Cervus elaphus), a 63 SNP set has been developed from a high-density IlluminaBovineHD BeadChip containing 777 962 SNPs after filtering of genotypes of 50stags. The successful genotyping rate was 38.6 % on the chip. The ratioof polymorphic loci among effectively genotyped loci was 6.5 %. Theselected 63 SNPs have been applied to 960 animals to perform parentagecontrol. Thirty SNPs out of the 63 had worked on the OpenArray platform. Theircombined value of the probability of identity and exclusion probability wasTo provide a cost-efficient parentage testing kit for red deer (A search for loci linked with antler quality was also performed on thegenotypes of the above-mentioned stags. Association studies revealed 14 SNPsassociated with antler quality, where low-quality antlers with short andthin main beam antlers had values from 1 to 2, while high-quality antlerswith long and strong main beams had values between 4 and 5. The chance for astag to be correctly identified as having high-value antlers is expected tobe over 88 %. Availability of SNP chip technology triggered its application inpopulation genetics  and inassociation studies .In deer, comprehensive studies have been conducted in search ofquantitative trait loci  and for the determination of theinbreeding effect on breeding success . Molecular datahave helped to confirm the genetic integrity of Carpathian red deer  and were used to seek key genes in antler development. The mechanism of antler development is alsodescribed by Gyurján et al. (2007) and Stéger et al. (2010).One aspect of methodologies is the application of a paternity test, which isalready routinely implemented for example in cattle and sheep in many countries. There is an obviousdemand from wildlife management and deer breeders to follow up the lineage,especially when important traits are logged in the herd book. The molecular genetic approach has already beenimplemented for completing this task previously. Bonnet et al. (2002)applied 11 microsatellites in three multiplex reactions, and Haanes et al. (2005) used 25 loci in six reactions for genotyping in deer.In Hungary, two groups have developed multiplex microsatellite tests forparentage testing. Zsolnai et al. (2009) used nine loci in one reaction,and Szabolcsi et al. (2014) applied 10 loci in two reactions. Theirobtained probability of identity was Here, we aimed to select and test markers suitable for parentage testing inred deer based on Illumina Bovine HD SNP chip and OpenArray platforms toreduce the costs and labour of microsatellite genotyping. We also aimed toperform a genome-wide association study, to look for loci associated withantler quality, which can contribute to a more effective breeding strategy.The selection of the Illumina Bovine SNP chip was based on its commercialavailability and the high similarity of the bovine and deer genomes .2We collected 49 red deer blood samples of stags from 10 places, includingBaltacím  reached a value from 1 to 2, while high-quality antlers(long and strong main beam) had values between 4 and 5. The low- andhigh-quality groups consisted of 24 and 25 animals, respectively. Thepreferred phenotype for the antler is to have long beams, high mass, andsymmetry. Samples were prepared and genotyped by Neogen Europe Ltd..https://webdata.illumina.com/downloads/productfiles/bovinehd/bovinehd-b1-annotation-file.zip (last access: 22 July 2022)and available in Supplement Table S1.For parentage testing, the hair roots of 960 animals were collected.DNA from hair root samples was extracted by the Chelex  procedure;8–12 hair root samples were immersed into a 100 After filtering (part a) 777 962 loci – presented on the Illumina BovineHDBeadChip – for a call rate Statistical analyses were performed by SVS SNP & Variation Suite 8.8.1.software .The performance of the SNPs selected for parentage testing was characterizedby GenAlEx 6.5 . For correction of populationstructure, the genomic kinship matrix was used in a multi-locusmixed model . The model wasSVS and the PLINK software v.1.9  were used to build amultidimensional scaling (MDS) plot using a genome-wide identity-by-statepairwise distance matrix (mds-plot 2 and cluster options). Populationstratification was calculated by SVS as described by Price et al. (2006).Python 3.6 programming language  and the matplotlib 3.2.1 library  were used for thevisualization of PCA data.Classification procedures were performed by Geneclass2 software .3To achieve the goal to select SNPs suitable for pedigree control, we tested777 962 SNPs on 50 red deer stags. After filtering for call rate and minorallele frequency, the number of retained loci was 103 562 and 25 919,respectively (filtering part a). After additional filtering (filteringpart b) for LD pruning and Hardy–Weinberg equilibrium , the SNPs proved to be adequate for statistical analyses.The genotypic principal component analysis of both 25 919 and 583 SNPs didnot reveal population stratification according to antler quality . Eigenvalues of axes 1, 2, and 3 were 5.422, 3.116, and2.339, respectively.We selected 63 SNPs for parentage testing. The probability level for theidentity of this set was In GWAS analysis date and place of birth and the father of the animalwere included in the model. A genomic kinship matrix was used to correctpossible relatedness, and 14 SNPs (Supplement Table S2) out of 25 919displayed significant differentiation between the two groups . Eigenvalues of PCA coordinates were 9.857, 5.637, and 5.004for axes 1, 2, and 3. The selected SNPs were able to position high- and low-antler-value animals into two distinct groups .Depending on the algorithm chosen within GeneClass2 software  the number of misclassified animals ranged from 3 to 6, which is 6 %–12 % of the total number of individuals.4Odocoileus hemionus and Odocoileus virginianus using Illumina Bovine BeadChip and they found 5 % polymorphic loci amongthe successfully genotyped SNPs on the Bovine BeadChip. In our case on Cervus elaphus, 13.3 % of the genotyped loci were effective, while the ratio ofpolymorphic loci among effectively genotyped loci was 6.5 %. The selected63 SNPs for parentage testing had an identity value (Haynes and Latch (2012) have achieved a 38.7 % successful genotyping rate onFor the genome-wide association study, we used data acquired from 50animals where antler quality was known. The genotypic principal componentanalysis of 583 SNPs did not divide the population by antler quality . The lambda value calculated by SVS was 1.02, indicating that therewas no hidden population stratification among the sampled animals.After filtering (part a) we found 14 SNPs that are associated withantler values (Supplement Table S2). Previously such a GWA-aided searchwas successfully applied in the case of Mangalitza pigs to findtrait-associated loci for checking quality and quantity parameters in themeat industry .Evaluating our 14 SNPs on red deer by PCA  and classificationalgorithms implemented in GeneClass2 software , we havefound that animals with a high antler value have never fallen into thelow-value group. However, several animals (4 %–10 %) from the low-valuegroup have been assigned to the high-value group.SENP1) markedly enhances androgen-receptor-mediated transcription in males, mediates cell growth anddifferentiation, and maintains male reproductive functions . Collagen alpha-1(II) chain (COL2A1) has key roles in chondrogenesisand osteogenesis ; it is upregulated in the main beams ofantlers . Vitamin D receptor (VDR) alleles are associatedwith postmenopausal osteoporosis susceptibility and bone mineral density. Dynamin 1 like (DNM1L) belongs to a superfamily ofGTPases, which are related to osteoclast differentiation and bone loss. Tyrosyl-TRNA synthetase 2 (YARS2) is coupled with theimpaired ability of the bone marrow to produce normal red blood cells . Plakophilin 2 (PKP2) overexpression was able to stop theproliferation of osteosarcoma . Zinc finger protein 518B(ZNF518B) is a putative ageing modulator . WDrepeat-containing protein 1 (WDR1) is part of a deer antler extract . Solute carrier family 2 member 9 (SLC2A9) plays a role in gout, aform of rheumatic arthritis , and has asignificant role in glucose homeostasis. Among the enriched targets ofmiRNAs there is zinc finger and BTB domain containing 49 (ZBTB49) after agrowth factor-beta 1 stimulation . The latter compound hasa high impact on bone formation and resorption .Molybdenum cofactor sulfurase (MOCOS) is involved in purine metabolism, and extracellular purine is vital for bonehomeostasis . Elongation protein 2 (ELP2)has a role in osteogenesis and osteogenic differentiation .Exocyst complex component 6B (EXOC6B) is associated with joint dislocationsyndrome . Nucleoporin 58 (NUP58) mediates moleculartrafficking to and from the nucleus and can be tied to delayed abscissionduring mitosis . Spermatogenesis associated 13(SPATA13) can weaken the migration of type I collagen via activation ofGTPase . Zinc finger homeobox 4 (ZFHX4) orchestratesendochondral bone formation . Lysine demethylase 4C(KDM4C) regulates condensin-dependent heterochromatin reorganization and isconnected with deterioration and premature bone ageing . Glycine decarboxylase (GLDC) locus is associated with overallsurvival in patients with osteosarcoma . Glutathioneperoxidase (GPX) level is higher at the upper sections of the velvet antlerand associated with lower Ca content . SMAD familymember 7 (SMAD7) directly interacts with microtubule actin crosslinkingfactor 1, initiating downstream osteogenic pathways .Dymeclin (DYM) is an important part of the extracellular matrix and has arole in bone development . Among the differentiallyabundant metabolites of ageing bone marrow mesenchymal stem cells, acetyl-CoAacyltransferase 2 (ACAA2) is related to senescence and lipid metabolism .Several candidate genes have been found in the close vicinity of the antler-quality-associated markers (Supplement Table S2). Among the candidategenes, we are mentioning those where bone- or antler-related functions areknown. Sentrin-specific protease 1  incorporating more animals into the study, (ii) developmentof a deer SNP chip based on red deer populations existing in Hungary and onexisting whole-genome sequencing , and (iii) collecting bloodat different stages of antler development and merging genotypic, phenotypic,and blood-metabolite data for deeper analysis.As for parentage testing, the identified 30 SNPs produce similardiscriminating power to that of a microsatellite set . The advantage of SNP typing lays in its cost-effectiveness comparedto microsatellites.As for antler quality predictions, the listed 14 SNPs coupled with antlerquality can be used to identify those animals at birth that are supposed todevelop good-quality antlers in their life. The chance that an animal(preselected by SNP typing) has a desirable high-valued antler isexpected to be over 88 %.10.5194/aab-65-267-2022-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/aab-65-267-2022-supplement.


CIMMYT maize lines (CMLs), which represent the tropical maize germplasm, are freely available worldwide. All currently released 615 CMLs and fourteen temperate maize inbred lines were genotyped with 180 kompetitive allele-specific PCR single nucleotide polymorphisms to develop a reference fingerprinting SNP dataset that can be used to perform quality control (QC) and genetic diversity analyses. The QC analysis identified 25 CMLs with purity, identity, or mislabeling issues. Further field observation, purification, and re-genotyping of these CMLs are required. The reference fingerprinting SNP dataset was developed for all of the currently released CMLs with 152 high-quality SNPs. The results of principal component analysis and average genetic distances between subgroups showed a clear genetic divergence between temperate and tropical maize, whereas the three tropical subgroups partially overlapped with one another. More than 99% of the pairs of CMLs had genetic distances greater than 0.30, showing their high genetic diversity, and most CMLs are distantly related. The heterotic patterns, estimated with the molecular markers, are consistent with those estimated using pedigree information in two major maize breeding programs at CIMMYT. These research findings are helpful for ensuring the regeneration and distribution of the true CMLs, via QC analysis, and for facilitating the effective utilization of the CMLs, globally. The development of a reference fingerprinting molecular marker dataset of all currently released CMLs is helpful in order to perform molecular marker-based quality assurance (QA) and quality control (QC) analysis on the increased seed stocks and to assure their genetic purity and identity. In addition, it is also useful for the breeders to better understand how to utilize the currently released CMLs in their breeding programs, based on the genetic diversity analysis and using the reference fingerprinting molecular marker dataset.Maize is one of the most important crops and is widely cultivated all over the world. Maize production of the tropically adapted germplasm occupied 30% of global maize production , and it The CMLs have been widely requested and used by both public and private institutions all over the world ; they arhttp://www.kbioscience.co.uk (accessed on 1 May 2022)) is a single-plex SNP genotyping platform that has been widely used for molecular marker-based QC analysis, linkage mapping, fine-mapping, and marker-assisted selection in maize [Compared to the traditional QC approaches of morphological comparison and isozymes electrophoresis, the molecular marker-based QC—i.e., DNA fingerprinting performed by genotyping at defined molecular markers that cover all 10 chromosomes of maize—is rapid, accurate, cost-effective, and independent to the environment. Over the past several decades, different molecular marker genotyping technologies, including restriction fragment length polymorphisms, simple sequence repeats, and single nucleotide polymorphisms (SNPs), have been applied in DNA fingerprinting and molecular marker-based QC analysis in maize ,8,9. Recin maize ,12. The in maize ,18,19,20in maize ,6. A QC in maize , and a tThe results of numerous germplasm characterization studies have shown that chip-based SNP genotyping platforms are promising and effective for performing genetic diversity analysis in collections of maize germplasms with different genetic backgrounds, although the cost per sample of chip-based SNP genotyping platforms is still high . Three gIn the present study, all of the currently released 615 CMLs (available till 2022) were genotyped with 180 KASPar QC SNPs, routinely used at CIMMYT, and two to five biological replications per CML were genotyped. The main objectives of the present study were to: (1) develop the reference fingerprinting SNP dataset of all currently released 615 CMLs; (2) implement QC analysis on two to five biological replications of each CML in order to identify suspicious CMLs in the CIMMYT gene bank with issues of genetic purity, identity, seed contamination or mislabeling during genotyping; (3) conduct genetic diversity analysis on all of the currently released 615 CMLs and 15 temperate maize inbred lines.All 180 KASP SNP markers were evenly anchored on the reference genome of B73_RefGen_V4, based on their flanking sequence information. In total, 179 SNP markers were anchored on the ten maize chromosomes, and one SNP marker was anchored on the B73_RefGen_V4_Contig104 . The numIn total, 360 alleles were detected for all the 180 KASP SNPs, with two alleles per SNP, as was expected. The SNP mutation type analysis indicated that 59.9% of the SNP mutations belong to the A/G, T/C type; 20.9% of the SNP mutations belong to the A/C, G/T type; and 19.2% of the SNP mutations belong to the A/T, C/G type. SNP is a type of mutation that occurs in a single nucleotide in the genome. In the present study, the number of KASP markers with the allelic types of A/G and T/C was higher than those with other allelic types. The summary information for all 180 KASP SNPs in the first round of genotyping dataset are shown in The genes associated with the KASP markers were used to perform GO analysis. In total, 164 of the 180 KASP SNP markers were associated with 208 genes, including 151 KASP SNP markers located in the genic region, 15 KASP SNP markers located within the two kb upstream intergenic region of a gene, and 34 KASP SNP markers located within the two kb upstream or downstream intergenic region of a gene. Among the 208 genes, 181 of them were annotated with potential function, such as Zm00001d018971, a regulatory protein of opaque-2 and Zm00001d034385, aldehyde oxidase 3, associated with drought stress tolerance . In totaThe genetic purity analysis revealed that the average heterozygosity rate across two biological replications of all the 603 CMLs was 2.29%. In total, 556 of the 1206 samples, i.e., 46.10%, had heterozygosity rates lower than 1%, and the seeds of these samples achieved the required purity level. In addition, 1058 of the 1206 samples, i.e., 87.73%, had heterozygosity rates lower than 5%, indicating that the seeds of these samples achieved the basic purity level. The 151 CMLs with a heterozygosity rate of at least one biological replication greater than 3% were used for the second round of genotyping, and are listed in The genetic identity analysis revealed that the average similarity rate between the two biological replications across all the 603 CMLs was 98.54%, indicating that most of the CMLs were essentially identical for the two biological replications genotyped in the first round of genotyping. The similarity rates between the two biological duplications of 485 CMLs were greater than 99%, indicating that these CMLs were essentially identical. The similarity rates between the two biological duplications of 93 CMLs were greater than 95%, indicating that these CMLs were identical, with slight genetic divergences caused by genetic drift. The rest of the 25 CMLs had similarity rates lower than 95% between the two independent biological replications, indicating either a genetic identity issue of the seeds or mislabeling during genotyping. Among these 25 CMLs, eleven CMLs had relatively high similarity rates, of greater than 85%, and relatively high heterozygosity rates, of greater than 5% in at least one biological replication; this result indicated that the genetic identity issue of these CMLs, including CML51B, CML95B, CML196A, CML208A, CML437A, CML473B, CML474A, CML487B, CML519B, CML521B, and CML523A, were probably caused by the issue of impurity seeds. The rest of the fourteen CMLs, including CML76A, CML80A, CML82A, CML83A, CML96B, CML100A, CML101A, CML107A, CML111A, CML112A, CML113A, CML116B, CML240A, and CML418B, had similarity rates lower than 85% between the two independent biological replications, and six of these fourteen CMLs, including CML76A, CML96B, CML100A, CML107A, CML111A, and CML240A, had relative high heterozygosity rates of greater than 5%. The genetic identity issue of these six CMLs was most likely caused by the issue of impurity seeds, and the genetic identity issue of the other eight CMLs was most likely caused by mislabeling during genotyping. In these 25 CMLs, the genetic distance between the two biological duplications of a few CMLs was greater than the between different CMLs, and they were selected in the second round of genotyping.Among the 540 samples of 180 CMLs genotyped in the second round, the heterozygosity rates of 482 samples, corresponding to at least one biological replication of 179 CMLs, were lower than 5%, including 323 samples with heterozygosity rates lower than 1%, and 111 samples with heterozygosity rates between 1% to 3%. The biological replications with heterozygosity rates lower than 3% from both rounds of genotyping were used to build the reference SNP dataset for the fingerprinting and QA/QC analyses. In addition, 13 of the 25 CMLs with similarity rates lower than 95% between the two biological replications in the first round of genotyping were identified with mislabeling issues in one biological replication in the first round of genotyping , based oThe low-density reference fingerprinting SNP dataset was developed for all of the currently released 615 CMLs through the integration of the reliable genotypic data of all the biological replications for each CML. For CML1A to CML603A, 152 SNPs were used to develop the reference SNP dataset; the SNPs in the first round of genotyping with a heterozygosity rate greater than 5%, a missing rate greater than 10%, and a MAF lower than 0.05, were excluded from the reference SNP dataset. For CML604A to CML615A, only one biological duplication for each CML was genotyped with 85 of the 152 SNPs to develop the reference SNP dataset. In total, the reference SNP dataset included 584 CMLs with a heterozygosity rate lower than 5% and an average similarity rate greater than 95% between the biological replications for each CML; 22 CMLs with a heterozygosity rate lower than 5%, and an average similarity rate lower than 95% between the biological replications for each CML; and CML179A with a heterozygosity rate of 10.08% and an average similarity rate lower of 97.94% between the biological replications . GenotypThe summary information of the 152 SNPs in the reference fingerprinting genotypic dataset in all the 584 reliable CMLs is shown in The genetic distance result for the 25 pairs of CML with pedigree-based close relationships is shown in The result of the principal component analysis in all of the currently released 615 CMLs, along with the fourteen temperate maize inbred lines, is shown in The pairwise genetic distance between all the 584 reliable CMLs varied between 0 and 0.63, with an average of 0.43 . The resThe average genetic distances between the different subgroups were calculated. The results indicated that the genetic distance between the tropical and temperate maize was greater than those among the three tropical subgroups. The genetic distance between the Temperate subgroup and each of the three tropical subgroups was 0.51. The genetic distance among each pair of all three tropical subgroups was 0.43 or 0.44.The analysis of the heterotic groups for each CIMMYT maize breeding program can facilitate the exchange and utilization of global germplasm resources. The 615 CMLs that are currently available have been divided into heterotic group A (HGA) and heterotic group B (HGB), according to the information of pedigrees and combining abilities. Within the Lowland tropical-Mexico (LLT) and East Africa (EA) CIMMYT maize breeding programs, a collection of key founder lines, including the most frequently used breeding lines and testers, were selected to investigate their genetic relatedness with each other . For LLTFor EA, 44 key founder CMLs, including 28 CMLs belonging to HGA and 16 CMLs belonging to HGB, were used to analyze the trends of heterotic patterns in this breeding program. The pedigree-based heterotic pattern was consistent with that estimated using the reference fingerprinting SNP dataset, with the exception of three CMLs mixed between the two heterotic groups, i.e., CML519B, CML544B, CML567A. This indicates that the CMLs from the same heterotic group were more closely related, and heterotic patterns are being developed in EA. In addition, a few key founder lines developed by the LLT breeding program, i.e., CML495A, CML494B, and CML576B, are also being used in EA to broaden the genetic diversity. The heterotic group information of these introduced lines is consistent with that of the EA lines, showing the feasibility of harmonization of heterotic patterns and utilization of founder lines across breeding programs, via the global germplasm exchange.The collection of currently released CMLs, which represent the diversity of improved tropical maize germplasm, has been distributed to various public and private sectors worldwide to improve a significant number of value-added traits in temperate and tropical maize. The reference fingerprinting SNP dataset for all the currently released CMLs developed in this study is helpful for the CIMMYT germplasm bank; in order to regenerate, maintain, and distribute the CML germplasm worldwide, they can genotype the regeneration seeds with the reference fingerprinting SNP dataset to assess their genetic purity and identity. Seed requesters can also use the reference fingerprinting SNP dataset to evaluate whether the obtained CML seeds are what they expect. Moreover, the reference fingerprinting SNP dataset developed by the present study is also useful for the breeders from both CIMMYT and partner institutes to better understand how to utilize the currently released CMLs in their breeding programs to select parental lines, replace testers, assign heterotic groups and create a core set of breeding germplasm .Previous studies only genotyped part of the currently released CMLs, using different genotyping platforms to perform QC and genetic diversity analyses ,6,21,29.Due to a large number of germplasm requests all over the world, regenerating, maintaining, and distributing the CML germplasms has become a significant challenge. During seed regeneration, there is a possibility of contamination with seeds or pollen . The QC Based on the first round of genotypic data, 25 CMLs were identified with the genetic identity issue, and eleven of these CMLs have both identity and purity issues; therefore, these CMLs need to first be purified, and then the reference fingerprinting SNP dataset of these CMLs need to be updated by sampling the purified new seed sources. Among these 25 CMLs, the genetic distance of seven pairs of CMLs, estimated with the reference fingerprinting SNP dataset, was zero, and most of these pairs of CMLs had similar genetic backgrounds and closer pedigree relationships. These results indicated that the CMLs with close pedigree relationships could not be distinguished by the low-density reference fingerprinting dataset of 152 SNPs and that, before release, all the CMLs candidates have to be genotyped with the same reference fingerprinting dataset as that of the currently released CMLs. In addition, the genetic relationships between the CML candidates and all currently released CMLs need to be estimated to avoid releasing any new CMLs that have close genetic relationships with the existing CMLs, and the seed purity of the CML candidates should be tested before being submitted to the CIMMYT maize germplasm bank for release. Moreover, the reference fingerprinting SNP dataset for all the CMLs can be updated as soon as the new CMLs are announced for release.The results of the genetic diversity analysis performed in the present study showed that the average genetic distance between all paired CMLs was 0.43, and 99.43% of pairs of CMLs had genetic distances greater than 0.30, indicating that high genetic diversity existed in the collection of the currently released CMLs, and most CMLs are distantly related. These results are similar to the previous studies . The resThe heterotic patterns in maize are discovered based on whether phylogenetic and geographical isolation exists, as well as being enhanced by breeders through long-term selection . The hetIn the present study, 615 CMLs and 14 temperate maize lines were used for genotyping in order to perform QC and genetic diversity analysis. Seeds of 615 CMLs were obtained from the CIMMYT Maize Germplasm Bank. The modified CTAB (cetyltrimethylammonium bromide) method was applied for DNA extraction from the leaf tissues . All of Based on the environmental adaptation, all of the 615 CMLs developed by the different breeding programs were classified into three categories: 336 CMLs adapted to the lowland tropical environment, 230 CMLs adapted to the subtropical/mid-altitude environment, and 37 CMLs adapted to the highland tropical environment . The maiIn total, 14 temperate maize inbred lines requested from the US Department of Agriculture (USDA)-Agricultural Research Service (ARS) North Central Regional Plant Introduction Station (NCRPIS) in Ames, Iowa, were used in the present study as controls to assess the genetic divergence between temperate and tropical maize. These inbred lines were primarily the Germplasm Enhancement Maize (GEM) accessions and the maize inbred lines, with expired plant variety protection. The fourteen temperate maize inbred lines, including 11 inbred lines from the Stiff Stalk Synthetic (BSSS) heterotic group, and 3 inbred lines from the non-Stiff Stalk Synthetic (NSSS) heterotic group, were referred to as the Temperate subgroup in the present study.https://excellenceinbreeding.org/toolbox/tools/kasp-low-density-genotyping-platform (accessed on 1 May 2022)) [A set of over 1200 KASPar SNP genotyping assays was developed from the mapping data against the B73 reference genome  and a SNy 2022)) , includiy 2022)) , kernel y 2022)) , qualityy 2022)) , and resy 2022)) ,37 etc.The summary statistics of each SNP in the genotypic dataset across 1206 samples of CMLs in the first round of genotyping, including PIC, MAF, missing rate, and heterozygosity rate, was analyzed using an in-house Perl script. To evaluate the uniformity of distribution, 180 SNPs were anchored to maize reference genome B73_RefGen_v4 using BLAST software and an in-house Perl script ,39. The http://bioinfo.cau.edu.cn/agriGO/index.php (accessed on 1 May 2022)) [p-value was adjusted using the hypergeometric test with a Benjamini-Hochberg false discovery rate (FDR). The visualization of GO enrichment was performed using the ggplot2 package in R.The gene ontology (GO) functional analysis was performed with the genes harboring the KASP markers using the singular enrichment analysis (SEA) with the online software AgriGO (y 2022)) . In the www.perl.org (accessed on 1 May 2022)). The genetic purity of each biological replication for all the CMLs was classified into three levels by their heterozygosity rates: purity level with a heterozygosity rate of less than 1%; basic purity level with a heterozygosity rate ranging from 1% to 5%; and heterozygosity with a heterozygosity rate greater than 5%. The CMLs with a heterozygosity rate of at least one biological replication greater than 3% were included in the second round of genotyping, with the leaf tissue collected from the individual plant and three biological replications per CML. For these CMLs, the reference fingerprinting SNP dataset was obtained by integrating the fingerprinting genotypic data of each CML from the five independent biological replications.The QC analysis was performed on 603 CMLs. First, the missing rate and the heterozygosity rate, representing the genetic purity, were calculated on each biological replication for CMLs using the in-house Perl scripts  the SNP site containing the consistent alleles in both biological replications, was merged directly; (2) the SNP site containing homozygous alleles in one biological replication and heterozygous alleles in the other biological replication kept the homozygous alleles; (3) the rest of SNP sites were set as missing. The missing rate and heterozygosity rate of each CML across the reference dataset were calculated using the in-house Perl scripts. The SNPs with heterozygosity rates greater than 5%, the missing rate greater than 10%, and the MAF  values lower than 0.05 were excluded to develop the reference fingerprinting SNP dataset and to perform further genetic diversity analyses.Principal component analysis (PCA) was performed with all of the reference fingerprinting SNPs, on all tropical and temperate maize inbred lines genotyped in the present study, using the TASSEL V5.0 software. The first three principal components were plotted using the Scatter3dplot package in R to show the genetic relationships among the subgroups adapted to different environments.To reveal the genetic relatedness among all CMLs and the temperate maize inbred lines, the identity by state (IBS) genetic distances were estimated with all the reference fingerprinting SNPs in the TASSEL V5.0 software. The average pairwise genetic distances were also estimated within each subgroup, adapted to different environments, i.e., Lowland Tropical subgroup, Subtropical/Mid-altitude subgroup, Highland Tropical subgroup, and Temperate subgroup.Based on the pedigree information and the breeding knowledge, a core set of CMLs used by the Lowland Tropical maize breeding program in Mexico and the Subtropical maize breeding programs in East Africa, including the most frequently used CMLs and testers, was selected to investigate the heterotic patterns developed by Lowland tropical-Mexico (LLT) and East Africa (EA) CIMMYT maize breeding program. The phylogenetic tree was constructed based on IBS’s genetic distance matrix for core CMLs in EA using the neighbor-joining algorithm in the ape package in R. The visualization of the phylogenetic tree was performed by MEGA7 software .In the present study, a set of 180 KASP SNPs was used to genotype all the currently released 615 CMLs to perform QC analysis, build the reference fingerprinting SNP dataset, and conduct the genetic diversity analysis. The QC analysis identified 25 CMLs having purity, identity, or mislabeling issues, further field observation, purification, and re-genotyping work on these CMLs are required. The reference fingerprinting SNP dataset was developed for all the currently released CMLs with 152 high-quality SNPs from 180 KASP SNP markers. Results of principal component analysis and average genetic distances between subgroups showed a clear genetic divergence between temperate and tropical maize, whereas the three tropical subgroups overlaid partially with each other. More than 99% of pairs of CMLs had genetic distances greater than 0.30, showing their high genetic diversity, and most CMLs distantly related. Heterotic patterns estimated with the molecular markers are consistent with those estimated with pedigree information in two major maize breeding programs at CIMMYT. These research findings are helpful to ensure the regeneration and distribution of the true CMLs via QC analysis, and facilitate the effective utilization of the CMLs globally.


Today’s consumers are increasingly aware of the beneficial effects of n-3 PUFA in preventing, delaying, and intervening various diseases, such as coronary artery disease, hypertension, diabetes, inflammatory and autoimmune disorders, neurodegenerative diseases, depression, and many other ailments. The role of n-3 PUFA on aging and cognitive function is also one of the hot topics in basic research, product development, and clinical applications. For decades, n-3 PUFA, especially EPA and DHA, have been supplied by fish oil and seafood. With the continuous increase of global population, awareness about the health benefits of n-3 PUFA, and socioeconomic improvement worldwide, the supply chain is facing increasing challenges of insufficient production. In this regard, microalgae have been well considered as promising sources of n-3 PUFA oil to mitigate the supply shortages. The use of microalgae to produce n-3 PUFA-rich oils has been explored for over two decades and some species have already been used commercially to produce n-3 PUFA, in particular EPA- and/or DHA-rich oils. In addition to n-3 PUFA, microalgae biomass contains many other high value biomolecules, which can be used in food, dietary supplement, pharmaceutical ingredient, and feedstock. The present review covers the health benefits of n-3 PUFA, EPA, and DHA, with particular attention given to the various approaches attempted in the nutritional interventions using EPA and DHA alone or combined with other nutrients and bioactive compounds towards improved health conditions in people with mild cognitive impairment and Alzheimer’s disease. It also covers the applications of microalgae n-3 PUFA in food and dietary supplement sectors and the economic and environmental sustainability of using microalgae as a platform for n-3 PUFA-rich oil production. Microalgae as a food source have been consumed for thousands of years in different geographic locations and across different cultures to provide nutrients with good safety records ,2. HowevRegarding PUFA, n-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have been extensively studied and demonstrated for their health benefits to many ailments ,5. It isThe present review attempts to provide a broad insight on the health benefits of n-3 PUFA and cover the issues with the food/feed applications of microalgal n-3 PUFA and their sustainability. The delivery systems of n-3 PUFA-rich oils are discussed, together with the technologies used to protect n-3 PUFA from oxidation. The authors are strongly inclined to a holistic approach towards the solutions concerning non-drug interventions for Alzheimer’s disease (AD) or pre-AD, which include but are not limited to nutrients supplementation together with lifestyle management, social activity engagement, as well as biomarker monitoring and control strategies. The importance of formulation in nutrition intervention is especially emphasized. In addition, sustainability and the environmental impact of the n-3 PUFA industry is critical to resolve the conflict between the limited supply and the rapid increase in the n-3 PUFA-rich oil market. However, there is limited information currently available in these areas. Bearing this in mind, we are interested in filling up this gap, at least in part, by looking into the three inter-related aspects of the sustainability of microalgal n-3 PUFA production and application industries, including techno-economic, socio-economic, and environmental impacts.A comprehensive literature search was conducted by accessing several databases, such as PubMed, ISI-Medline, and Google Scholar. The keywords were chosen based on the key objectives of this review paper that are reflected in the title, subtitles, and contexts. Published papers in the past 20 years have primarily been selected, mirroring the health benefits, food applications, and sustainability of microalgae-derived n-3 PUFA. A total of 287 references have been included in this review. In each section, the selected references were summarized and analyzed to support our statements or help to explain the differences or discrepancies between different studies. We also attempted to draw our conclusions and point out the strengths and weaknesses, and accordingly the areas or directions that can be addressed by future research and technology/product developments.Growing evidence provided by numerous scientific and clinical studies demonstrates the health benefits of n-3 PUFA, particularly EPA and DHA. The major health benefits of these fatty acids are centered on the reduction of cardiovascular disease (CVD) risks, including high triglyceride level, high blood pressure, inflammation, as well as the improvement of cardiac and vascular functions ,15,16,17Early research dating back to the 1970s revealed a low rate of cardiovascular diseases in the Greenland Inuit and other ethnic groups, such as Japanese, who eat a fair amount of fish and other marine species ,26,27. SIn the early 2010s, several clinical trials in patients with CVD failed to replicate the previous positive findings and ignited debates over the health benefits of n-3 PUFA to CVD events . For exaInteresting results were obtained in two trials published in 2018, which include (1) the Vitamin D and Omega-3 Trial (VITAL) , and (2)® in which EPA was the sole source of n-3 PUFA, provided in an ethyl ester form [The 2019 REDUCE-IT trial set up a milestone with the high dose 4 g/day) prescription drug Vascepater form . Despiteter form . These r g/day prter form .In 2021, two meta-analyses provided new insights into the benefits of EPA and DHA on cardiovascular system, the effective dosages, and the effect of EPA alone verses the combination of EPA and DHA ,40. The Microalgae have advantages over fish as the sources of n-3 PUFA, in particular the production of singular EPA or DHA-rich oils. Microalgae are potentially excellent sources of singular EPA or DHA, avoiding the costly separation and purification processes involved in purifying or concentrating EPA or DHA from fish oil [Numerous epidemiological studies have indicated that a long-term intake of the Mediterranean diet  is positively correlated with cognitive functions in aged populations ,43,44,45Accumulative evidence suggests that the brain pathophysiological changes may begin 10 to 20 years before AD is diagnosed . Once pa® is a typical example of such formulae. It is also called Fortasyn Connect®) and is a formulated dairy nutritional beverage, developed commercially as a nutritional intervention against AD and dementia based on the research work conducted by Wurtman in 2006 [® was designed to support synapse formation and function in early AD [®, and other nutrients include uridine monophosphate, choline, vitamins , phospholipids, and selenium. Several clinical trials with Souvenaid® have shown inconsistent results, with insignificant effects being seen in patients with mild to moderate AD, but certain improvements noted in subjects with mild AD dementia or MCI due to AD pathology [It has become widely accepted that neurodegeneration leads to mild cognitive impairment and AD. This neurodegeneration process is very sophisticated and involves many potential mechanisms in which an effective cure with a single therapeutic agent has yet to be found ,66. This in 2006 . Souvenaearly AD . DHA andathology ,71.The Europe LipiDiDiet trial was designed to investigate the effects of Fortasyn Connect on cognition and related measures in prodromal AD in patients with MCI due to AD pathology . The expIn addition to multi-nutrients intervention, researchers have investigated a multi-domain approach for the prevention and treatment of AD and other neurodegenerative diseases . A MultiMany inventions have combined n-3 PUFA with other nutrients by targeting an array of brain health benefits in recent years. Fortier et al.  reportedIt is known that DHA is highly concentrated in the human brain and retina, while only little EPA can be found in these tissues . TherefoSeveral randomized controlled trials in adolescents with attention deficit hyperactivity disorder have shown improvements after n-3 PUFA supplementation ,92,93. HDepression is another area where n-3 PUFA has shown beneficial effects . One stuThe effect of n-3 PUFA on cancer prevention and treatment is complicated, with some studies showing a decreased risk, while others demonstrate an increased risk ,111,112.Regarding colorectal cancer, several reviews and meta-analyses have concluded a negative association between fish or n-3 PUFA intake and colorectal risk ,120,121.The effects of n-3 PUFA on prostate cancer are controversial between studies, although a negative association was seen between n-3 PUFA intake and prostate cancer risk in some studies ,122,123.In a large clinical trial, no association was found between n-3 PUFA intake and reduction of the overall risk of cancers, including breast cancer, prostate cancer, or colorectal cancer ,128,129.In general, n-3 PUFA has been of great research interest since the late 1980s. To date, numerous studies have been conducted to determine the beneficial effects of n-3 PUFA on nutritional homeostasis, metabolic and physiological functions, health conditions, and diseases . ResearcIt should be recognized that n-3 docosapentaenoic acid (n-3 DPA) and n-6 DPA are also important PUFA but less studied, especially the latter. N-3 DPA is the metabolic intermediate of EPA and DHA and evidence suggests that n-3 DPA is an important contributor to total n-3 PUFA intake and imparts unique benefits . SeveralAs mentioned above, n-3 PUFA play crucial metabolic and physiological roles in different stages of life with significant health benefits and are indispensable to human nutrition and health ,150. TheNeither human nor fish can synthesize EPA and DHA efficiently from their precursor ALA . Marine Crythecodinium cohnii was realized commercially [Schizochytrium sp. and Ulkenia sp., of which Schizochytrium sp. is known for its ability of fast lipid production. This microalgae can increase the cell density to over 200 g/L within 72 h, with the lipid content over 40% of the biomass [Schizochytrium sp. can produce a high yield of n-6 DPA (up to 17%), with n-3 EPA up to 19% of the total lipids [Although the discovery of a valuable n-3 PUFA producing species dates back to the 1980s, it was not until the early 2000s when DHA-rich  oil from microalgae ercially ,166. The biomass . Schizocl lipids . Dependil lipids . Infant l lipids .life’sDHA™ oils were sold to 24 infant formula producers and occupied more than 70% of the world infant formula market [https://www.gminsights.com/request-sample/detail/4832; accessed on 17 March 2022).It should be realized that DHA can be retro-converted to EPA by enzymes in the human body, which offsets the health benefits of DHA in the brain development of infants, whereas ARA can suppress this conversion . As sucha market . AccordiApart from infant formulae, microalgal n-3 PUFA-rich oils are used to enrich or fortify dairy products, such as liquid milk and yogurt , meat prN-3 PUFA are highly unstable due to the presence of multiple double bonds in the backbone of the molecules. Oxidative degradation can result in poor product quality, such as off-flavors and odors ,178, dueWhile n-3 PUFA can be enriched by adding n-3 PUFA-rich oil to food, the bioavailability of n-3 PUFA is critical in achieving the expected nutritional and physiological functions and the associated health benefits. In general, the delivery form determines the bioavailability of a nutrient or bioactive compound. For example, a randomized controlled trial was conducted for a period of 28 days to assess the bioavailability of DHA in microalgal DHA-rich oils from two different algal species in a form of capsules in comparison to a microalgal DHA-fortified food . It was Nostoc, Arthrospira , and Aphanizomenon are protein-rich microalgae and have long been used to supplement protein in the human diet [Arthrospiraplatensis, Arthrospira maxima) from Texcoco salted lakes around AD 1300 [Arthrospira fusiformis) from Lake Kossorom at the northeast fringe of Lake Chad and using it for food on a daily basis. This activity is believed to date back to perhaps the 9th century [Nostoc flagelliforme, N. muscorum, and Nostoc sphaeroides have been used by the Chinese as a food delicacy and for their herbal values for hundreds of years [Aphanotheca sacrum is found in Japan and during the Edo period, the domain of Hosokawa and Akizuki conserved Aphanotheca sacrum as a material of rare country dishes [Spirogyra and Oedogonium are used as a dietary component in Burma, Thailand, Vietnam, and India [Human consumption of microalgae can be dated back to thousands of years ago when some species were demonstrated to be safe as human food ,2. Nostoman diet . In the man diet ,202. Azt AD 1300 ,204. Peo century . The har century ,207. Nosof years . These sy dishes , and they dishes . The filnd India . HoweverConsumption of microalgae has been steadily rising. Adding microalgae to food products is a great strategy to enrich diet with n-3 PUFA and many other nutrients and beneficial components. Numerous examples exist in the incorporation of microalgae in food, which are demonstrated to be safe and effective in providing n-3 PUFA and other nutrients including protein and antioxidants . AmongstNannochloropsis gaditana [Although microalgae added to food have numerous health benefits, it is important to understand the digestion, absorption, and distribution of algal nutrients. Wells et al. 2017) examined the release mode of nutrients in algal food, their transports in digestive epithelium, the influence of the gut microbiome, transformations during the digestion process, and the final fraction that reaches the body system in a strain-specific manner. These effects can be manifested via translocation across the small intestinal epithelial cells into the blood stream, interactions with the digestive epithelia, altering uptake of other substances, regulating the microbial consortium, or contacting with colonic epithelial cells of the large intestine 017 exami,219, as gaditana . If a biTM is one of the algal oil supplements in the market which is rich in EPA and DHA and sold as the capsules. Source oil Algae Omega 3, extracted from Schizochytrium sp., is another n-3 PUFA supplement approved by the FDA for human consumption [www.algarithm.ca; accessed on 15 March 2022).Dietary supplements have been gaining notable momentum in recent years for many reasons, and the main driving force is the increased concerns about chronic diseases and their associations with diet habits. Consequently, consumers are increasingly taking various supplements to promote their health and reduce the risk for developing diseases, aiming to improve life quality and lifespan ,222. By sumption . Nordic sumption . AlgaritIn addition to capsules and softgels, several studies were conducted to develop powder forms suitable for specific applications in tablet products of n-3 PUFA oils ,226. CurDunaliellatertiolecta for scallop larvae [Lobosphaeraincisa for Zebrafish [Nannochloropsis sp. for Kuruma shrimp [Schizochytrium sp. for pacific white shrimp [Microalgae have been used for many years as a feed ingredient in fish and animals . The potp larvae , Lobosphebrafish , Nannocha shrimp , and Sche shrimp , common e shrimp , tilapiae shrimp , and Atle shrimp . The ince shrimp . Microale shrimp ,241. In Schizochytrium sp., Crypthecodinium cohnii, and Phaeodactylum tricornutum have been tested successfully on gilthead seabream diet to replace fish oil [Schizochytriu sp. oil was found to be an effective substitute for fish oil in the diet of Atlantic salmon and increased the amount of DHA in the muscle [Schizochytrium sp. algal meal at 11% in the diet of Atlantic salmon post-molts resulted in similar DHA levels, but a low level of EPA in the muscle [The replacement of fish oil n-3 PUFA in fish diet is achieved primarily by substituting fish oil with microalgal biomass, while oils extracted from microalgae have also been tested. For example, oils extracted from fish oil . Thrauste muscle . The repe muscle . The cele muscle , which mAurantiochytrium sp. in dairy cows increased n-3 PUFA in milk [Isochrysis galbana and Nannochloropsis sp. in laying hen diet increased n-3 PUFA content in eggs. The inclusion of Schizochytrium sp. in lamb [Similar phenomena were observed in livestock feeding with n-3 PUFA-rich microalgae  Table 3Table 3.  in milk , Isochry in lamb , goat [2 in lamb , pig [24 in lamb , and boa in lamb  diets imPresently, due to limited production and high cost of microalgal biomass or extracted oils, the application of microalgae as a source of n-3 PUFA in terrestrial animal diet is not currently feasible. It is promising to use microalgae or extracted oil from microalgae in fish diet to meet fish n-3 PUFA requirements and enrich muscle lipid with n-3 PUFA for human consumption. From the point of view of production cost, it is more feasible to include microalgal biomass rather than extracted oil as a means of replacing fish oil. In addition to enriching fish muscle with n-3 PUFA, microalgae-based feed has also been reported to help fish develop faster and produce better fillets . The unpThe consistent rise in the global population is pressing for developing novel and alternative sources of foods and functional ingredients to meet the rising market demand for high quality edible oils, especially n-3 PUFA. It is well understood that terrestrial crops are not good sources of n-3 PUFA as the major n-3 PUFA in plant sources are ALA, LA, or ARA. The terrestrial animal products contain n-3 PUFA with better profiles, for example, containing EPA and DHA, but the contents are low or quite low. Naturally, marine species dominate the supply of EPA and DHA. Global marine fisheries catches have been stable or slightly decreasing in the past decades due to the maximal or over harvesting and the climate changes. Inversely, aquaculture has been increasing fast in the past two decades to fill up the shortage of food supply. Aquaculture species, particularly fish, also require a high amount of n-3 PUFA in their diets for health and productivity, while enriching the products with n-3 PUFA for human consumption. As such, aquaculture actually competes with humans and animals, especially pets, for n-3 PUFA or n-3 PUFA-rich oils, conventionally from fish. To overcome this challenge, microalgae have been picked up as a promising source, and this scenario is supported by the available information on the suitability and sustainability of microalgae as solutions of producing n-3 PUFA-rich oils and many other nutritional compounds for food and feed products ,253,254.2. Photobioreactors have a higher cost, while being more efficient and having a higher productivity. At present, there are a few commercial manufacturers in the world that produce high value products using microalgae. Heterotrophic fermentation system is widely used by industry because of its higher productivity and is, therefore, the main culture system for the production of algal n-3 PUFA-rich oil. Tabernero et al. (2012) assessed the economic sustainability of an industrial fermentation system and reported that the cost for the production of Chlorella protothecoides biomass was about 1.23 € kg−1 [−1, while an open raceway pond (ORP) system yields biomass of the same species at $673 tonne−1 [Nannochloropsis sp. at the industrial scale in Central Germany over a time span of 30 years [Salmosalar) raised in aquaculture, EPA from Nannochloropsis sp. resulted in about half the cultivation cost (44–60%). Major expenditures comprised photobioreactor infrastructure, maintenance, and labor cost. Higher net present value and return-on-investment are expected to increase substantially when the cultivation occurs in a region with warm climates and extended cultivation seasons [To date, most research on the techno-economic sustainability of microalgae production focuses on the production of microalgae biofuel oil ,257,258.3 € kg−1 . Hoffman tonne−1 . A recen30 years . The cul seasons .Further analysis covering the whole production processes/steps from cultivation to the final oil product indicates that the cost of microalgae oil is strongly impacted by the energy-intensive processes, from harvesting to oil extraction and, further, to oil purification. A quantitative analysis of algae harvesting–dewatering concluded that 3–30% of the total production costs come from this step ,263, whiThe cost for the production of microalgae oil is still high compared with vegetable oil and fish oil. However, the consumers of high-value food supplements (such as n-3 PUFA capsules) are willing to pay more for the same product and prefer products obtained from sources that are suitable for their particular eating habits , greater environmental awareness, and perhaps to a lesser extent, the allergic issues. Microalgae-derived n-3 PUFA-rich oil supplements meet these particular consumer requirements, and this advantage has been taken into consideration in the investment in the large scale production and exploiting whole microalgae as food ingredients. Heterotrophic cultivation is a promising mode of operation for cost reduction that involves a lower requirement of land and investment due to the smaller ratio of surface area to volume, wide range of carbon and energy sources, and reduced downstream processing costs . It is aThe algae industry is not older than 70 years and significant advancements have been made in the past decade. This industry mainly produces extracts for processed foods and other industries, such as cosmetics and medicine, while the ready-to-use of raw biomass has been widely generated as well. Similar to other industries, a sustainable algal industry can create a great value chain, which is an important step towards blue-green bioeconomy ,267. TheIt is well known that disease not only affects life quality and lifespan but also leads to substantial economic loss through different ways, particularly productivity, treatment cost, and even mortality. The impact of clinical and subclinical diseases on labor availability and quality, production efficiency and economic returns may be greater than the losses as a result of mortality. This specific socioeconomic impact could be ignored to certain extent until the severe conditions occur, for instance, when chronic conditions progress to a level that results in illness, disability, and death. Nevertheless, the negative socioeconomic impact actually happens over a long time course until being recognized. To reduce the socioeconomic impact of chronic health conditions and diseases, the availability and affordability of nutritional and functional products are crucial by means of offering disease prevention and treatment through diet habit or intake of healthy food and other forms of health products. Among a number of naturally-occurring health products, n-3 PUFA, especially EPA and/or DHA, have been intensively examined for their health benefits . As an ecofriendly, nutrient-rich dietary supplement for humans and animals, Spirulina has been promoted as a possible solution to food insecurity and malnutrition in developing countries. The increased investment occurs not only in the research funding support, but also in the infrastructure for the commercial production of algal products. Among many examples is the opening of a new plant in Nebraska of the United States by Veramaris, a joint venture of DSM and Evonik in 2019, costing $200 million, to produce n-3 PUFA oil using microalgae Schizochytrium sp. Pond Technologies, and its Stelco Lake Erie Works facility in Ontario, Canada received funds in 2019 from the Inventiv Captical Management for a total of $16 million to build/expand the algal production facility .Investment or funding support for microalgal research and technology development is another important factor to the socioeconomic impact of algal industry, including n-3 PUFA production. The current European political priorities favor a transition towards a more sustainable economy where developing algal sector has become its green and bioeconomy strategies . The EurTo date, few studies have been conducted to systemically evaluate the socioeconomic impact of algal n-3 PUFA industry, while a number of studies have been carried out to assess the socioeconomic impact of algal industry for biofuel ,273. RecThere are an increasing number of studies supporting the use of microalgae for the production of n-3 PUFA and other value-added ingredients and biomolecules, which also bring various socioeconomic benefits ,275. AltIn the past few decades, wild fish have been the most common source of n-3 PUFA. However, with the increase of global population and consumption per capita linked to the increased awareness of health benefits, apparently fish oil cannot meet the market requirement. Fisheries catches are maximized or over-exercised, limiting further increase of fish oil production from marine sources. In the meantime, aquaculture has its own issue to solve in competing for sources of DHA and EPA as feed. The reality is that the global average intake of DHA and EPA is still lower than the recommended levels ,277. In 2 in the atmosphere and various organic compounds as carbon sources for the photosynthesis of fatty acids and other biomolecules. It is apparent that microalgal cultivation can help to reduce CO2 in the air and alleviate the intensification of greenhouse impacts [Microalgae have been considered sustainable sources of n-3 PUFA and other special fatty acids because of their ability to grow and accumulate high contents of fatty acids under adverse conditions . They ca impacts ,281. At  impacts ,283. Was impacts . It has  impacts . In hete impacts ,286. Whe impacts . The furCrytthecodiniumcohnii, Schizochytrium sp., and Phaeodactylumtricornutum are some of the microalgal species that have high productivity rates of n-3 PUFA-rich oil. Several n-3 PUFA delivery strategies have been developed, including food fortification, dietary supplementation, and the addition of n-3 PUFA-rich microalgae in food products. Feeding farmed animals with microalgae can enrich animal foods with n-3 PUFA. With the advances of scientific research and technological development, it is expected that new microalgal species will be discovered that are able to produce n-3 PUFA-rich oil with better productivities and economic values. When the cost of microalgal PUFA-rich oil is reduced and becomes comparable with fish oil or other sources of n-3 PUFA-rich oils, microalgal n-3 PUFA will be used increasingly by adults, in addition to infants.Today’s consumers are increasingly aware of the beneficial roles of n-3 PUFA in preventing, delaying, and intervening various diseases, such as coronary artery disease, hypertension, diabetes, inflammatory and autoimmune disorders, neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases, depression, and many other ailments. The benefits of n-3 fatty acids on aging and cognitive functions are also one of the hot topics in scientific research and clinical studies. However, numerous longitudinal surveys in many countries revealed that a large portion of population worldwide does not have a sufficient intake of n-3 PUFA through diet. To resolve the insufficiency of n-3 PUFA intake, recommendations have been provided and promoted globally to increase the consumption of fatty fish and other seafood that contain high contents of n-3 PUFA, such as those from wild catches or aqua-farming, and also dietary supplementation of fish oil or n-3 PUFA-rich oils. With the increase of global population, the awareness of the health benefits associated with n-3 PUFA, and socioeconomic improvement worldwide, the market demand for n-3 PUFA-rich oils have been continuously increasing, especially EPA and DHA. The fisheries’ catches and aquaculture cannot meet such a fast-expanding market, and consequently a strong demand is rising in seeking alternative sources of n-3 PUFA-rich oils. In this regard, microalgae have been receiving increased academic and industrial interests due to their abilities in producing high yields of EPA- and/or DHA-rich oils and the advantages of high productivity, techno- and socio-economic sustainability, and environmental sustainability. Although the questions that we were trying to answer in this paper were identified prior to the search of literatures as mentioned in


Bemisia tabaci, which is a serious agricultural pest in the world. However, the interaction mechanism between ToCV and its whitefly vector is still poorly understood. Our previous transcriptome analysis demonstrated that the expression level of an immune-related gene, prophenoloxidase (PPO), in B. tabaci increased after ToCV acquisition, which indicates that the PPO may be involved in the interaction mechanism between the ToCV and its vector. To determine the role of the PPO in the acquisition and retention of ToCV by B. tabaci, we cloned the complete Open Reading Frames (ORF) of the BtPPOs (BtPPO1 and BtPPO2), and then structure and phylogenetic analyses were performed. BtPPOs were closely related to the PPO genes of Hemiptera insects. Spatial-temporal expression detection was qualified by using reverse transcription quantitative PCR (RT-qPCR), and this revealed that BtPPOs were expressed in all tissues and developmental stages. We found that only BtPPO1 was significantly upregulated after B. tabaci acquired ToCV for 12 and 24 h. According to the paraffin-fluorescence probe-fluorescence in situ hybridization (FISH) experiment, we verified that ToCV and BtPPO1 were co-located in the thorax of B. tabaci, which further revealed the location of their interaction. Finally, the effects of the BtPPOs on ToCV acquisition and retention by B. tabaci were determined using RNA interference (RNAi). The results showed that the RNAi of the responsive gene (BtPPO1) significantly increased the titer of ToCV in B. tabaci. These results demonstrate that BtPPO1 participates in ToCV acquisition and retention by B. tabaci.Tomato chlorosis virus (ToCV) has seriously impacted tomato production around the world. ToCV is semi-persistently transmitted by the whitefly,  Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a destructive agricultural pest worldwide. It poses a threat to crop production through direct feeding and honeydew secretion [B. tabaci can also transmit more than 300 plant viruses and lead to virus outbreaks [Crinivirus) has spread from north central Florida (USA) into many countries, including China, accompanied by the outbreak of B. tabaci [B. tabaci in many places [B. tabaci MED , and its incidence was enhanced with the increasing B. tabaci populations [B. tabaci.The sweet potato whitefly, ecretion ,2,3,4. Butbreaks ,6,7. Tom. tabaci ,9,10. Toy places ,12,13,14B. tabaci was upregulated after ToCV acquisition by B. tabaci [B. tabaci. Insect PPO is a type 3 copper-containing protein, which is involved in defense against bacteria, fungi, and viruses [Frankliniella occidentalis after acquisition of tomato spotted wilt virus (TSWV) [BtPPOs may play a role in ToCV acquisition and retention by B. tabaci, which needs to be verified.The mechanism of plant virus transmission by insects is complex and involves interactions among plants, insects, and plant viruses . Many ge. tabaci , which s viruses . Similars (TSWV) . Thus, wBtPPOs in B. tabaci, we cloned the complete ORF of BtPPOs in B. tabaci and examined its expression characteristics in different stages and tissues. We qualified the changes of the expression of these PPO genes after ToCV acquisition using RT-qPCR. We then determined the location of the ToCV and BtPPO1 by fluorescence in situ hybridization (FISH). Finally, we used the RNA interference (RNAi) method to reveal the effects of BtPPOs on ToCV acquisition and retention by B. tabaci.To study the role of the BtPPO1 and BtPPO2 were 2148 and 2151 bp  and 1.37-fold (p < 0.05) after ToCV acquisition at 12 and 24 h, respectively  (expression decreased by 30%) compared with the level in adults after feeding on a diet containing dsEGFP (p < 0.01) (B. tabaci treated with dsBtPPO1 was significantly (p < 0.001) higher than the titer in the control treated with dsEGFP  C. The vih dsEGFP B. Howevereatment D.BtPPOs in B. tabaci adults significantly decreased  compared with the level in adults after feeding on a diet containing dsEGFP (B. tabaci treated with dsBtPPO1 was significantly (p < 0.05) higher than the titer in the control treated with dsEGFP  exposed to Barley yellow dwarf virus (BYDV) [B. tabaci can be induced by Tomato yellow leaf curl virus (TYLCV) [B. tabaci [B. tabaci was upregulated after ToCV acquisition [Drosophila melanogaster increases survival after infection with Gram-positive bacteria and fungi and promotes melanization in insect host defense [BtPPOs in B. tabaci during ToCV acquisition and retention remains poorly understood.Many genes in the vector insects are involved in plant virus transmission. Meanwhile, many immune-related genes in vector insects are involved in the transmission of plant viruses ,23. For s (BYDV) . Similar (TYLCV) . ToCV ha (TYLCV) ,14. Howe. tabaci ,26. The uisition . In inseuisition ,28,29. F defense ,32,33,34BtPPOs, in B. tabaci. Alignments of the deduced amino acid sequences of BtPPOs with other insects from Hemiptera showed that the BtPPOs have three domains, namely, CuA, CuB, and a thiol ester region-like motif [B. tabaci and other Hemiptera insects indicates that BtPPOs may perform a vital function similar to other insects.We cloned the complete ORF of the immune genes, ke motif , which wB. tabaci that have significantly different expressions in various development stages and tissues. BtPPO1 has the highest expression in the abdomen, while BtPPO2 has the highest expression in the head. The expression of BtPPO1 fluctuated during all life stages of B. tabaci, and had the lowest expression in eggs, which suggests that BtPPO1 has the most important role in the life stages from larva to adult, and the second- to third-stage nymphs had greater amounts than the other developmental stages. Interestingly, the expression of a PPO, PXPPO2, in Plutella xylostella was also higher in the third instar larvae than in other stages [BtPPO1 may play an important role during larval development.Our results identified two PPO genes in r stages . These rBtPPOs, BtPPO1, could respond to ToCV acquisition , where LIYV virions were found to be retained in the foreguts of new world (NW) species of Bemisia tabaci [B. tabaci may be involved in the interaction between plant virus and vector insects.The RNAi of  foregut . The sama tabaci . Howevera tabaci . This fiBtPPO1 in the interaction between B. tabaci and ToCV, but the connection between the PPO cascade and signaling pathway requires further study. For example, there is a relationship between the PPO cascade and the Toll signaling pathway [BtPPO1 may participate in the Toll signaling pathway to control ToCV acquisition and retention by B. tabaci.We determined the function of  pathway , and upr pathway . Hence, BtPPO1 gene in B. tabaci and confirmed that the BtPPO1 gene participates in ToCV acquisition and retention by B. tabaci. The results indicated that BtPPO1 was not conducive to acquire and retain ToCV, which provides new insight into the interaction between the plant virus and the vector whitefly. However, the interaction between PPO and B. tabaci should be further studied to reveal the mechanism of the acquisition and retention of ToCV by B. tabaci.In summary, we identified and characterized the B. tabaci used in this study were initially collected in a field in Shandong Province in 2017 and maintained on cotton plants: at 27 ± 1 °C, 60% ± 5% relative humidity (RH), and a 16:8 h (L:D) photoperiod. The B. tabaci adults were identified as a MED species by using the mitochondrial cytochrome oxidase I genes (mtCOI) PCR-RFLP method [Gossypium hirsutum L. , and the tomato plants (host plant of ToCV), Solanum lycopersicum L. , were cultivated using the formula soil in a greenhouse under the conditions: 27 ± 1 °C, 60% ± 5% RH, and a 16:8 h (L:D) photoperiod.P method . The cotB. tabaci adults were used to extract total RNA with TRIzol reagent following the manufacturer’s instructions . The purity and concentration of the RNA were determined using a NanoDrop spectrophotometer . A 1 μg quantity of the total RNA was reverse transcribed to produce cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser . Based on the genome [BtPPO1 and BtPPO2). The genes were purified using a SteadyPure Agarose Gel DNA Purification Kit AG21005  and then cloned into the pMD-18T vector .About 30 e genome , gene-sphttp://web.expasy.org/translate/ (accessed on 5 May 2022)) was used to deduce the protein sequence, and the isoelectric point (pI) and molecular weight (MW) were predicted by the ExPASy proteomics tool Compute pI/MW [BtPPO1 with other PPO subfamily genes using the neighbor-joining method in MEGA X  with 1000 bootstrap replications. Amino acid alignment with other sequences was conducted with the DNAMAN v.6.03 program .The ExPASy translation tool  , first-stage nymphs (500), second- to third-stage nymphs (500) (the second and third stages are difficult to distinguish), fourth-stage nymphs (300), 50 adult females and adult males, and 100 of each tissue as a biological replicates, were used for each sample, and the expression was quantified using RT-qPCR.To investigate the expression of the BtPPOs after the acquisition of ToCV, the B. tabaci adults were fed on ToCV-infected tomato leaves for 12 and 24 h, respectively. These ToCV-infected tomato plants were used as viral sources 4 weeks post-inoculation with the virus. After B. tabaci acquired the virus, we detected the relative BtPPOs mRNA level changes by RT-qPCR. Non-virus feeding was used as a control (CK), and virus feeding for 12 and 24 h were used as treatment. Four replicates (n = 30 adults per replicate) were performed for each treatment.To understand the expression of the B. tabaci adults (acquired ToCV for 48 h) were immediately placed into the fixed fluid after 12 h. The B. tabaci was dehydrated by gradient alcohol and paraffin-embedded. The paraffin was sliced, and slices were dewaxed and dehydrated. Proteinase K (20 μg/mL) working solution  was added to cover objectives, incubated at 37 °C for 15 min, then washed three times (5 min each) in PBS (pH 7.4) . After washing in PBS for 15 min, the slices were rinsed in hybridization buffer  sodium dodecyl sulfate, 30% (vol/vol) formamide)  for the pre-hybrid (without the probe). Then, 1 μmol of the fluorescent BtPPO1 probe (conjugated with Cy3) was added into the slices. The slices were rinsed in hybridization buffer again. After that, 1 μmol of the fluorescent ToCV probe (conjugated with FAM) was added into the slices. Then, the hybridized slices were rinsed three times in hybridization buffer and incubated with DAPI  for 8 min in the dark, then mounted before taking photos with a positive fluorescence microscope . Probe sequences are listed in BtPPOs in samples that received different treatments (® Premix Ex Taq™ (Tli RNaseH Plus) , 0.8 μL of each primer, and 6.4 μL of DEPC-treated water. The optimized real-time PCR program consisted of an initial step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. After the cycling protocol, the double-stranded DNA was denatured by increasing the temperature from 60 to 95 °C (0.6 °C s−1) to obtain the melting curves. The RT-qPCR amplifications were carried out in 96-well plates , and the assays were run on a JENA qTOWER 2.2 system . Quantification of the transcript level of the genes was conducted using the 2−ΔΔCt method [The relative transcription levels of the PPO mRNA were examined by RT-qPCR. The cDNA synthesis was performed as described above. The expression stabilities of five genes, namely, β-Actin, elongation factor 1 alpha (EF-1α), nicotinamide adenine dehydrogenase (NADH), heat shock protein 90 (HSP90), and succinate dehydrogenase complex subunit A (SDHA), were also evaluated. We selected EF-1α and SDHA as endogenous reference genes for the expression of eatments . The reat method .BtPPO1, BtPPO2, and enhanced green fluorescent protein gene (EGFP) were amplified by reverse transcription PCR (RT-PCR) using specific primers conjugated with 20 bases of the T7 RNA polymerase promoter  for each treatment, dsEGFP and dsBtPPO1, and six replicates (n = 30 adults per replicate) for dsBtPPO2 and dsEGFP, were exposed to ToCV-infected tomato leaves for 24 h and used to extract total RNA to detect the virus titer in B. tabaci and determine potential knockdown of targeted genes. The effect of BtPPOs on the ability of B. tabaci to acquire the virus was verified by the above steps.Whitefly adults (incubated within 72 h) were collected and placed into a 50 mL tube . Both sides of the tube were open, and one side was separated by a layer of PTFE film that was filled with 2.5 mL of 800 ng/μL of dsBtPPOs (dsBtPPO1 and dsBtPPO2). The control group was fed with an equivalent concentration of dsEGFP. After continuously ingesting dsEGFP- and dsBtPPOs-containing diets for 72 h, the adults were fed on ToCV-infected tomatoes for 24 h. Eight replicates (n = 30 adults per replicate) for each treatment, dsEGFP and dsBtPPO1, and six replicates for dsEGFP and dsBtPPO2, were used to extract total RNA to determine potential knockdown of targeted genes and the virus titer in B. tabaci. The effect of BtPPOs on the ability of B. tabaci to retain the virus was verified by the above steps.Firstly, whitefly adults (incubated within 72 h) were exposed to ToCV-infected tomato leaves to acquire ToCV for 48 h, and then the adults continuously ingested dsEGFP- and dsBtPPOs-containing diets for 72 h. Nine replicates . The statistical significance of the gene expressions in different stages and tissues of BtPPO1 gene in B. tabaci and confirmed that the BtPPO1 gene participates in ToCV acquisition and retention by B. tabaci. The results indicated that BtPPO1 was not conducive to acquire and retain ToCV, which provides new insight into the interaction between the plant virus and the vector whitefly. We used RNAi to reveal the role of PPO in the transmission of ToCV by B. tabaci. BtPPO1 can inhibit the acquisition and retention ability of ToCV by B. tabaci. These results provide a reference for revealing the molecular mechanism of interactions between B. tabaci and ToCV.In summary, we cloned and identified the


The long-distance transport of messenger RNAs (mRNAs) has been shown to be important for several developmental processes in plants. A popular method for identifying travelling mRNAs is to perform RNA-Seq on grafted plants. This approach depends on the ability to correctly assign sequenced mRNAs to the genetic background from which they originated. The assignment is often based on the identification of single-nucleotide polymorphisms (SNPs) between otherwise identical sequences. A major challenge is therefore to distinguish SNPs from sequencing errors. Here, we show how Bayes factors can be computed analytically using RNA-Seq data over all the SNPs in an mRNA. We used simulations to evaluate the performance of the proposed framework and demonstrate how Bayes factors accurately identify graft-mobile transcripts. The comparison with other detection methods using simulated data shows how not taking the variability in read depth, error rates and multiple SNPs per transcript into account can lead to incorrect classification. Our results suggest experimental design criteria for successful graft-mobile mRNA detection and show the pitfalls of filtering for sequencing errors or focusing on single SNPs within an mRNA. The The vegetative material that is grafted onto another plant is referred to as the scion, the plant onto which the scion is grafted is referred to as the stock, see Homologous genes from closely related organisms often have highly similar sequences. Sequences that are identical cannot be distinguished. We assume that distinguishable homologous mRNAs from the two grafted plants with different genetic backgrounds (genotypes) differ in a number of nucleotide positions, single-nucleotides polymorphisms (SNPs). Other differences, such as insertions or deletions, are possible but not considered here.Given that the numbers of transported transcripts are often low compared with the number of endogenous copies  and thatOur goal is thus to develop a framework for assessing from which genotype transcripts have originated. If we sample tissue from one of the grafted genotypes , we may find SNPs associated with the other genotype . A challenge is to assess whether the RNA-Seq reads for these positions that could be indicative of transcripts that have crossed the graft junction can be explained by expected biological and technical variation in the local genotype.Here, we construct a probabilistic description of how to distinguish sequencing errors from graft-mobile transcripts using a Bayesian formalism for which we derive exact  solutions. Bayesian approaches ,22 have . 2. 2.1A and B. Different graft combinations may result in homografts, i.e. stock:scion is A:A or B:B, and heterografts, i.e. stock:scion is A:B or B:A, see In the following, genotype can be a species, an accession, an ecotype or a cultivar. We denote the two genotypes used for grafting as A and B, we can expect to find numbers of reads that correspond to A and/or B for each SNP. See Thieme et al. [N and the total number of reads that contain SNPs that correspond to the genotype that is different from the genotype of the sampled tissue by n (for short we will say n reads map to the other genotype). For instance, if we perform RNA-Seq on tissue taken from a scion of genotype A, then those reads that map best to genotype B at a certain SNP will be denoted by n. If the sample was taken from a homograft A:A and n reads map to genotype B, then these n must be viewed as ‘errors’ that occurred during the process. Potential error sources include biological variation due to, for instance, mutations or the occurrence of different splice variants, or technical issues during library preparation or polymerase chain reaction (PCR) amplification steps, mapping errors to the reference genome and errors therein, as well as bioinformatic tools and the choice of associated parameters. In electronic supplementary material, figure S1, we describe the relationships between the numbers from RNA-Seq data and the actual reads from each genetic background. The key question is whether the data support the presence of transcripts from the other genotype (e.g. from B) in the tissue of the sampled genotype (e.g. A) and how many reads we can attribute as being graft-mobile.We wish to identify homologous transcripts from the grafted plants that differ in a number of nucleotide positions (SNPs). If we perform RNA-Seq on material from the scion and stock of grafted plants and map the reads onto reference genomes of e et al.  for detaA or B). We assume that a transcript has a set of SNPs, S = {s}, for which the RNA-Seq analysis results in data in the form D = {Ds} = {Ns, ns}, where Ns is the total number of reads for SNP s, of which ns map to the other, non-sampled tissue of another genetic background, the distal genotype (denoted by ‘2’).We denote the genetic background of the sampled tissue, the local genotype, by ‘1’ /P(H1|D) [H1 and H2, are equally likely a priori, P(H1) = P(H2), the posterior odds ratio becomes equal to the marginal likelihood ratio (Bayes factor), BF21 = P(D|H2)/P(D|H1), where P(D|H1) and P(D|H2) are the marginal likelihoods, also known as evidences. If the ratio of graft-mobile transcripts to non-graft-mobile transcripts were known, then this information could be used as the prior ratio for P(H2)/P(H1). Here, we will assume a 1:1 ratio and use Bayes factors (BFs). Following Jaynes [21 of 0 means that there is an equal probability for both hypotheses, whereas logBF21 > 0 favours hypothesis 2 (graft-mobile) and logBF21 < 0 favours hypothesis 1 (errors), see Hypothesis 1 thus posits that RNA-Seq reads from only one genetic background are sufficient to explain the data, whereas hypothesis 2 requires that two genetic backgrounds are present. If statistical evidence for two genetic backgrounds is found in the data, then a plausible inference is that transcripts have moved across the graft junction. We can compare hypotheses using the posterior odds ratio, /P(H1|D) ,22. When. 2.2P(D|H1), requires integration over all parameters associated with H1, p′. We assume that these parameters can be specific to each SNP s in a transcript, S| is the cardinal number of S, i.e. the number of SNPs in the transcript. Assuming the parameters are independent between SNPs, the likelihood can be expressed as the product of the likelihoods for each SNP. The evidence for H1 over all data related to the transcript can thus also be expressed as a product, P(D|H1) = ∏s∈SP(Ds|H1), where P(Ds|H1) is the evidence for H1 for data Ds associated with SNP s. Analogously we can derive equations for H2, whereby there may be a different number of parameters associated with each SNP for H2, H1. The parameters are explained in the following sections and the electronic supplementary material, appendix. Following the aforementioned procedure, we can express the evidence P(D|H2) as a product of SNP-based evidence, P(D|H2) = ∏s∈SP(Ds|H2). The log BF of hypothesis H2  over H1  can thus be written as the sum over log BFs for each SNP s within a transcript ,The computation of the evidence, H2) or not (H1).This factorization into contributions from each SNP allows us to focus on deriving equations for a single SNP. We then sum these contributions to obtain an overall log BF for a transcript being graft-mobile .As mentioned earlier, we denote the total number of reads over a SNP by u1, u2), which results in the posterior having the same functional form,u1 and u2 are hyperparameters of the prior and α and β are the updated Beta distribution parameters of the posterior, see electronic supplementary material, appendix and figure S2.The conjugate prior to the binomial distribution is the Beta distribution, Beta. From this posterior distribution, the expected ratio of reads from transported transcripts can be computed, r2 = 〈N2〉/N for each SNP s. For a transcript, the expected ratio is given by averaging over all SNPs. n and how well N2 can be estimated for different homograft read depths.As described earlier, we can focus on each SNP individually and then combine the contributions from all SNPs in a transcript, equation . Given N. 2.5P(H1|D) and P(H2|D) can be computed from the above described distribution P(N2|D). Under hypothesis H1, any reads with SNPs associated with the distal genotype must be considered errors, i.e. there are no transcripts from genotype 2 in the data, N2 = 0. Under hypothesis H2, there are reads from transcripts from genotype 2 and N2 is a natural number. For N2 > 0, there are N possible values for N2, and we can compare the evidence for each of them against H1. The uniform prior distribution over N2  ensures that each instance of N2 > 0, which corresponds to H2, will have the same prior probability as N2 = 0 that corresponds to H1, thus resulting in an equal prior for H1 and H2 and the posterior ratio being equal to the BF. The maximum log posterior odds ratio over all possible N2 values for a transcript with |S| SNPs can now be computed from P(N2|D),N2. Given that this approach has P(H1) = P(H2), the aforementioned equation is equal to the sum of SNP-specific BFs, P(N2|Ds) is given in the electronic supplementary material, appendix. The aforementioned equation can be used to assess whether a transcript is graft-mobile from RNA-Seq data, We now show how both . 2.6As most predicted graft-mobile mRNAs from RNA-Seq data have not been validated, there is uncertainty regarding their labelling, making an evaluation of the classification performance problematic. We circumvent this problem by creating datasets for which we know exactly which transcripts are assigned to be graft-mobile and which not (see §3). The use of simulated data gives us full control over important parameters such as error rates and read depths while testing the accuracy of our method.. 2.6.1θ〉, plotted against the true error rate q for a range of q from 0 to 1 and for read depth N from 10 to 10 000. For low read depths (N = 10), the possible number of outcomes is small, leading to a visibly discretized set of inferred error rates with significant variation. For N = 100, the inferred error rates match already well to the true error rates. For N = 1000 and above, the estimates are accurately defined with high precision. This suggests that with suitably high RNA-Seq read depths (relative to the error rate), we can expect the inferred error rate per SNP to be a fair reflection of the true error rate.A first question was how well we can infer the true error rate from simulated data (see §3). Electronic supplementary material, figure S5 shows the inferred error rate, 〈. 2.6.210BF21, meaning that a value of zero arises when the data give no evidence either way, a negative value when the data is consistent with expected error rates and a positive value if graft-mobile transcripts are present, We compare hypotheses by evaluating the evidence for one hypothesis over another. Here, we use logarithm base 10 of the BF for hypothesis 2 over hypothesis 1, logq = 0.01 will not be well represented by a read depth of 100 or less.To investigate whether the BF calculation would correctly assign a negative value for cases where there are only errors (no graft-mobile transcripts), we generated datasets (see §3) for different error rates and different read depths. Data were generated that represent both homograft combinations to infer their posterior distributions for the error rates. Separate data were generated from the same process with the same error rates for each SNP and treated as heterograft data. We found that the BFs from most simulated datasets correctly favour hypothesis 1 that the observed data arose from a process with consistent error rates between homo- and heterograft data. However, as expected, there are several exceptions due to the stochastic nature of the simulations, electronic supplementary material, figure S5. The variability in BFs depends on how well the read depth captures the underlying error rate, for instance an error rate of q, then we would expect to have on average qN reads that are errors. The standard deviation of this value is θ, the expectation of which, 〈θ〉, is our best estimate for q. The detection of reads from graft-mobile transcripts is therefore limited by the available data through the variation (precision) in the inferred error rate. We conclude that the BFs perform well at the individual SNP level, but that false assignments can be expected, in particular for low read depths that fail to represent the underlying error rates.After confirming that the BFs perform satisfactorily on negative controls, we next validated the approach on positive controls. We used simulated data for which additional reads from genotype 2 were added (graft-mobile transcripts), thus making them less consistent with the inferred homograft error rates. As anticipated, the closer the number of reads from genotype 2 is to the expected number of errors, the more challenging it is to distinguish graft-mobile transcripts from errors, electronic supplementary material, figure S7. If the error rate for a SNP is . 2.7A major advantage of the proposed framework is its ability to combine the evidence across multiple SNPs within a transcript, equation . If the . 2.8per se but instead evaluating the evidence for them being graft-mobile, or not. The BFs are thus used to rank our confidence in a transcript being graft-mobile given the data. The strength of the evidence can be assessed using well-established ranges [One key advantage of using BFs is that we are not classifying transcripts d ranges ,22,35, fTo compare the presented Bayesian approach with alternative criteria for defining mobile mRNA, we defined and implemented two approaches, Methods A and B, inspired by previous publications ,18,36. Met al. [10BF21 ≥ 1 (see §3) to classify transcripts as graft-mobile and analysing the same dataset with the Bayesian method delivers both high TPRs and low FPRs, reflected in high accuracy  and a value for q. A range of values for N and q were used and are given in the individual figures. For each read, from 1 to N, a uniform random number is drawn, and if this number is greater than q, then the read is assigned to genotype 1, otherwise to genotype 2, electronic supplementary material, figure S6. The generated reads per SNP thus represent a discrete realization of a stochastic process with a defined error rate, q, with N reads assigned to genotype 1 and n reads assigned to genotype 2. The heterograft data are generated using the same process, but with the addition of further reads, N2, from genotype 2 that represent mobile transcripts. Different numbers of added mobile transcripts, N2, are used to evaluate the sensitivity of the method. For measuring the classification accuracy (see below), we use balanced datasets throughout to not distort any of the performance metrics.Simulated datasets are generated based on a binomial distribution with an error rate . 3.1.2Arabidopsis Col-0 and Ped-0 accessions [p) × Col-0 + p × Ped-0, where p is the blending proportion. For instance, p = 0.1 would include 10% of the RNA-Seq reads for each SNP from Ped-0 and add them to the reduced (1 − p) reads in Col-0. If a SNP in the Ped-0 dataset had 100 reads assigned to the Ped-0 genotype, then for p = 0.1, we would add 10 of these reads to create a graft-mobile transcript. We selected SNPs that had a comparable read depth in Col-0 and Ped-0, electronic supplementary material, figure S6. Note that this approach creates data homograft and heterograft data of similar read depths, providing favourable conditions for Methods A and B. Given the spread and limited data compared with the simulations, we used adaptive binning to provide approximately equal-sized sets to evaluate the performance of the method as a function of read depth N.While the simulated data are inherently noisy, they follow an underlying statistical model which may not capture the variability inherent in experimental data. We therefore generated labelled data based on existing RNA-Seq datasets. We took RNA-Seq homograft data from the cessions . We then. 3.221 ≥ 1 to select hypothesis 2 over hypothesis 1. It is worth noting that we could also determine transcripts for which there is strong evidence for them not being graft-mobile (e.g. those with logBF21 ≤ −1). This latter point is important for curating high-confidence datasets for training using positive and negative examples.The Bayes factor denotes the evidence provided by the data that supports one hypothesis over another ,22. We u. 3.3The classification performance is evaluated using accuracy, and true and false positive rates. Each mRNA is given one of four different labels: an mRNA that is correctly classified as graft-mobile is a true positive (TP); an mRNA incorrectly classified as graft-mobile is a false positive (FP); an mRNA correctly classified as non-graft-mobile is a true negative (TN) and an mRNA that is incorrectly determined to be non-graft-mobile is a false negative (FN). From this, we can compute the true positive rate (TPR) as the proportion of the graft-mobile mRNAs that are classified correctly from all the graft-mobile mRNAs (TP/(TP + FN)), and the false positive rate (FPR) is the proportion of the non-graft-mobile mRNAs that are incorrectly assigned out of all of the non-graft-mobile mRNAs (FP/(FP + TN)). The accuracy is calculated as (TP + TN)/(TP + TN + FP + FN). The accuracy is 0 if no classes are identified correctly  and 1.0 for error-free classification .. 4Long-distance transport of mRNAs has been shown to be important for plant development and recently also in mammalian systems . A populHere, we have presented an approach for distinguishing between expected variation  in RNA-Seq pipelines and putative graft-mobile transcripts. We set up the problem as an hypothesis-testing framework founded in Bayesian inference for which we derived an exact analytical solution. A key, yet unverified, assumption inherent to the current method and other approaches that compare with homograft data is that the biological and technical variance of the RNA-Seq analysis in homografts and heterografts will be similar.For equal prior probabilities for the hypotheses 1 and 2, the posterior probabilities ratios are equivalent to Bayes factors. Bayes factors are computed for each SNP position based on the associated read counts and then combined into a Bayes factor per transcript. Replicates can be handled analogously. Bayes factors allow transcripts to be ranked based on data supporting their graft-mobility. Higher Bayes factors are indicative of more translocated individual transcripts .We show that RNA-Seq error rates can be accurately estimated using the presented methodology  and how the read depth influences the precision  and the inferred number of graft-mobile transcripts . MultiplThe evaluation of different mobile mRNA detection methods showed that approaches using absolute number thresholds of reads per SNP on selected positions lead to high rates of mis-classified transcripts, figures The approach could be extended in several ways. We have simplified the outcome at each SNP position to map to the two genetic backgrounds used for grafting, resulting in a binomial likelihood. Including errors for all four nucleotides could provide a better description of the overall nucleotide variability, leading to a replacement of the bionomial likelihood by a multinomial distribution. The conjugate prior of the multinomial distribution is the Dirichlet distribution; however, we have not explored how tractable this approach would be to solve analytically in full. A key assumption of the presented approach is that the error rates per SNP are comparable between experiments. This assumption will need to be checked for real data. If necessary the inferred error rate distributions may need to be adjusted to take large deviations into account (e.g. by increasing the spread by reducing the Beta function parameters). Another addition would be to include sequencing quality scores within the framework. These extensions will be the subject of future developments after the careful analysis of existing datasets to assess shortcomings in the presented developments.To summarize, this contribution presents a Bayesian framework that takes account of read depths, error rates, replicates and multiple SNPs per transcript, providing a powerful means for distinguishing graft-mobile mRNA from RNA-Seq errors. As graft-mobile mRNA are often rare compared with endogeneous mRNA , RNA-Seq