Molecular and genetic insights into secondary metabolic regulation underlying insect-pest resistance in legumes.

Insect pests pose a major threat to agricultural production, resulting in significant economic losses for countries. A high infestation of insects in any given area can severely reduce crop yield and quality. This review examines the existing resources for managing insect pests and highlights alternative eco-friendly techniques to enhance insect pest resistance in legumes. Recently, the application of plant secondary metabolites has gained popularity in controlling insect attacks. Plant secondary metabolites encompass a wide range of compounds such as alkaloids, flavonoids, and terpenoids, which are often synthesized through intricate biosynthetic pathways. Classical methods of metabolic engineering involve manipulating key enzymes and regulatory genes to enhance or redirect the production of secondary metabolites in plants. Additionally, the role of genetic approaches, such as quantitative trait loci mapping, genome-wide association (GWAS) mapping, and metabolome-based GWAS in insect pest management is discussed, also, the role of precision breeding, such as genome editing technologies and RNA interference for identifying pest resistance and manipulating the genome to develop insect-resistant cultivars are explored, highlighting the positive contribution of plant secondary metabolites engineering-based resistance against insect pests. It is suggested that by understanding the genes responsible for beneficial metabolite compositions, future research might hold immense potential to shed more light on the molecular regulation of secondary metabolite biosynthesis, leading to advancements in insect-resistant traits in crop plants. In the future, the utilization of metabolic engineering and biotechnological methods may serve as an alternative means of producing biologically active, economically valuable, and medically significant compounds found in plant secondary metabolites, thereby addressing the challenge of limited availability.

Gene-allele system of shade tolerance in southern China soybean germplasm revealed by genome-wide association study using gene-allele sequence as markers.

KEY MESSAGE: Fifty-three shade tolerance genes with 281 alleles in the SCSGP were identified directly using gene-allele sequence as markers in RTM GWAS, from which optimized crosses, evolutionary motivators, and gene-allele networks were explored. Shade tolerance is a key for optimal cultivation of soybean inter/relay-cropped with corn. To explore the shade tolerance gene-allele system in the southern China soybean germplasm, we proposed using gene-allele sequence markers (GASMs) in a restricted two-stage multi-locus model genome-wide association study (GASM-RTM-GWAS). A representative sample with 394 accessions was tested for their shade tolerance index (STI), in Nanning, China. Through whole-genome re-sequencing, 47,586 GASMs were assembled. From GASM-RTM-GWAS, 53 main-effect STI genes with 281 alleles (2-13 alleles/gene) (totally 63 genes with 308 alleles, including 38 G × E genes with 191 alleles) were identified and then organized into a gene-allele matrix composed of eight submatrices corresponding to geo-seasonal subpopulations. The population featured mild STI changes (1.69 → 1.56-1.82) and mild gene-allele changes (92.5% alleles inherited, 0% alleles excluded, 7.5% alleles emerged) from the primitive (SAIII) to the derived seven subpopulations, but large transgressive recombination potentials and optimal crosses were predicted. The 63 STI genes were annotated into six biological categories (metabolic process, catalytic activity, response to stresses, transcription and translation, signal transduction and transport and unknown functions), interacted as gene networks. From the STI gene-allele system, 38 important alleles of 22 genes were nominated for further in-depth study. GASM-RTM-GWAS performed powerful and efficient in germplasm population genetic study comparing to other procedures through facilitating direct and thorough identification of its gene-allele system, from which genome-wide breeding by design could be achieved, and evolutionary motivators and gene-allele networks could be explored.

The Identification of a Quantative Trait Loci-Allele System of Antixenosis against the Common Cutworm (Spodoptera litura Fabricius) at the Seedling Stage in the Chinese Soybean Landrace Population.

Common cutworm (CCW) is an omnivorous insect causing severe yield losses in soybean crops. The seedling-stage mini-tray identification system with the damaged leaf percentage (DLP) as an indicator was used to evaluate antixenosis against CCW in the Chinese soybean landrace population (CSLRP) under three environments. Using the innovative restricted two-stage multi-locus genome-wide association study procedure (RTM-GWAS), 86 DLP QTLs with 243 alleles (2-11/QTL) were identified, including 66 main-effect loci with 203 alleles and 57 QTL-environment interaction loci with 172 alleles. Among the main-effect loci, 12 large-contribution loci (R2 ≥ 1%) explained 25.45% of the phenotypic variation (PV), and 54 small-contribution loci (R2 < 1%) explained 16.55% of the PV. This indicates that the CSLRP can be characterized with a DLP QTL-allele system complex that has not been found before, except for a few individual QTLs without alleles involved. From the DLP QTL-allele matrix, the recombination potentials expressed in the 25th percentile of the DLP of all possible crosses were predicted to be reduced by 41.5% as the maximum improvement and 14.2% as the maximum transgression, indicating great breeding potential in the antixenosis of the CSLRP. From the QTLs, 62 candidate genes were annotated, which were involved in eight biological function categories as a gene network of the DLP. Changing from susceptible to moderate plus resistant varieties in the CSLRP, 26 QTLs had 32 alleles involved, in which 19 genes were annotated from 25 QTL-alleles, including eight increased negative alleles on seven loci and 11 decreased positive alleles on 11 loci, showing the major genetic constitution changes for the antixenosis enhancement at the seedling stage in the CSLRP.

An Improved Genome-Wide Association Procedure Explores Gene-Allele Constitutions and Evolutionary Drives of Growth Period Traits in the Global Soybean Germplasm Population.

In soybeans (Glycine max (L.) Merr.), their growth periods, DSF (days of sowing-to-flowering), and DFM (days of flowering-to-maturity) are determined by their required accumulative day-length (ADL) and active temperature (AAT). A sample of 354 soybean varieties from five world eco-regions was tested in four seasons in Nanjing, China. The ADL and AAT of DSF and DFM were calculated from daily day-lengths and temperatures provided by the Nanjing Meteorological Bureau. The improved restricted two-stage multi-locus genome-wide association study using gene-allele sequences as markers (coded GASM-RTM-GWAS) was performed. (i) For DSF and its related ADLDSF and AATDSF, 130-141 genes with 384-406 alleles were explored, and for DFM and its related ADLDFM and AATDFM, 124-135 genes with 362-384 alleles were explored, in a total of six gene-allele systems. DSF shared more ADL and AAT contributions than DFM. (ii) Comparisons between the eco-region gene-allele submatrices indicated that the genetic adaptation from the origin to the geographic sub-regions was characterized by allele emergence (mutation), while genetic expansion from primary maturity group (MG)-sets to early/late MG-sets featured allele exclusion (selection) without allele emergence in addition to inheritance (migration). (iii) Optimal crosses with transgressive segregations in both directions were predicted and recommended for breeding purposes, indicating that allele recombination in soybean is an important evolutionary drive. (iv) Genes of the six traits were mostly trait-specific involved in four categories of 10 groups of biological functions. GASM-RTM-GWAS showed potential in detecting directly causal genes with their alleles, identifying differential trait evolutionary drives, predicting recombination breeding potentials, and revealing population gene networks.

RSC3 K of soybean cv. Kefeng No.1 confers resistance to soybean mosaic virus by interacting with the viral protein P3.

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens of soybean (Glycine max (L.) Merr). In total, 22 Chinese SMV strains (SC1-SC22) have been classified based on the responses of 10 soybean cultivars to these pathogens. However, although several SMV-resistance loci in soybean have been identified, no gene conferring SMV resistance in the resistant soybean cultivar (cv.) Kefeng No.1 has been cloned and verified. Here, using F2 -derived F3 (F2:3 ) and recombinant inbred line (RIL) populations from a cross between Kefeng No.1 and susceptible soybean cv. Nannong 1138-2, we localized the gene in Kefeng No.1 that mediated resistance to SMV-SC3 strain to a 90-kb interval on chromosome 2. To study the functions of candidate genes in this interval, we performed Bean pod mottle virus (BPMV)-induced gene silencing (VIGS). We identified a recombinant gene (which we named RSC3 K) harboring an internal deletion of a genomic DNA fragment partially flanking the LOC100526921 and LOC100812666 reference genes as the SMV-SC3 resistance gene. By shuffling genes between infectious SMV DNA clones based on the avirulent isolate SC3 and virulent isolate 1129, we determined that the viral protein P3 is the avirulence determinant mediating SMV-SC3 resistance on Kefeng No.1. P3 interacts with RNase proteins encoded by RSC3 K, LOC100526921, and LOC100812666. The recombinant RSC3 K conveys much higher anti-SMV activity than LOC100526921 and LOC100812666, although those two genes also encode proteins that inhibit SMV accumulation, as revealed by gene silencing in a susceptible cultivar and by overexpression in Nicotiana benthamiana. These findings demonstrate that RSC3 K mediates the resistance of Kefeng No.1 to SMV-SC3 and that SMV resistance of soybean is determined by the antiviral activity of RNase proteins.

Differential SW16.1 allelic effects and genetic backgrounds contributed to increased seed weight after soybean domestication.

Although seed weight has increased following domestication from wild soybean (Glycine soja) to cultivated soybean (Glycine max), the genetic basis underlying this change is unclear. Using mapping populations derived from chromosome segment substitution lines of wild soybean, we identified SW16.1 as the causative gene underlying a major quantitative trait locus controlling seed weight. SW16.1 encodes a nucleus-localized LIM domain-containing protein. Importantly, the GsSW16.1 allele from wild soybean accession N24852 had a negative effect on seed weight, whereas the GmSW16.1 allele from cultivar NN1138-2 had a positive effect. Gene expression network analysis, reverse-transcription quantitative polymerase chain reaction, and promoter-luciferase reporter transient expression assays suggested that SW16.1 regulates the transcription of MT4, a positive regulator of seed weight. The natural variations in SW16.1 and other known seed weight genes were analyzed in soybean germplasm. The SW16.1 polymorphism was associated with seed weight in 247 soybean accessions, showing much higher frequency of positive-effect alleles in cultivated soybean than in wild soybean. Interestingly, gene allele matrix analysis of the known seed weight genes revealed that G. max has lost 38.5% of the G. soja alleles and that most of the lost alleles had negative effects on seed weight. Our results suggest that eliminating negative alleles from G. soja led to a higher frequency of positive alleles and changed genetic backgrounds in G. max, which contributed to larger seeds in cultivated soybean after domestication from wild soybean. Our findings provide new insights regarding soybean domestication and should assist current soybean breeding programs.

Growth period QTL-allele constitution of global soybeans and its differential evolution changes in geographic adaptation versus maturity group extension.

Soybean (Glycine max (L.) Merr.) has been disseminated globally as a photoperiod/temperature-sensitive crop with extremely diverse days to flowering (DTF) and days to maturity (DTM) values. A population with 371 global varieties covering 13 geographic regions and 13 maturity groups (MGs) was analyzed for its DTF and DTM QTL-allele constitution using restricted two-stage multi-locus genome-wide association study (RTM-GWAS). Genotypes with 20 701 genome-wide SNPLDBs (single-nucleotide polymorphism linkage disequilibrium blocks) containing 55 404 haplotypes were observed, and 52 DTF QTLs and 59 DTM QTLs (including 29 and 21 new ones) with 241 and 246 alleles (two to 13 per locus) were detected, explaining 84.8% and 74.4% of the phenotypic variance, respectively. The QTL-allele matrix characterized with all QTL-allele information of each variety in the global population was established and subsequently separated into geographic and MG set submatrices. Direct comparisons among them revealed that the genetic adaptation from the origin to geographic subpopulations was characterized by new allele/new locus emergence (mutation) but little allele exclusion (selection), while that from the primary MG set to emerged early and late MG sets was characterized by allele exclusion without allele emergence. The evolutionary changes involved mainly 72 DTF and 71 DTM alleles on 28 respective loci, 10-12 loci each with three to six alleles being most active. Further recombination potential for faster maturation (12-21 days) or slower maturation (14-56 days) supported allele convergence (recombination) as a constant genetic factor in addition to migration (inheritance). From the QTLs, 44 DTF and 36 DTM candidate genes were annotated and grouped respectively into nine biological processes, indicating multi-functional DTF/DTM genes are involved in a complex gene network. In summary, we identified QTL-alleles relatively thoroughly using RTM-GWAS for direct matrix comparisons and subsequent analysis.

Identification of QTLs and joint QTL segments of leaflet traits at different canopy layers in an interspecific RIL population of soybean.

KEY MESSAGE: A leaflet trait on different canopy layers may have different QTLs; leaflet trait QTLs may cluster to form joint QTL segments; all canopy layer QTLs form a complete QTL system for a leaflet trait. As the main part of the plant canopy structure, leaf/leaflet size and shape affect the plant architecture and yield. To explore the leaflet trait QTL system, a population composed of 199 recombinant inbred lines derived from Changling (annual wild, narrow leaflet) and Yiqianli (landrace, broad leaflet) with their parents was tested for leaflet length (LL), width (LW) and length to width (LLW). The population was genotyped with specific-locus amplified fragment sequencing (SLAF-seq) and applied for linkage mapping of the leaflet traits. The results showed that the leaflet traits varied greatly even within a plant, which supported a stratified leaflet sampling strategy to evaluate these traits at top, middle and bottom canopy layers. Altogether, 13 LL, 10 LW and 9 LLW in a total of 32 plus 3 duplicated QTLs were identified, in which, 17 QTLs were new ones, and 48.6%, 28.6% and 22.8% of QTLs were from the top, middle and bottom layers, respectively, indicating the genetic importance of the top layer leaves. Since a leaflet trait may have layer-specific QTLs, all layer QTLs form a complete QTL system. Five QTL clusters each with their QTL supporting intervals overlapped were designated as joint QTL segments (JQSs). In JQS-16, with its linkage map further validated using PCR markers, two QTLs, qLW-16-1 and qLLW-16-1 of the top layer leaflet, were identified six QTL·times. Six candidate genes were predicted, with Glyma.16G127900 as the most potential one for LW and LLW. Three PCR markers, Gm16PAV0653, BARCSOYSSR_16_0796 and YC-16-3, were suggested for marker-assisted selection for LW and LLW in JQS-16.

CRISPR-Cas9 based stress tolerance: New hope for abiotic stress tolerance in chickpea (Cicer arietinum).

Plants are subjected to biotic and abiotic stresses regularly, which irreparably harm agricultural production. Eco-friendly and sustainable technology to deal with this challenge is to breed abiotic stress tolerant cultivars. To generate crop plants conferring resistance against stresses, conventional breeding was used in the past, but because of the complex heredity of abiotic stress tolerance traits, such techniques remain insufficient in making greater enhancement. Genome-engineering based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein9) has shown enormous potential in developing climate-resilient cultivars. Likewise, the development of chickpea transgenic lines by knockout of 4CL and REV7 genes exhibits drought tolerance which establishes a foundation for future studies in chickpea. In addition, the CRISPR-Cas9 system can boost yield potential under abiotic stress situations by producing non-transgenic plants having the required characteristics. This review article discusses the validation of gene function based on the CRISPR-Cas9 for the development of abiotic stress-tolerant crop plants, emphasizing the chickpea to open the new ventures of generating abiotic stress-tolerant chickpea varieties.

Evolutionary QTL-allele changes in main stem node number among geographic and seasonal subpopulations of Chinese cultivated soybeans.

The main stem node number (MSN) is a trait related to geographic adaptation, plant architecture and yield potential of soybean. The QTL-allele constitution of the Chinese Cultivated Soybean Population (CCSP) was identified using the RTM-GWAS (restricted two-stage multi-locus genome-wide association study) procedure, from which a QTL-allele matrix was established and then separated into submatrices to explore the genetic structure, evolutionary differentiation, breeding potential and candidate genes of MSN in CCSP. The MSN of 821 accessions varied from 8.8 to 31.1, with an average of 16.3 in Nanjing, China (32.07° N, 118.62° E), where the MSNs of all the materials could be evaluated in a standardized manner. Among the six geo-seasonal subpopulations, the MSN varied from 21.7 in a southern summer-autumn-sowing subpopulation (SA-IV) down to 13.5 in a northeastern spring-sowing subpopulation (SP-I). The materials were genotyped with restriction site-associated DNA-sequencing. Totally 142 main-effect QTLs (73.24% new) with 560 alleles contributing 72.98% to the phenotypic variance were identified. The evolutionary QTL-allele changes in MSN from SA-IV through SP-I showed that inheritance (78.93% of alleles) was the primary factor influencing the evolution of this trait, followed by allele emergence (19.64% alleles), allele exclusion (1.43% alleles), and recombination among retained alleles. In the evolutionary changes, 70 QTLs, including 12 newly emerged QTLs, with 118 alleles were involved. An increase potential of 2-8 nodes was predicted and 112 candidate genes were annotated and preliminarily verified with χ2-tests in the CCSP. The RTM-GWAS showed powerful in detecting QTL-allele system, assessing evolution factors, predicting optimal crosses and identifying candidate genes in a germplasm population.

Comprehensive Identification of Drought Tolerance QTL-Allele and Candidate Gene Systems in Chinese Cultivated Soybean Population.

Drought is one of the most important factors affecting plant growth and productivity. The previous results on drought tolerance (DT) genetic system in soybean indicated a complex of genes not only few ones were involved in the trait. This study is featured with a relatively thorough identification of QTL-allele/candidate-gene system using an efficient restricted two-stage multi-locus multi-allele genome-wide association study, on two comprehensive DT indicators, membership index values of relative plant weight (MPW) and height (MPH), instead of a single biological characteristic, in a large sample (564 accessions) of the Chinese cultivated soybean population (CCSP). Based on 24,694 multi-allele markers, 75 and 64 QTL with 261 and 207 alleles (2-12/locus) were detected for MPW and MPH, explaining 54.7% and 47.1% of phenotypic variance, respectively. The detected QTL-alleles were organized into a QTL-allele matrix for each indicator, indicating DT is a super-trait conferred by two (even more) QTL-allele systems of sub-traits. Each CCSP matrix was separated into landrace (LR) and released cultivar (RC) sub-matrices, which showed significant differentiation in QTL-allele constitutions, with 58 LR alleles excluded and 16 new ones emerged in RC. Using the matrices, optimal crosses with great DT transgressive recombinants were predicted. From the detected QTL, 177 candidate genes were annotated and validated with quantitative Real-time PCR, and grouped into nine categories, with ABA and stress responders as the major parts. The key point of the above results is the establishment of relatively full QTL-allele matrices composed of numerous gene functions jointly conferring DT, therefore, demonstrates the complexity of DT genetic system and potential of CCSP in DT breeding.

Efficient QTL detection of flowering date in a soybean RIL population using the novel restricted two-stage multi-locus GWAS procedure.

KEY MESSAGE: Eighty-six R1 QTLs accounting for 89.92% phenotypic variance in a soybean RIL population were identified using RTM-GWAS with SNPLDB marker which performed superior over CIM and MLM-GWAS with BIN/SNPLDB marker. A population (NJRIKY) composed of 427 recombinant inbred lines (RILs) derived from Kefeng-1 × NN1138-2 (MGII × MGV, MG maturity group) was applied for detecting flowering date (R1) quantitative trait locus (QTL) system in soybean. From a low-depth re-sequencing (~ 0.75 ×), 576,874 SNPs were detected and organized into 4737 BINs (recombination breakpoint determinations) and 3683 SNP linkage disequilibrium blocks (SNPLDBs), respectively. Using the association mapping procedures "Restricted Two-stage Multi-locus Genome-wide Association Study" (RTM-GWAS), "Mixed Linear Model Genome-wide Association Study" (MLM-GWAS) and the linkage mapping procedure "Composite Interval Mapping" (CIM), 67, 36 and 10 BIN-QTLs and 86, 14 and 23 SNPLDB-QTLs were detected with their phenotypic variance explained (PVE) 88.70-89.92% (within heritability 98.2%), 146.41-353.62% (overflowing) and 88.29-172.34% (overflowing), respectively. The RTM-GWAS with SNPLDBs which showed to be more efficient and reasonable than the others was used to identify the R1 QTL system in NJRIKY. The detected 86 SNPLDB-QTLs with their PVE from 0.02 to 30.66% in a total of 89.92% covered 51 out of 104 R1 QTLs in 18 crosses in SoyBase and 26 out of 139 QTLs in a nested association mapping population, while the rest 29 QTLs were novel ones. From the QTL system, 52 candidate genes were annotated, including the verified gene E1, E2, E9 and J, and grouped into 3 categories of biological processes, among which 24 genes were enriched into three protein-protein interaction networks, suggesting gene networks working together. Since NJRIKY involves only MGII and MGV, the QTL/gene system among MG000-MGX should be explored further.

An innovative procedure of genome-wide association analysis fits studies on germplasm population and plant breeding.

KEY MESSAGE: The innovative RTM-GWAS procedure provides a relatively thorough detection of QTL and their multiple alleles for germplasm population characterization, gene network identification, and genomic selection strategy innovation in plant breeding. The previous genome-wide association studies (GWAS) have been concentrated on finding a handful of major quantitative trait loci (QTL), but plant breeders are interested in revealing the whole-genome QTL-allele constitution in breeding materials/germplasm (in which tremendous historical allelic variation has been accumulated) for genome-wide improvement. To match this requirement, two innovations were suggested for GWAS: first grouping tightly linked sequential SNPs into linkage disequilibrium blocks (SNPLDBs) to form markers with multi-allelic haplotypes, and second utilizing two-stage association analysis for QTL identification, where the markers were preselected by single-locus model followed by multi-locus multi-allele model stepwise regression. Our proposed GWAS procedure is characterized as a novel restricted two-stage multi-locus multi-allele GWAS (RTM-GWAS, https://github.com/njau-sri/rtm-gwas ). The Chinese soybean germplasm population (CSGP) composed of 1024 accessions with 36,952 SNPLDBs (generated from 145,558 SNPs, with reduced linkage disequilibrium decay distance) was used to demonstrate the power and efficiency of RTM-GWAS. Using the CSGP marker information, simulation studies demonstrated that RTM-GWAS achieved the highest QTL detection power and efficiency compared with the previous procedures, especially under large sample size and high trait heritability conditions. A relatively thorough detection of QTL with their multiple alleles was achieved by RTM-GWAS compared with the linear mixed model method on 100-seed weight in CSGP. A QTL-allele matrix (402 alleles of 139 QTL × 1024 accessions) was established as a compact form of the population genetic constitution. The 100-seed weight QTL-allele matrix was used for genetic characterization, candidate gene prediction, and genomic selection for optimal crosses in the germplasm population.

Detecting the QTL-allele system of seed isoflavone content in Chinese soybean landrace population for optimal cross design and gene system exploration.

KEY MESSAGE: Utilizing an innovative GWAS in CSLRP, 44 QTL 199 alleles with 72.2 % contribution to SIFC variation were detected and organized into a QTL-allele matrix for cross design and gene annotation. The seed isoflavone content (SIFC) of soybeans is of great importance to health care. The Chinese soybean landrace population (CSLRP) as a genetic reservoir was studied for its whole-genome quantitative trait loci (QTL) system of the SIFC using an innovative restricted two-stage multi-locus genome-wide association study procedure (RTM-GWAS). A sample of 366 landraces was tested under four environments and sequenced using RAD-seq (restriction-site-associated DNA sequencing) technique to obtain 116,769 single nucleotide polymorphisms (SNPs) then organized into 29,119 SNP linkage disequilibrium blocks (SNPLDBs) for GWAS. The detected 44 QTL 199 alleles on 16 chromosomes (explaining 72.2 % of the total phenotypic variation) with the allele effects (92 positive and 107 negative) of the CSLRP were organized into a QTL-allele matrix showing the SIFC population genetic structure. Additional differentiation among eco-regions due to the SIFC in addition to that of genome-wide markers was found. All accessions comprised both positive and negative alleles, implying a great potential for recombination within the population. The optimal crosses were predicted from the matrices, showing transgressive potentials in the CSLRP. From the detected QTL system, 55 candidate genes related to 11 biological processes were χ (2)-tested as an SIFC candidate gene system. The present study explored the genome-wide SIFC QTL/gene system with the innovative RTM-GWAS and found the potentials of the QTL-allele matrix in optimal cross design and population genetic and genomic studies, which may have provided a solution to match the breeding by design strategy at both QTL and gene levels in breeding programs.

Establishment of a 100-seed weight quantitative trait locus-allele matrix of the germplasm population for optimal recombination design in soybean breeding programmes.

A representative sample comprising 366 accessions from the Chinese soybean landrace population (CSLRP) was tested under four growth environments for determination of the whole-genome quantitative trait loci (QTLs) system of the 100-seed weight trait (ranging from 4.59g to 40.35g) through genome-wide association study (GWAS). A total of 116 769 single nucleotide polymorphisms (SNPs) were identified and organized into 29 121 SNP linkage disequilibrium blocks (SNPLDBs) to fit the property of multiple alleles/haplotypes per locus in germplasm. An innovative two-stage GWAS was conducted using a single locus model for shrinking the marker number followed by a multiple loci model utilizing a stepwise regression for the whole-genome QTL identification. In total, 98.45% of the phenotypic variance (PV) was accounted for by four large-contribution major QTLs (36.33%), 51 small-contribution major QTLs (43.24%), and a number of unmapped minor QTLs (18.88%), with the QTL×environment variance representing only 1.01% of the PV. The allele numbers of each QTL ranged from two to 10. A total of 263 alleles along with the respective allele effects were estimated and organized into a 263×366 matrix, giving the compact genetic constitution of the CSLRP. Differentiations among the ecoregion matrices were found. No landrace had alleles which were all positive or all negative, indicating a hidden potential for recombination. The optimal crosses within and among ecoregions were predicted, and showed great transgressive potential. From the QTL system, 39 candidate genes were annotated, of which 26 were involved with the gene ontology categories of biological process, cellular component, and molecular function, indicating that diverse genes are involved in directing the 100-seed weight.

Exploration of presence/absence variation and corresponding polymorphic markers in soybean genome.

This study was designed to reveal the genome-wide distribution of presence/absence variation (PAV) and to establish a database of polymorphic PAV markers in soybean. The 33 soybean whole-genome sequences were compared to each other with that of Williams 82 as a reference genome. A total of 33,127 PAVs were detected and 28,912 PAV markers with their primer sequences were designed as the database NJAUSoyPAV_1.0. The PAVs scattered on whole genome while only 518 (1.8%) overlapped with simple sequence repeats (SSRs) in BARCSOYSSR_1.0 database. In a random sample of 800 PAVs, 713 (89.13%) showed polymorphism among the 12 differential genotypes. Using 126 PAVs and 108 SSRs to test a Chinese soybean germplasm collection composed of 828 Glycine soja Sieb. et Zucc. and Glycine max (L.) Merr. accessions, the per locus allele number and its variation appeared less in PAVs than in SSRs. The distinctness among alleles/bands of PCR (polymerase chain reaction) products showed better in PAVs than in SSRs, potential in accurate marker-assisted allele selection. The association mapping results showed SSR + PAV was more powerful than any single marker systems. The NJAUSoyPAV_1.0 database has enriched the source of PCR markers, and may fit the materials with a range of per locus allele numbers, if jointly used with SSR markers.

A new fluorescent probe with high selectivity and sensitivity for Cys detection in bovine serum.

Cysteine (Cys) is one of the most basic mercaptans in the human body. As an important endogenous small molecule mercaptan, Cys plays a vital role in various physiological processes and can participate in maintaining redox balance to ensure homeostasis. Abnormal Cys levels can lead to a variety of diseases. However, the detection of cysteine may be interfered with by other small molecule biothiols. Therefore, the design of fluorescent probes based on the structural characteristics and reactivity of cysteine has become the focus of current research. In this paper, a fluorescent probe (3-(2H-benzo[d][1,2,3]triazol-2-yl)-2-oxo-2H-benzo[g]chromen-8-yl acrylate, BTAB) for Cys detection was synthesized with acrylic ester as the reaction site. Under the conditions of gradual optimization, BTAB can achieve selectivity and anti-interference ability for Cys detection. The linear range of Cys was 0.3-10 µM, and the detection limit was 0.154 µM. Finally, this probe was applied to detect the Cys content in bovine serum samples with satisfactory results.

Effects of age on differential resistance to duck hepatitis A virus genotype 3 in Pekin ducks by 16 S and transcriptomics.

Duck hepatitis A virus genotype 3 (DHAV-3) is the major cause of viral hepatitis in ducks in Asia. Previous studies have shown that ducklings younger than 21 days are more susceptible to DHAV-3. To elucidate the mechanism by which age affects the differential susceptibility of Pekin ducks to DHAV-3, intestinal (n = 520), liver (n = 40) and blood (n = 260) samples were collected from control and DHAV-3-infected ducks at 7, 10, 14, and 21 days of age. Comparisons of plasma markers, mortality rates, and intestinal histopathological data showed that the resistance of Pekin ducks to DHAV-3 varied with age. 16 S sequencing revealed that the ileal microbial composition was influenced by age, and this correlation was greater than that recorded for caecal microbes. Candidatus Arthromitus, Bacteroides, Corynebacterium, Enterococcus, Romboutsia, and Streptococcus were the differntially abundant microbes in the ileum at the genus level after DHAV-3 infection and were significantly correlated with 7 differentially expressed genes (DEGs) in 7- and 21-day-old ducklings. 3 immunity-related pathways were significantly different between 7- and 21-day-old ducklings, especially for IFIH1-mediated induction of the interferon-alpha/beta pathway, which induces differential production of CD8(+) T cells and was influenced by a combination of differentially abundant microbiota and DEGs. We found that microbes in the ileum changed regularly with age. The intestinal microbiota was associated with the expression of genes in the liver through IFIH1-mediated induction of the interferon-alpha/beta pathway, which may partially explain why younger ducklings were more susceptible to DHAV-3 infection.

Effects of DHAV-3 infection on innate immunity, antioxidant capacity, and lipid metabolism in ducks with different DHAV-3 susceptibilities.

The aim of the experiment was to evaluate the status of innate immunity, oxidative status and lipid accumulation in ducklings exhibiting varying susceptibilities to DHAV-3 infection. In the experiment, ducklings with different DHAV-3 susceptibilities were used. Samples were collected at 6, 12, 15, and 24 h post infection (hpi), with 5 samples per time point. Plasma biochemistry, antioxidant enzyme activities, lipid content of liver and kidney were detected in the experiment. Elevated plasma level of total bilirubin, direct bilirubin, and creatinine indicated the injury of liver and kidney in susceptible ducklings (P < 0.05). The histopathological sections showed the injury in kidney. During the infection time, there was an increase in the concentrations of reactive oxygen species and oxidative damage markers (malondialdehyde and nitric oxide) in plasma of susceptible ducklings, particularly at 24 hpi (P < 0.05). Compared with the resistant ducklings, DHAV-3 infection resulted in a significant increase in the plasma total triglyceride (TG) level and a decrease in glucose level in susceptible ducklings. Gene expression of the innate immune response was both investigated in liver and kidney. In resistant ducklings, the expressions levels of pattern recognition receptors RIG-I, MDA5 remained constant. In contrast, the gene expressions peaked at 24 hpi in the susceptible ducklings. DHAV-3 infection promoted the expression of IFN, IL6, IL12ß, caspase-8 or caspase-9 in both liver and kidney of susceptible ducklings. In conclusion, DHAV-3 infection led to the mobilization of antioxidant defenses, alterations in lipid metabolism, and oxidative stress in susceptible ducklings during DHAV-3 infection.