestication with selection for oil, at the cost of fixing a low-protein phenotype across modern breeding populations . Reads were realigned by GATK  was compared to the whole reference genomes of 13 legume species and 2 outgroup Rosids species: Glycine max , Phaseolus lunatus {Garcia, 2021 #554}, Vigna unguiculata {Lonardi, 2019 #555}, Vigna radiata {Kang, 2014 #556}, Vigna angularis {Yang, 2015 #557}, Lupinus albus {Hufnagel, 2020 #558}, Lotus japonicus {Sato, 2008 #559}, Cicer arietinum {Varshney, 2013 #560}, Medicago truncatula {Pecrix, 2018 #561}, Pisum sativa {Kreplak, 2019 #562}, Vitis vinifera (accessed in release 52 at http://ftp.ensemblgenomes.org) {Jailon, 2007 #563} and Arabidopsis thaliana {Lamesch, 2012 #564}. Nucleotide-Nucleotide BLAST 2.12.0 + {Zhang, 2000 #578} was used with parameters -outfmt 6 -word_size 11. After running with the entire Glyma.20G085100 genic region as the query, the results were classified as part of the 304 bp SV region if Qrystart > 4000 and Qryend > 4400. Results with an E-value less significant than E-10 and bitscore < 149 were filtered out.The Wm82.a4.v1 reference fasta sequence for the Glyma.20G085100 genic region using mosdepth v0.2.6 (-b 1 -Q 10) ; the most significant site was at 31,604,127 bp with a p-value of 3.15E-07, with 13 of the top 20 sites all within the 104 kb range of 31,604,126–31,708,981 bp (Table S1). GWAS for oil content was conducted on the same dataset using the FarmCPU method and 945 individuals (Table S3). A significant site corresponding to decreased seed oil (p = 3.4e-09) was detected at 31,687,912 bp, at which the 183 individuals with the alternate allele displayed a mean oil content 32.7% lower (6.1 g oil per 100 g seed weight) than the 743 individuals conforming to the reference.A genome-wide association study (GWAS) for protein content was conducted on chromosome 20 using 985 accessions, including 131 wild lines, 708 landraces, 44 old cultivars and 102 modern cultivars, for which phenotypic data was available from the UWA SoyPan dataset (soybase.org). GWAS was tested using different combinations of unfiltered, filtered, unimputed and imputed data . The results using the FarmCPU method and the unimputed dataset indicate a highly significant site associated with protein content located at position 31,649,589 in the Wm82.a4.v1 reference genome , though R2 linkage is only present above 0.8 across a 330 kb region from 31,590,913 to 31,920,542 bp, and above 0.91 across a 173 kb region from 31,604,127 to 31,777,346 bp. D' for sites that have an R2 greater than 0.7 decay precipitously as distance increases from the site, before D' plateaus below 0.967 beyond the same range designated by the 0.91 R2 cut-off: 31,604,127 to 31,777,346 bp . The 173 kb region is hereafter defined as the genomic context of cqProt-003 within which SNPs are tightly linked with the GWAS-SNP, and therefore the range within which causal variants are likely contained.Linkage with the GWAS-SNP assessed using R2 ≥ 0.91) across the region . Within the 173 kb region are three gene structures: Glyma.20G084500 (pre-mRNA-processing factor 17-like isoform X1), Glyma.20G085250 (unknown protein), and Glyma.20G085100 , potentially indicating structural diversity, problematic read alignments or mis-assembly in the Wm82.a4 reference.The 173 kb linkage region spans seven significant blocks (> 10 SNPs) of linkage defined by the confidence interval approach , with a mean increase of 3.32 g/100 g seed weight for landrace individuals with the alternate allele for marker group M01; conversely, oil content displays a mean decrease of 2.66 g/100 g seed weight . These high-protein/low-oil haplotypes (B-G) are comprised mostly of wild and landrace individuals, compared to haplotype group A which is predominantly domesticated  that remains consistent with the high-protein phenotype seen in other haplotype populations that contain a mixture of wild and landrace individuals  that contained different combinations of six groups of markers Fig. , which i−3 among individuals with the alternate allele for the M01 marker group, compared to π = 3.24E−4 with the reference allele. This represents a significant drop of diversity in the 173 kb region for the population with the M01 reference allele that possess an average diversity (π) of 1.74E−3 across the entire genome. In contrast, the population of individuals with the alternate allele at M01 has only slightly elevated genetic diversity in the region relative to the mean across the whole genome (π = 3.60E3). Increased genetic diversity in the region appears to be contained in the predominantly wild populations; of the alternate M01 haplotypes, only the landrace group C possesses lower levels of diversity consistent with the primarily domesticated haplotypes A and B (Table S7). Furthermore, divergence in the 173 kb region between groups A-C is far lower than groups D-G with the alternate M04 marker group , which includes 157 SNPs, though has a slightly lower association with seed protein than M01  with the GWAS-SNP, warranting their inclusion in the high-protein haplotype (Table S9). Of the 39 small- and medium-sized variants classified as in the cqProt-003 haplotype, 33 were in intergenic regions and five were adjacent to genes (Table S9). There were no variants detected from the haplotype in coding regions for two of the three genes in the 173 kb region: Glyma.20G084500 and Glyma.20G085250. However, for the final gene, Glyma.20G085100.1, a multiallelic insertion at 31,727,019 bp is located in the second exon (Table S10). The mutations at this locus appear to be predominantly trinucleotide tandem repeat expansions of ‘CAA’, which are conservative in-frame insertions of Asparagine; however, there are four individuals who reported either disruptive in-frame insertions or frameshift variants (Table The haplotype of 40 variants in linkage with the GWAS-SNP (Table S9) was further characterized. Four insertions and a single deletion were also found to be in tight linkage R2 > 0.8 with theGlyma.20G085100 gene, with a 304 bp deletion (relative to Wm82) . The deletion spans from 31,728,619 to 31,728,923 bp, truncating 94.8% of the fourth exon, and 56.4% of the first of two 3' UTRs (Table S10). This deletion is present in 194 lines that have a mean protein content 7.9% greater than those with the reference sequence (Table S6), consistent with the high-protein haplotype. There is significant overlap between the trinucleotide insertions and the 304 bp deletion: 152 of the 194 individuals with the 304 bp deletion possess a homozygous trinucleotide insertion, and all but one individual with a homozygous trinucleotide insertion had the 304 bp deletion (Table S6). The 304 bp deletion is linked to the GWAS-SNP with R2 = 0.918 and D' = 0.958, and it is most tightly linked with the SNP at position 31,680,574 bp . The start position for the 304 bp deletion, 31,728,619 in Wm82.a4.v1 . This dataset similarly contains trinucleotides with 3–8 ‘CAA’ inserted repeats across 5.3% of individuals; however, they do not identify the remaining six other rare alternate alleles (Table Glyma.20G085100 (Table S11). The W05, W01, W02, W03 and SoyC12 accessions contain a 318 bp (302 bp for W03) deletion relative to ZH13 and WM82 cultivars, significantly truncating the upstream portion of the fourth exon for ZH13_20G075300/Glyma.20G085100 (Table S10-11). Individuals with 318 bp deletions in the Liu et al. dataset are present in our data with 304 bp deletions; however, no deletion in the Glyma.20G085100 gene was identified for W05 using the same genomic data from Liu et al. aligned to the Wm82.v1.a4 reference.No significant (MAF > 0.05) InDel sites were identified in the syntenic cqProt-003 region from a Korean collection of 855 soybean individuals . In addition, more distant homologs for the 304 bp sequence were identified in abundance across the common pea (Pisum sativum) and barrel medic (Medicago truncatula) genomes, and less frequently in the chickpea (Cicer arietinum) genome (Table S12). Excluding the 304 bp region, the rest of the Glyma.20G085100 gene possessed fragments with homologous sequences in all of the 13 legumes aligned against, though no homologous sequences in Arabidopsis thaliana and grape (Vitis vinifera) (Table S13). The previously reported paralog for Glyma.20G085100 gene, Glyma.10G13440 {Brown, 2021 #549}, does not contain a homologous sequence for the 304 bp SV (Table S13); whilst the second exon of Glyma.10G13440 does contain six CAA repeats, starting at position 36,191,376 in Wm82.a4.v1, we could not identify InDel variation across the population at this locus indicative of expansion.Local alignment searches for the nucleotide sequence of the gene containing both the trinucleotide insertions and SV, Whole-genome resequencing datasets from global domesticated and wild soybean populations have provided an opportunity for the detailed characterization of the cqProt-003 protein-associated region, to identify patterns of variation and identify candidate causative variants. We have refined the genomic interval underlying significant protein and oil variability to a 173 kb region. The region defined in our study sits in the centre of a broader 550 kb ‘fourth block’ previously identified using USDA 50 k-SNP chip genotyping  appear to display variation only when the alternate alleles are present in the candidate marker group that is most strongly associated with protein content (M01). The lack of variation in individuals containing the Williams 82-like alleles for the M01 group appears to be the result of a strong domestication bottleneck for the high oil/low-protein phenotype in domesticated lines. Further functional characterization of the variation across the different haplotype populations (B-G) is needed to explore how different allelic combinations modulate the effect of cqProt-003 on seed compositional traits.Glyma.20G085100, providing additional evidence that this gene is likely involved in the high-protein phenotype. We located a 304 bp SV in the fourth exon that is associated with the low-protein phenotype. This represents an insertion in modern lines relative to wild progenitor populations, and this insertion is likely to modify the expression and/or function of the Glyma.20G085100 gene. Previous research that first identified this SV focused in the structural variation between two lines, PI468916 (HNY.50) and Williams 82, before screening a population of 302 individuals using PCR with markers targeting a CCT domain  are likely due to different assemblies, Wm82 compared to ZH13  as is widely found in low-protein individuals (five). The lack of copy number variation at the repeat locus in Glyma.10G13440 suggests this may represent a threshold for mutability, providing additional evidence that the capacity for expansion has been lost for Glyma.20G085100 in most domesticated soy populations. Our finding may represent the first case of causal short tandem repeat variation in a coding region underlying phenotypic effect in soybean. We hypothesize that prior to domestication there may have been balancing selection to maintain a higher number of trinucleotide repeats that may be needed for a function that results in a high level of protein or oil. The repressed repeat count seen across most domesticated populations may be a significant loss for the adaptive potential of this gene that likely underpins a large degree of trait variability.We identified two candidate variants located in n Fliege . They coGlyma.20G085100 gene, demand validation for impacts on plant phenotype. Furthermore, a key gap in our understanding still remains regarding the role in seed morphology and development of the additional proteins produced by individuals with the high-protein haplotype. A deeper understanding of the functional pathways involving the Glyma.20G085100 gene could open the door to further optimization of the cqProt-003 locus for agronomic gain using gene editing technology such as CRISPR. These findings draw attention to the lack of diversity in modern breeding lines at this important locus for seed composition, and the potential to exploit the natural variability remaining in exotic landraces and wild populations (haplotype populations B-G) to provide breeders with additional tools for producing protein-rich soy in a world with increasing nutritional demands.We have refined the linked region associated with the high-protein phenotype, defined haplotype structures within this region, thoroughly examined the high-protein haplotype, assessed the untapped variability within the population with this haplotype and identified likely causal genomic candidates. The variants in the high-protein haplotype act as high confidence markers for the high-protein haplotype, which can support low-cost genomic inference of the cqProt-003 trait. The causal candidates, trinucleotide insertions and structural variation in the Supplementary file1 (XLSX 356 kb)Supplementary file2 (DOCX 1828 kb)Below is the link to the electronic supplementary material.


It is well-established that child maltreatment practiced by parents is associated with adolescent aggression. Emerging evidence has suggested that higher levels of mindful parenting are associated with fewer negative parenting practices. However, the relationships among mindful parenting, child maltreatment, and adolescent aggression remain unclear.To examine the association between mindful parenting, child maltreatment, and adolescent aggressive behavior among Chinese parent-adolescent dyads.Survey data from 554 Chinese parent-adolescent dyads were used for the analysis. Parents reported mindful parenting, and adolescents reported three forms of child maltreatment  by their parents and aggressive behavior. Path models were used to analyze whether higher levels of mindful parenting were associated with decreased likelihood of parent-to-adolescent maltreatment that were further related to lower levels of adolescent aggression.interaction with full attention and compassion and acceptance) were associated with lower likelihood of physical abuse and psychological aggression, which were related to decreased levels of adolescent aggressive behavior. Stratified analyses by parent gender showed that the path from interaction with full attention to adolescent aggression through psychological aggression was also significant or marginally significant in both mother-adolescent and father-adolescent dyads. Stratified analyses by adolescent gender demonstrated that the paths from interaction with full attention to physical abuse and psychological aggression were significant, which were also significantly correlated with adolescent aggression among male adolescents, whereas the mindful parenting-child maltreatment-adolescent aggression paths were not significant among female adolescents.Mindful parenting and its two factors (i.e., The findings contributed to the existing literature by assessing mindful parenting as a multifaceted construct and exploring the gender differences in the relationships. Gender-tailored interventions to improve mindful parenting, specifically focusing on the parents’ ability of interacting with adolescents providing full attention, compassion, and acceptance may work towards preventing child maltreatment and promoting adolescent behavioral health.The online version contains supplementary material available at 10.1186/s13034-022-00507-5. Aggression is one of the most prevalent and destructive behaviors among adolescents . AdolescMindful parenting can be an important parental factor that may be protective against child maltreatment. As a construct of growing interest, mindful parenting is defined as the parenting style that involves paying full attention in parent–child interactions, adopting an attitude of non-judgmental acceptance and compassion toward the self as a parent and the child, developing emotional awareness of the self and the child, and exerting self-regulation in the parenting relationship , 13. MinIn line with this, Duncan et al. proposed a theoretical model of mindful parenting’s effects, which depicted that mindful parenting can promote parents to adopt reasonable parenting practices and establish positive parent–child interactions, and in turn, such positive parent–child interactions ultimately decrease negative outcomes for children . SimilarTo our best knowledge, two empirical studies tested this relationship empirically. Both studies reported that higher mindful parenting was associated with fewer negative parenting practices , which were related to lower levels of internalizing and externalizing symptoms among adolescents , 18. HowBased on the existing theoretical models of mindful parenting , 17, theIn addition, considering that mothers and fathers exhibited different mean levels of mindfulness in parenting , and thaThe study used data from a large survey conducted in 2018, which purposively recruited adolescents from a local vocational secondary school located in a disadvantaged county in eastern China to investigate the relationship between childhood adversities and adolescent mental and behavioral health and its possible psychosocial mechanisms. According to the local and national statistical yearbook of 2018 , 30, thet =  − 6.20, p < 0.001), more likely to be female  and only child , less likely to experience paternal physical abuse , and exhibit a lower level of aggression , and were included in the analysis. Of the 554 dyads, 228 were mother-adolescent and 326 were father-adolescent dyads. A comparison between the adolescents included in the study and those excluded showed that the included adolescents were older .Adolescents reported their aggressive behavior using the Aggressive Behavior Subscale of the Youth Self Report . The 17-item Aggressive Behavior Subscale was rated on a 3-point scale . Higher scores indicate more aggressive behavior. The YSR has been validated in Chinese adolescents, showing acceptable to good reliability , 33. In interaction with full attention , compassion and acceptance , self-regulation , and emotional awareness of child , which were different from the factor structure of the original 31-item IM-P. This was because during the validation process by Pan et al., one item was deleted since it was easily misunderstood with Chinese parents, and six items mainly referring to compassion and acceptance of self were further deleted due to poor corrected item-total correlation and item discriminability : t =  −3.00, p = 0.003; father-adolescent dyads [FA]: t = − 2.92, p = 0.004) or psychological aggression  reported higher levels of aggressive behavior regardless of perpetrator. Aggression score did not differ significantly among adolescents with and without neglect. Parents perpetrating physical abuse reported lower scores on the total scale of mindful parenting , the compassion and acceptance subscale , and the interaction with full attention subscale . Parents perpetrating psychological aggression reported lower scores on the interaction with full attention subscale  than their counterparts. Neglectful parents did not differ from non-neglectful parents on mindful parenting and its factors.Compared to non-maltreated counterparts, adolescents who experienced physical abuse . Analyses among the father-adolescent dyads revealed that physically abusive fathers exhibited low levels of mindful parenting , interaction with full attention , and compassion and acceptance , and psychologically aggressive fathers showed lower levels of interaction with full attention . See Table Analyses among the mother-adolescent dyads showed that physically abusive mothers had lower levels of interaction with full attention subscale score , whereas its correlations with the total score for mindful parenting and the other two subscale scores were not statistically significant .As presented in Table OR = 0.59; 95% CI = ; p = 0.012], which was related to low levels of adolescent aggressive behavior  = 0.54, p = 0.030). Repetitive models using scores of different factors of mindful parenting as the exogenous variable showed that higher interaction with full attention subscale score was negatively associated with both psychological aggression  and physical abuse , which were positively related to lower levels of adolescent aggression . Additionally, higher compassion and acceptance subscale score was related to low levels of adolescent aggression through decreased likelihood of physical abuse , and psychological aggression . In addition, among the father-adolescent dyads, compassion and acceptance also related to adolescent aggression through psychological aggression . See Table Among both the mother-adolescent dyads and father-adolescent dyads, the path from interaction with full attention score was related to lower risks of physical abuse  and psychological aggression  and further lower levels of adolescent aggression  among male adolescents. The mindful parenting-child maltreatment-adolescent aggression paths were not significant among female adolescents. See Additional file High parental interaction with full attention and compassion and acceptance factors were negatively associated with physical abuse and psychological aggression, which were positively related to adolescent aggression,. The pathway from higher interaction with full attention to lower levels of adolescent aggression through reduced risks of psychological aggression was also evident in both mother-adolescent and father-adolescent dyads. In addition, among father-adolescent dyads, compassion and acceptance was related to adolescent aggression through psychological aggression. Stratified analyses by adolescent gender demonstrated that the relationship between interaction with full attention and adolescent aggression was mediated by physical abuse and psychological aggression among male adolescents, whereas the mindful parenting-child maltreatment-adolescent aggression paths were not significant among female adolescents. To the best of our knowledge, this is one of the first studies to examine the relationship of mindful parenting and its factors with child maltreatment and adolescent aggression, using data from parent-adolescent dyads.This study revealed that after adjusting adolescent sociodemographic characteristics, mindful parenting, especially its interaction with full attention factor was significantly and negatively correlated with aggression. Further path analysis also found that after adjusting adolescent sociodemographic covariates, parents with higher levels of mindful parenting, especially interaction with full attention and compassion and acceptance, were less likely to engaged in physical abuse and psychological aggression towards adolescents. Furthermore, non-maltreated adolescents exhibited low levels of aggressive behavior, which supported the models developed by Duncan et al. [Although we did not find significant correlation between the total score of mindful parenting and aggression in the zero-order correlation analysis, the n et al.  and Ahemn et al. , and wasn et al. , 18. When et al. , which mn et al. . Furthern et al. , which fn et al. . In addin et al. , and, heNevertheless, mindful parenting and its different factors were not significantly associated with neglect. In this study, the classification of neglect was driven by the item “showing or telling me that he/she loves me” . Culturally, Chinese parents tend to refrain from showing direct affection for their children, and choose to express their affect through providing instrumental support and making sacrifices , 46. Expself-regulation factor of mindful parenting and child maltreatment, indicating that not all mindful parenting factors are equally important in reducing child maltreatment. This is, to some extent, consistent with previous findings that different factors of mindful parenting were differentially associated with parenting practices [We did not find a significant relationship between the ractices , 23. Forinteraction with full attention on psychological aggression and adolescent aggression was evident in both mother-adolescent and father-adolescent dyads, while the protective role of compassion and acceptance was only evident in the father-adolescent dyads. Furthermore, the paths from interaction with full attention to adolescent aggression through physical abuse and psychological aggression were significant in male adolescents, but not in female adolescents. This is inconsistent with the findings from a previous study showing that the protective role of mindful parenting on parenting practices and child externalizing problems was equivalent for mothers and fathers, and for boys and girls [interaction with full attention and compassion and acceptance components of mindful parenting, and male adolescents are more likely to benefit from increasing parental ability of interacting with children with full attention.Stratified analysis by parent and adolescent gender showed that the protective role of nd girls . The incnd girls , culturand girls , and thend girls . Taken tThe findings should be interpreted cautiously because of limitations. First, the cross-sectional design did not allow the establishment of causal links between the variables. The direction of the association between parenting practices and adolescent behavioral development remained inconsistent, including both reciprocal and unidirectional links , 50. Futinteraction with full attention and compassion and acceptance factors, were associated with decreased likelihood of physical abuse and psychological aggression, and further related to low levels of adolescent aggression. The protective role of interaction with full attention against psychological aggression and adolescent aggression was evident in both mother-adolescent and father-adolescent dyads, while the protective role of compassion and acceptance was only evident in the father-adolescent dyads. These findings should be interpreted with caution given the unadjusted covariates. Despite this, the findings contributed to the existing literature by assessing mindful parenting as a multifaceted construct and exploring the gender differences in the relationships. In addition, the findings shed light on the importance of gender-tailored interventions on increasing parents’ ability of interacting with adolescents with full attention, compassion, and acceptance, in preventing child maltreatment and promoting adolescent behavioral health.This study revealed the protective role of mindful parenting and its factors against child maltreatment and adolescent aggressive behavior in a sample of Chinese parent-adolescent dyads. More specifically, high levels of mindful parenting, specifically its Additional file 1: Table S1. Comparisons between adolescents included in the study and those excluded. Table S2. Comparisons between the mothers/fathers included in the study and those excluded. Table S3. The paths from mindful parenting and its different factors to adolescent aggressive behavior through child maltreatment among female adolescents and male adolescents, respectively.


Penaeusvannamei is the most important economic shrimp in the world. Many selective breeding programs are carried out to improve its production and performance traits. Although significant differences in the reproductive ability of female P. vannamei under artificial breeding conditions have been reported, the genome-wide adaption of the reproductive ability of domesticated female P. vannamei is less investigated. In this study, whole-genome analysis was performed along with pooled DNA sequencing on two fecundity separated bulks, high fecundity bulk (HB), and low fecundity bulk (LB). Each bulk contained 30 individuals from 3 commercial populations. A sequencing depth of >30× was achieved for each bulk, leading to the identification of 625,181 and 629,748 single nucleotide polymorphisms (SNPs) in HB and LB, respectively. Fixation index (Fst) combined with p ratio allowed for the identification of 145 selective sweep regions, with a sequence length of 14.5 Mb, accounting for 0.59% of the genome. Among the 145 selective sweep regions, a total of 64,046 SNPs were identified, and further verification was performed by genotyping 50 candidate SNPs on 60 samples from the offspring of the three populations. Furthermore, 121 genes were screened from the sweep regions. GO annotation and KEGG enrichment analyses showed that partial genes were essential for fecundity regulation. This study provides important information for in-depth investigation of genomic characteristics for long-term selective breeding on the fecundity of female P. vannamei and will also be important for genome-assisted breeding of P. vannamei in the future. Penaeus vannamei) is the most important economic shrimp in the world, with a global aquaculture production of 5,446,216 tons and a value of 32,190,978 dollars in 2019 . Single nucleotide polymorphism (SNPs) and genes associated with reproductive traits were identified. This work will contribute to the deep investigation of the molecular mechanisms of reproduction and the development of molecular breeding for high reproductive traits of P. vannamei.Fecundity is one of the noninvasive predictive criteria to identify females with high spawning frequency . HoweverP. vannamei were introduced to BLUP Aquabreed Co., Ltd . The three populations that contained 65 families were selected for years and labeled A, B, and C, respectively, among which A and B were characterized by strong resistance and C by fast growth. In 2019, the shrimps were cultivated as candidate parents. By the age of 10 months, 8–10 females and 8–9 males from each family were selected. Females subjected to unilateral eyestalk ablation to promote the synchronous development of ovaries and a number-coded ring were placed on the remaining eyestalk. At the same time, the parent shrimp started to undergo nutrition fortification for about 20 days. A total of 604 females and 500 males underwent nutrition fortification . Sexually matured females were placed into the tank with males for natural mating at 9:00 a.m. and collected at 7:00 p.m. every day . The mated females were put into the 120-L spawning tank (recording number-coded ring), whereas the unmated females were put back to the original tank. Male shrimp of one family mated with female shrimp from two different families at most at 1 day. The family construction period was from September 4 to 29 September 2019.yi is the fecundity index of the ith individual of the 87 spawning individuals; aelfi and aaeni are the ELF and AEN of the ith individual, respectively; μelf and μaen are the average of the ELF and AEN of the 87 spawning individuals; and σelf and σaen are the standard error of the ELF and AEN of the 87 spawning individuals.The ELF of 604 females during this period and the average egg number (AEN) from each spawn per female was recorded. Among these individuals, 179 had spawning records and 87 had offspring that successfully developed to juvenile stage. We only selected high reproductive individuals from the 87 females with offspring, because the vitality of offspring can also reflect the reproductive performance of females to some extent, although not absolutely. According to the ELF and AEN, we developed a fecundity index to measure the fecundity of female shrimp. The calculation formula was as follows:According to the reproductive index, a total of 30 females with high reproductive index and 30 individuals with no sexual maturity record were selected from the three populations to comprise a high reproductive bulk (HB) and a low-reproductive bulk (LB), respectively.Muscle samples of 30 high- and 30 low-reproductive shrimp were collected and frozen in liquid nitrogen. For genotyping, the total genomic DNA of each individual was isolated using cetyltrimethylammonium bromide (CTAB). Equal DNA content quantified by Qbit 2.0  was pooled separately to generate a “high” pool and a “low” pool.Paired-end sequencing libraries with an insert size of about 500 bp were constructed using the Paired-End DNA Sample Prep Kit . The libraries were sequenced on an Illumina HiSeq 2500 system using standard protocol . The sequence depth of two pools was 30× on average.Raw reads were processed to obtain high-quality clean reads according to three stringent filtering standards: 1) removing reads with ≥10% unidentified nucleotides (N); 2) removing reads with >50% bases having Phred quality scores of ≤20; and 3) removing reads aligned to the barcode adapter.To identify SNPs, the Burrows–Wheeler Aligner BWA,  was usedp ratio  (p ratios (p < 0.01) and significantly high Fst values (p < 0.01) of the empirical distribution were regarded as regions with strong selection signals along the genome. All related graphs were drawn using R scripts   and p ra, πA/πB) . A 50-kb, πA/πB) . In brie scripts .Candidate SNPs and genes within the sweep regions were extracted for further analysis. To evaluate the reliability of SNP prediction using pooled DNA-seq data, 50 candidate SNPs were genotyped in 60 samples based on individual DNA-seq data using the MALDI-TOF method. The 60 samples were obtained from the offspring of the three populations with contrasting reproduction traits, among which 30 were of high fecundity and 30 were of low fecundity. The SNP index of each locus and the frequency difference distribution in the two bulks were calculated. The direction was as follows: Δ(index) = index (HB) − index (LB). Only significant SNPs were selected as candidates for validation. The locus with Δ index close to 1 or to −1 was used as the preferred sites for verification.http://www.geneontology.org/). Gene numbers were calculated for every term, and significantly enriched GO terms were defined using hypergeometric test with p < 0.05. KEGG is a major public pathway–related database. The calculation formula is the same as that in GO analysis. Pathways with p < 0.05 were defined as significantly enriched pathways in genes.All candidate genes were mapped to GO terms in the Gene Ontology database . A total of 625,181 SNPs were shared by HB and LB based on Fst, and 629,748 SNPs were shared based on the p ratio (p < 0.01). Among them, 377,714 and 376,872 were transitions (Ts) and 247,467 and 252,876 were transversions (Tv), with Ts/Tv ratios of 1.53 and 1.49, respectively  were identified as significantly enriched pathways . p ratio were used to determine the selective sweep region. Fst was calculated using the Bayesian method, which might result in a high degree of false positives . In the selective sweep regions, the shared sites exhibited a Ts/Tv ratio of 1.49 to 1.53, which is lower than another ratio reported in P. vannamei  and predict the genomic estimated breeding value (GEBVs) of candidate populations in the future GS programs  family and was a membrane-related enzyme. The bioactive substances produced by the PLC family participate in a variety of cellular processes through intercellular and extracellular signal transduction pathways  gene, which is a scaffold protein and plays an important role in regulating cell polarity, proliferation, migration, and tight junction  super protein family, which may be involved in the optimal growth and maturation of follicles (Lvcyp2L1 is a member of the cytochrome P450 enzyme system, is widely distributed in the endoplasmic reticulum and mitochondria in the cell membrane system, and is one of the key enzymes for steroid transformation before oocyte maturation (P450c17 gene was highly expressed in the prophase of rainbow trout ovary and oviposition (P450c17 gene was at a high level in the middle stage of oocyte development (Among the 121 identified genes in the selective sweep region, pathways . PLC gen oocytes . The hom oocytes . Lvpard3junction . Khadar junction . Dong etsa cells . Lvtrx2 ollicles . Lvcyp2Lturation . Sakai sposition . In chanelopment . It is iP. vannamei, 145 selective sweep regions in the genome were identified combining two parameters, namely, Fst and p ratio. A total of 64,046 SNPs and 121 genes were screened from the sweep regions, which need further verification. GO annotation and KEGG enrichment analyses showed that partial genes were essential for fecundity regulation. These results would provide important clues for the differences in fecundity of female Pacific white shrimp at the genomic level.In conclusion, in two fecundity separated populations of


Fruit tree yield and fruit quality are affected by the tree’s growth type, and branching angle is an important agronomic trait of fruit trees, which largely determines the crown structure. The weeping type of peach tree shows good ventilation and light transmission; therefore, it is commonly cultivated. However, there is no molecular marker closely linked with peach weeping traits for target gene screening and assisted breeding.First, we confirmed that the peach weeping trait is a recessive trait controlled by a single gene by constructing segregating populations. Based on BSA-seq, we mapped the gene controlling this trait within 159 kb of physical distance on chromosome 3. We found a 35 bp deletion in the candidate area in standard type, which was not lacking in weeping type. For histological assessments, different types of branches were sliced and examined, showing fiber bundles in the secondary xylem of ordinary branches but not in weeping branches.This study established a molecular marker that is firmly linked to weeping trait. This marker can be used for the selection of parents in the breeding process and the early screening of hybrid offspring to shorten the breeding cycle. Moreover, we preliminary explored histological differences between growth types. These results lay the groundwork for a better understanding of the weeping growth habit of peach trees.The online version contains supplementary material available at 10.1186/s12870-022-03840-1. Plant growth structure plays an important role in the economic value of crops, and it can indirectly affect yield and fruit quality. A tree structure is mainly determined by height, the growth direction of the branches, and the size and angle of the branches , which tTiller angle control 1) gene elicit vertical-like growth in plants such as rice (Oryza sativa) [Zea mays) [LAZY1 (Regulator of nonsense transcript protein) causes Arabidopsis [TAC1) and the two zinc finger proteins LPA1 (Tetratricopeptide repeat-containing protein) and PROG1 (PROSTRATE GROWTH 1) (Zinc finger protein GIS3) [LPA1 and its Arabidopsis homolog SGR5 (C2H2-like zinc finger protein) regulate geotropism upstream of amyloid deposition [Due to the advantages of short growth cycles and simple handling, considerable progress has been made regarding research on the morphological development of herbaceous plants. Such studies have shown that mutations in the TAC1 ( sativa) , and maiea mays) . Mutatiobidopsis , rice [1bidopsis , and maibidopsis  to produin GIS3) , 12. LPAposition , 14. HowPrunus persica) originates from China and is one of the largest groups of deciduous fruit trees. There are abundant peach genetic resources of different growth types in China, including standard type, pillar type, dwarf type, semi-dwarf type, and spur type. Weeping type peach trees were suggested to exhibit improved light transmission, which is conducive to fruit coloring and quality formation [Br in peaches, which promotes horizontal branch growth. This gene is highly homologous to Tiller angle control 1 (TAC1) in rice and was thus named PpeTAC1. Molecular identification showed that the standard genotypes are BrBr, upright (Brbr), and pillar (brbr). Columnar (br) peach trees produce branches that grow nearly vertically, caused by a functional mutation of Tiller angle control 1 (TAC1). This gene encodes a protein belonging to the IGT gene family, which also includes LAZY1 and DRO1 (Deeper rooting 1) which regulate the orientation of lateral branches and roots, respectively [TAC1) in plums results in a branching orientation similar to that of columnar peach, however, with a narrower canopy. By contrast, overexpression of PpeTAC1 in plum trees results in wider branching angles, more horizontal branching orientation, and formation of a larger crown [PpeGID1c) on the distal end of chromosome 6 is associated with peach dwarfing traits, and nonsense mutations in this gene lead to peach dwarfing [PI) had an epistatic effect, relative to the weeping genotype (pl), and an arching growth form (AR) appeared in the F2 generation of these two genotypes [The weeping branch is one of the most important architectural plant traits, which is frequent in ornamental plants and is valued by breeders. Among Rosaceae, many tree species such as cherry blossoms, cherries, plums, begonias, and peach trees may occur as weeping branch types. Such trees are valued for landscaping and also important in scientific research on woody plant morphology . Peach (ormation . Peach tectively . RNAi sier crown . Moreovedwarfing . A tempedwarfing . Moreoveenotypes .Lagerstroemia indica L.) [Castanea mollissima Bl.) [Prunus mume) [The genetic characteristics and regulatory mechanisms of weeping traits have been studied in crape myrtle (dica L.) , chestnuima Bl.) , and Japus mume) , whereasCross and longitudinal sections of different growth types fixed in paraffin collected during the same period were compared Fig. , showingThe raw sequence data  of the standard and weeping pools amounted to 31.21 and 28.35 GB, respectively; after trimming the barcodes, the respective amounts of clean data were 31.14 and 28.28 GB. A total of 197,372,658 reads (104.24 folds average sequencing depth) and 174,956,782 reads (93.86 folds average sequencing depth) from the standard and weeping pools, respectively, were mapped to the reference genome (Table 2 parent (genotype Aa) as a reference, and calculating the SNP-index (frequency of SNP) of the two gene pools respectively. SNP-index(weeping) = aa/Aa = 1,SNP-index(Standard) = AA/(AA + 2Aa) = 1/3,Δ(SNP-index) = SNP-index(weeping)-SNP-index(Standard) = 1–1/3 = 2/3 = 0.667. A total of 1,000 permutation tests were performed, and a 95% confidence level was used as a screening threshold. Through analyzing the SNP index of each pool, we calculated the Δ(SNP-index) of the entire genome from chromosomes 1–8  of 641,993 marker sites between the two offspring types were examined. Trait mapping was performed by calculating the Δ(SNP-index) value between different progeny pools, selecting the FPp3G198900 and Pp3G201500  in the standard type  and weeping2 (we2) associated with stalk droop were found in morning glory mutants, which are highly homologous to SCR and SHR (GRAS family transcription factor) in Arabidopsis, respectively, and are found in different plants. The GRAS  family of transcription factors plays a crucial role in the development of the plant endothelium and the normal tropism response [1 (Gibberellin 1) correlates with the elongation of secondary xylem fibers [A systematic analysis of the role of auxin in the formation of plant type suggested apical dominance in plant growth and development which is related to auxin anabolism, polar transport, and signal transduction . Branch response . Celluloresponse . Plant sm fibers . A studym fibers .3 (Gibberellin 3) treatment could rescue the weeping shoot phenotype. Transcriptional analysis and co-expression network analysis of differentially expressed genes indicated that gibberellin synthesis and signal transduction pathways play important roles in weeping traits. Virus-induced gene silencing of LfiGRAS1, a negative element of plant gibberellin signaling, leads to new branches growing in a negative direction, and gibberellin, interacting with other factors, strongly affects the growth type of crape myrtle [Xiang et al.(2008) examined the levels of endogenous hormones in branches of different types of peach trees, and they found that the lignin content of straight branches was higher than that of drooping branches, and the lignin content of the underside and upside of the branches also differed between growth types. The data showed that the content of gibberellin on the upside of drooping peach branches was higher than that on the underside, which was consistent with the distribution of lignin. It was speculated that the uneven distribution of gibberellin may explain the unbalanced development of the secondary xylem . Anothere myrtle .In the present study, the cell morphology of two peach growth types was compared and analyzed, showing that the internal structure of the branches was dynamic, and the internal morphology of branches differed between growth types. Considering previous studies, we suggest that such differences are related to endogenous hormones; lignocellulose is an important substance providing support for branches, and a lack of lignocellulose in drooping peach branches is the main reason for branch drooping.2 hybrid population of weeping peach, and the segregation ratio conformed to the 1:3 segregation ratio (χ2 = 2.2) of single-gene recessive traits according to Mendelian inheritance law. These results are consistent with the conclusions drawn from the preliminary population segregation of the present study.Monet described the weeping growth of peach trees as early as 1988, and Bassi later also studied weeping peach trees . It was pl) of peach was located between two markers MA039a  and SSRLG3_16m46  on LG3, with a physical distance of approximately 4 Mb [Ppa013325 (Prupe.3G200700) was determined as the single candidate gene. The expression site of the gene was examined, showing that the gene was mainly expressed in the peach phloem, encoding a protein containing a sterility alpha motif domain, and the protein contained a total of 129 amino acids [The peach weeping gene  that the gene was mainly expressed in the phloem of shoots; however, considering previous results on weeping branch-related endogenous hormones and morphological examination as performed in the current study, the main factor causing drooping in peach branches is a difference in the development of the xylem. In conclusion, the regulatory mechanism of the peach weeping trait requires further systematic study.In the current study, based on BSA-seq, the candidate genes of the peach weeping branch were found to be located within a physical distance of 159 kb  on chromosome 3, which was consistent with previous results , 37, howTraditional breeding methods rely on the phenotype, the development of which, however, may take considerable time, thus impeding the breeding efficiency. Molecular genomic means may thus help improve and accelerate breeding selection. Molecular markers are not affected by the external growth environment and can be detected during the entire growth and development period of plants. Therefore, molecular markers closely related to late-stage plant development and growth traits are very important for the early selection of seedlings, which can greatly improve the breeding efficiency and shorten breeding time , 38. HolIn the present study, based on fine mapping of genes associated with the weeping trait, sequence variation of candidate genes was further compared and analyzed. Primers were designed for confirmation in different populations, and a molecular marker was identified that is strongly linked to the candidate gene. This molecular marker can be used for early seedling identification or later gene function verification in the identification of target seedlings, thus representing a foundation for the development of new weeping peach varieties.2 generation populations were obtained. The target gene was located between 18–21 Mb on chromosome 3 based on BSA-seq, and fine-mapped in the range of 159 kb with SNP markers, using the 395 F2 individuals of this population. A candidate Indel in the gene Prupe.3G200000 was obtained based on the resequencing data of other cultivars (lines). The Indel marker was able to identify the phenotypes in 72 cultivars (lines). By genotyping analysis, the Indel was co-segregated with the weeping phenotype and used for identification of stander or weeping type with 100% accuracy.Using the hybrid progeny ‘05–2-144’ of ‘97 dwarfing’ and ‘yuanyang weeping’, a total of 395 F2 generation were obtained, which were planted in the peach breeding garden of Zhengzhou Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences. Accession ‘05–2-144’ , samples were stored at 4 °C after vacuum drying.The samples were dehydrated using the following treatments: (1) 75% ethanol for 4 h (2) 85% ethanol for 2 h (3) 90% ethanol for 2 h (4) 95% ethanol for 1 h (5) absolute ethanol for 1 h (6) transition into histo-clear xylene for 20 min (7) embedding in paraffin using TKY-BMB . Thereafter, the samples were sliced (4 μm thickness) using a rotary microtome . Three samples were examined per replicate.2 population was determined by measuring the branching angle of each plant. The inbred progeny of ‘05–2-144’ had two types: standard type and weeping type. Statistical analysis of two single trees showed that the segregation ratio of the progeny population was close to 3:1 (standard type to weeping type), which was in line with Mendelian inheritance and with the expected ratio of the single-gene recessive control , suggesting that the weeping phenotype was controlled by a single recessive gene.In 2016, the phenotype of 395 offspring of the FYoung leaves were collected and placed on the bottom of 1.2 mL eight-row centrifuge tubes using tweezers. Total genomic DNA was isolated using the CTAB method, with minor modifications . Quantithttps://www.rosaceae.org/species/prunus_persica/genome_v2.0.a1) using a BWA aligner, then SAMTOOLS was used to convert alignment files to SAM/BWA files and to perform SNP calling [Based on the phenotypic evaluation, we selected 15 individual plants of the standard type and weeping type in 2017 to extract DNA. Fifteen samples of each phenotype were mixed in equal amounts, and SNP markers were developed through BSA-seq resequencing to preliminarily locate the target loci. Briefly, clean reads were first aligned to the peach reference genome  sequencing depth below seven-fold and SNP index < 0.3 were discarded, and (2) Only one pool exists SNP missing were discarded. Therefore, a weeping candidate region had an SNP index of 1, and the standard SNP index = 1/3. The Δ(SNP-index) was obtained by subtracting the SNP index of the weeping pool from that of the standard pool, which was approximately 0.333.http://primer3.ut.ee).Based on BSA-seq, the candidate regions associated with the weeping trait were validated using the indel markers as confirmed through PAGE. For fine mapping, SNP markers were used for polymorphism screening in the parent of ‘05–2-144’ and the offspring. As for referencing the genome, IGV software was used to detect SNPs in the bam files. Primers were designed using Primer3 (available at PCR amplification for Sanger sequencing was performed using a reaction volume of 40 μL, and with a 15 μL reaction volume for indel analysis. For indel markers, the PCR products were examined using PAGE and were Sanger sequenced for SNP analysis.Additional file 1: Table S1. Candidate region of SNP_index. Additional file 2: Table S2. Genome-wide Delta_SNP_index. Additional file 3: Table S3. Locus information of candidate gene. Additional file 4: Table S4. Fine-mapping results based on segregated populations.