Beak deformity, typically expressed as the crossing of upper and lower mandibles, is found in several indigenous chicken breeds, including the Beijing-You chickens studied here. Beak deformity severely impairs the birds’ growth and welfare. Although previous studies shed some light on the genetic regulation of this complex trait, the genetic basis of this malformation remains incompletely understood.P-value was corrected by a Bonferroni adjustment based on linkage disequilibrium pruning. The single SNP-based association study identified one associated SNP with 5% genome-wide significance and seven suggestively associated SNPs. Four high-confidence genes, LOC421892, TDRD3, RET, and STMN1, were identified as the most promising candidate genes underlying this complex trait in view of their positions, functions, and overlaps with previous studies. The pathway-based association study highlighted the association of six pathways with beak deformity, including the calcium signaling pathway.In this study, single SNP- and pathway-based genome-wide association studies (GWASs) were performed using ROADTRIPS and SNP ratio test (SRT), respectively. A total of 48 birds with deformed beaks (case) and 48 normal birds (control) were genotyped using Affymetrix 600 K HD genotyping arrays. As a result, 95 individuals and 429,539 SNPs were obtained after quality control. The Potentially useful candidate genes and pathways for beak deformity were identified, which should be the subject of further functional characterization.The online version of this article (10.1186/s12864-018-4882-8) contains supplementary material, which is available to authorized users. Loxia are characterized by the mandibles with crossed beaks [ALX1 [HMGA2 [FGF8 [Shh [BMP4 [CaM [HOXA1 and HOXD3 genes may result in beak deformity in chicks [LOC426217) and pathways (e.g. unsaturated fatty acid biosynthesis and glycerolipid metabolism) [The beak is an external anatomical structure of birds, which consists of an upper and a lower mandible . In a sied beaks , which ied beaks –9. Accored beaks . In addiks [ALX1 , HMGA2 [1 [HMGA2 , FGF8 [1A2 [FGF8 , Shh [14hh [BMP4 , 16, andMP4 [CaM . The oven chicks . Previouabolism) , 19 thatWith the advance of high-throughput genotyping platforms and computing technology, molecular markers and genes related to complex traits or diseases have been identified using genome-wide association studies (GWASs) –23. ThisIn the present study, with the aim of identifying the genetic backgrounds of beak deformity in chickens, 48 BJY birds with deformed beaks and 48 normal ones were genotyped using the 600 K high-density (HD) genotyping array . ROADTRIIn this study, a total of 48 birds with deformed beaks (case) and 48 normal birds (control) were genotyped using the Affymetrix Chicken 600 K HD genotyping arrays. According to the quality control (QC) criteria, one individual that had > 10% missing genotypes was removed. Additionally, 4505 SNPs with call rate < 90% and 120,261 SNPs with minor allele frequency (MAF) < 5% were excluded. As a result, 95 individuals and 429,539 SNPs were carried forward for subsequent analyses. The distribution of SNPs that passed QC in the chicken genome is presented in Table P-values ) for all SNPs was represented by a Manhattan plot, as shown in Fig. ROADTRIPS was used for the traditional SNP-based association analysis. In total, one associated SNP ) on GGA 3 (chicken chromosome 3), and seven suggestively associated SNPs  on GGAs 1, 3, 5, 6, 10, and 23 were detected. The detailed information of the SNP reaching 5% genome-wide significance and the seven suggestively associated SNPs is shown in Tables P < 0.05, Additional file The same genotyping dataset was used in this analysis. Based on the QC and SNP selection criteria see , a totalPEMP < 0.05), including the ribosome pathway, the oocyte meiosis pathway, the pantothenate and CoA biosynthesis pathway, the pyruvate metabolism pathway, the glycine, serine, and threonine metabolism pathway, and the calcium signaling pathway  using the SNP data in this study. This indicated that this trait is genetically determined. However, previous studies did not validate the role of any candidate genes for beak shapes and beak length in beak malformation [Excluding other environmental contributing factors, beak deformity is observed in each generation of BJY chickens and cannot be eliminated based solely on the phenotype. The heritability of beak deformity was estimated at 0.12 (SE = 0.17) by ASReml-R 3 , KLHL31 (Kelch like family member 31), GCLC , and ELOVL5 (ELOVL fatty acid elongase 5) genes, respectively, and 26 kb and 0.21 Mb downstream of LRRC1 (Leucine rich repeat containing 1) and TINAG  genes, respectively. This SNP is also located in the intron of LOC107053134, which is a predicted gene. The seven suggestively associated SNPs, rs313002111, rs16327528, rs317906090, rs316010119, rs317725514, rs14944750, and rs313486014, are located on GGAs 1, 3, 5, 6, 6, 10, and 23, respectively. They are located within, downstream or upstream of genes including TDRD3 (Tudor domain containing 3), RET (Ret proto-oncogene), and STMN1 (Stathmin 1). Two genes identified here, LOC421892 and TDRD3, were also highlighted in our former transcriptome study with a false discover rate (FDR) < 0.01 and |log2-Ratio | ≥ 1.5 [LOC421892 was down-regulated and TDRD3 was up-regulated in the deformed beaks. The LOC421892 gene is a homolog of TCEB3  gene, which is related to von Hippel-Lindan (VHL) [TDRD3, located 0.19 Mb downstream of a suggestively significant SNP (rs313002111), is related to chromatin binding and methylated histone binding [TDRD3 is associated with human Fragile-X syndrome [RET, located 0.26 Mb upstream and 92 kb downstream of two suggestively significant SNPs, rs316010119 and rs317725514, respectively, might also play an important role in the beak shaping. Its gene product is involved in neural crest development [RET can undergo oncogenic activation in vivo and in vitro by cytogenetic rearrangement [RET are also associated with the disorders such as multiple endocrine neoplasia, type IIA, multiple endocrine neoplasia, type IIB, Hirschsprung disease, and medullary thyroid carcinoma [LRIG2, was also identified as one of the most promising candidate genes underlying this trait in our previous CNV study within the same genotyped data used here [RET and LRIG2 genes might jointly contribute to the development of deformed beaks. Similarly, STMN1, also named Oncoprotein 18, located 0.35 Mb downstream of another suggestively significant SNP (rs313486014), is involved in myelodysplastic syndromes [In the single SNP-based study, ROADTRIPS was used to increase the power of the association study . PLINK wan (VHL)  and nonsan (VHL)  in human binding . Mutatiosyndrome  and primsyndrome . RET, loelopment . RET canangement , the orised here . Thus, tyndromes , and is In contrast to the single-SNP GWAS, pathway-based GWAS can identify the biological pathways that are useful to interpret the genetic basis of complex traits. The limits of pathway annotation and the power of the GWAS dataset, mean that adequate SNP coverage is essential for a pathway to be effectively tested . This meSix associated pathways were obtained, of which, the calcium signaling pathway , were used for a random mating experiment to produce 921 offspring. The incidence of beak deformity birds was 7.82%, which was higher than that observed previously in the normal population (up to 3%). Forty-eight birds with deformed beaks and 48 normal birds were randomly selected from the offspring at 20 days of age, when the crossed beaks could be clearly identified, and used for genotyping. All of these birds were observed until 90 days of age before blood collection to make sure that the phenotype was stable. Blood samples were collected from the brachial vein by venipuncture. Genomic DNA (gDNA) was isolated from blood samples using the phenol-chloroform method. The purity and concentration of the gDNA samples were measured using a Nanodrop ND-2000 spectrophotometer . The final concentration was adjusted to 50 ng/μL. gDNA samples with an A260/280 ratio of 1.8–2.0 were submitted for genotyping.http://zzz.bwh.harvard.edu/plink/). First, individuals with > 10% missing genotypes were excluded (n = 1). Second, out of the initial full set of 554,305 effective SNPs in this study, we discarded those with a call rate < 90% (n = 4505) and those having a minor allele frequency (MAF) < 0.05 in all birds . The Hardy-Weinberg equilibrium (HWE) was not used to filter data in view of the small population.gDNA samples were genotyped using 600 K Affymetrix Axiom HD genotyping arrays containing 580,954 SNPs based on the Affymetrix GeneChip platform . The gen2 < 0.2; (b) the pairwise identical-by-state (IBS) distance among the 95 individuals was calculated using the remaining 21,984 SNPs; (c) the first two MDS dimensions were extracted via the “MDS-plot” command and visualized in R .To evaluate the existence of a population substructure among the individuals, the classical MDS was performed in PLINK using the following procedures: (a) autosomal SNPs were subjected to LD-based pruning to ensure that uncorrected LD did not distort the analysis. Thus, the remaining SNPs within a window size of 50 SNPs and a step of 10 SNPs had pairwise rRM test was performed for the association analysis in ROADTRIPS (Version 1.2) [P-value was corrected using a strict Bonferroni adjustment based on LD pruning [P-value thresholds, including 5% genome-wide significance  and suggestive association . The Q-Q plot and the Manhattan plot of GWAS for this trait were produced using the “gap” package [The ion 1.2) . An impo pruning . The sum pruning . Finally package  in R.http://www.genome.jp/kegg/) to identify pathways that potentially contribute to the beak deformity trait in the chicken. A total of 162 annotated pathways were collected for analysis. Only SNPs located within or 50 kb upstream or downstream of a gene were selected to create a file linking pathways and SNP information  was employed for the analysis. The SRT compared the proportion of the significant SNPs  to all the SNPs that are part of a pathway and computed an empirical P-value (PEMP) based on comparisons to ratios in simulated datasets where the assignment of case/control status had been randomized. The simulated datasets were constructed from the original dataset, preserving the original case/control ratio, but randomizing the assignment of case/control status among individuals. The SRT accepts files in the PLINK binary format and allows the user to prepare randomized phenotype datasets. The PEMP for a particular pathway, = (s + 1)/(N + 1), where s is the number of simulated datasets that produce a ratio greater than or equal to the original ratio and N is the number of permutations [We retrieved all the pathways in chicken ) versus: (a) the number of significant SNPs in the pathways, (b) the number of SNPs in the pathways, (c) the number of genes in the pathways, (d) total length (kb) of genes in the pathways, and (e) average length (kb) of the genes in the pathways. The P = 0.05 cut-off is highlighted by a vertical red line. (JPG 105 kb)Additional file 2:Figure S2. Manhattan plots showing the association of all SNPs with beak deformity trait using PLINK. SNPs are plotted on the x-axis according to their positions on each chromosome against their association with this trait on the y-axis ). The red dashed line indicates suggestive genome-wide significance . (TIF 2715 kb)Additional file 3:Figure S3. The calcium signaling pathway. (TIF 27 kb)Additional file 4:Figure S4. The pipeline of SRT . (TIF 100 kb)Additional file 5:Table S1. Raw results of the single SNP-based association study. (XLSX 21782 kb)Additional file 6:Table S2. Single SNP-based association analysis of the selected SNPs used in the pathway-based association study. (XLSX 5391 kb)Additional file 7:Table S3. The detailed information of each pathway based on the pathway-based association analysis. (XLSX 15 kb)Additional file 8:Table S4. The detailed lists of all the chicken pathways used in the pathway-based association analysis. (XLSX 14 kb)Additional file 9:Table S5. The detailed lists of pathways and SNPs information used in the pathway-based association analysis. (XLSX 3841 kb)


BMPR1 and one SNP in COX-2 were found significantly associated with the total number of piglets born in Landrace. In Duroc, two SNPs in PLCz, one SNP in VWF and one SNP in ZP3 were found significantly associated with total number of piglets born. These SNPs explained between 0.27% and 1.18% of the genetic variance. These effects are too low for being used directly for selection purposes but can be of interest in SNP-panels used for genomic selection.Finding effective predictors of traits related to boar fertility is essential for increasing the efficiency of artificial insemination systems in pig breeding. The objective of this study was to find associations between single-nucleotide polymorphisms (SNPs) within candidate genes and fertility in the breeds Landrace and Duroc. Animals with breeding values for total number of piglets born, were re-sequenced for exonic regions of 14 candidate genes related to male and female fertility using samples from 16 Landrace boars and 16 Duroc boars  to detect genetic variants. Genotyping for the detected SNPs was done in 619 Landrace boars and 513 Duroc boars. Two SNPs in  The traditional measurements of boar fertility are pregnancy rate and litter size. However, these measurements are retrospective and highly influenced by the breeding management and the genetic potential of the sows. Finding effective predictors of traits related to boar fertility would allow for excluding the less fertile boars from pig production and improving the breeding system.PLCz) and cyclooxygenase isoenzyme type 2 (COX-2) [BMP15), bone morphogenic protein receptor 1B (BMPR1B), and growth differentiation factor 9 (GDF9) [ESR1 and ESR2) have proved to be involved in both male and female reproduction [Several genes have been proposed as candidate genes for male fertility, including phospholipase C zeta ( COX-2) . Also, w . Also, 9 (GDF9) . The estoduction . Samples2(parity)) and litter and animal  as random effects were included in the following model:In this study, we analysed male fertility of the boars as sire of their litters, and female fertility measured as fertility of the boars’ daughters for detection of genetic variants. The variance components were estimated and the heritabilities were estimated by dividing female or male variance by the total variance. The estimated variance components and estimated breeding values (EBVs) for total number of piglets born (TNB) were calculated using a univariate animal repeatability model and included both the direct genetic effect  and sire effect based on daughter fertility . In addition, herd × year, parity to the mother (parity_m), parity of the sow (parity), season as fixed effects, age of sow at farrowing within parity as fixed regression effects (age(parity), ageVWF) was added to the analysis based on previous results [The candidate genes were chosen based on previous reported involvement in fertility or in pathways related to reproduction. In total, 14 candidate genes were selected  with accession number PRJEB23828.For the association study of male and female fertility, 619 Landrace boars and 513 Duroc boars were included. All the boars in this study have been used in AI during the last 10 years, with 107,640 litters in Landrace (mean TNB = 13.7 ± 3.6) and 16,849 litters in Duroc (mean TNB = 9.3 ± 2.9). The EBVs of direct genetic effect  and maternal genetic effect  are the phenotypes used for association. Genomic DNA was extracted from blood or semen using BioSprint DNA Kit , assay design was done using Assay Design Suite software , and genotyping was done using the Sequenom massARRAY platform (Agene Biosciences) at CIGENE, Norwegian University of Life Sciences. Newly detected SNPs were submitted to the European Variant Archive (EVA) : rs331082315 in BMPR1 and rs337596396 in COX-2      has been reported to initiate the sperm binding and to induce the acrosome reaction in sperm cells in humans [ZP3 gene was found associated with male fertility in Duroc.The zona pellucida protein 3 (n humans . In thisVWF in Duroc. VWF has been suggested to have an effect in the angiogenesis in the porcine oviduct in response to seminal plasma, to be involved in the early stage of endothelium activation and to be sign of vascular bed remodelling in the oviduct [A significant SNP was found associated to male fertility in  oviduct , 15. No  oviduct .The SNPs explained between 0.27 and 1.18% of the genetic variance for TNB. The low genetic effects for TNB is supported by other association studies for litter size in pigs , 17. In BMPR1, COX-2, PLCz, VWF and ZP3 in total number of piglets born. The SNPs explain between 0.27 and 1.18% of the genetic variance for TNB, suggesting a limited contribution to the total genetic variation of the trait. Furthermore, significant SNP-effects from fixed effects models are generally overestimated, which probably means that the explained variation is overestimated. Further studies are needed to discover the functional mutations and to find other genes contributing to the genetic variation of litter size. The SNPs obtained might, however, be of interest in SNP-panels used for genomic selection, since they are likely more associated to these traits than randomly selected SNP-markers.The current study suggests a role of the genes Additional file 1. Overview over 14 genes used for association between SNP within candidate genes and fertility in Landrace and Duroc pigs.Additional file 2. Primer sequences.


Pinus radiata D.Don), capable of capturing in excess of 80,000 single nucleotide polymorphism (SNP) markers with a minor allele frequency above 0.03 in the population tested. This represents approximately 29,000 gene models from a core set of 48,914 probes. A set of 704 SNP markers capable of pedigree reconstruction and differentiating individual genotypes were tested within two full-sib mapping populations. While as few as 70 markers could reconstruct parentage in almost all cases, the impact of missing genotypes was noticeable in several offspring. Therefore, 60 sets of 110 randomly selected SNP markers were compared for both parentage reconstruction and clone differentiation. The performance in parentage reconstruction showed little variation over 60 iterations. However, there was notable variation in discriminatory power between closely related individuals, indicating a higher density SNP marker panel may be required to elucidate hidden relationships in complex pedigrees.Development of genome-wide resources for application in genomic selection or genome-wide association studies, in the absence of full reference genomes, present a challenge to the forestry industry, where longer breeding cycles could benefit from the accelerated selection possible through marker-based breeding value predictions. In particular, large conifer megagenomes require a strategy to reduce complexity, whilst ensuring genome-wide coverage is achieved. Using a transcriptome-based reference template, we have successfully developed a high density exome capture genotype-by-sequencing panel for radiata pine ( Pinus radiata D.Don) is the most widely planted exotic conifer species in the world [Radiata pine  . Estimatkilobase ; in P. tu et al.  reportedve exons . Even thve exons . In addiP. radiata is in excess of 20 gigabases [The implementation of multiple molecular based technologies in forest tree breeding requires a sufficiently large genome-wide set of DNA markers. Such resources have only been realised through the advent of new DNA sequencing and genotyping technologies, which have allowed for cheaper and effective high throughput marker discovery. Development of a genome-wide SNP panel ideally involves whole genome resequencing of an appropriate reference population , 30, andigabases , distribigabases .Pseudotsuga menziesi Mirb.) [Pinus pinaster Aiton) [Pinus radiata D.Don) [P. taeda [Picea abies) [P. menziesii) [Transcriptome-based SNP discovery has proven to be a successful alternative to whole genome resequencing. Such an approach assumes that gene-based SNPs will cover a sufficient proportion of the genome to enable the capturing of haplotype blocks that are needed for genomic predictions. It may even improve predictive ability either because the panel includes SNPs that cause trait variation, or a sufficient number of SNPs are in linkage disequilibrium with causative DNA sequence variants –43. To di Mirb.) , maritimr Aiton)  and radia D.Don) . There aa D.Don) , Affymeta D.Don) ) and gena D.Don) , 49. We a D.Don) . This meP. taeda , and suca abies)  and Dougnziesii) .P. radiata. While some studies have published SNP resources for this species [P. radiata, and initial evaluation of the efficacy of a subset of SNPs for pedigree reconstruction. This includes the development of a data processing pipeline that identified large numbers of high quality SNPs, suitable for application in several downstream genetic analyses. This SNP panel is now enabling the implementation of genomics in many facets of the New Zealand radiata pine breeding programme, to accelerate both advanced generation breeding and the development of improved clonal varieties.To date, no commercially available medium to high density SNP genotyping resources have been available for  species –55, thesP. radiata. In the first instance a small pilot set of samples (set 1) were selected to determine if the genotyping platform, Exome-capture GBS, could reproducibly identify biologically real, single-locus SNP variants, distinguishable from spurious sequencing errors. Experimentally, we made use of genotypes where variants had already been independently identified through RNA sequencing. Biologically, we also made use of parent/progeny trios to track Mendelian segregation and a maternally-derived haploid tissue, known as the megagametophyte, to confirm single-locus variants. The larger set 2, was designed to test and capture SNP variants across the New Zealand breeding populations of radiata pine and included linkage mapping populations, training populations, and putatively unrelated progenitor material.We undertook a two-step design process to first test the efficacy of exome capture in A first set of 30 samples was used to obtain some preliminary performance metrics for the pilot probe panel, and consisted of: a) genotypes used to generate the exome templates  for captP. radiata production population; and d) a single haploid megagametophytes from each of 104 of the 135 putatively unrelated trees.A second set of 1,345 samples was selected to evaluate more rigorously the final exome capture probe panel and the resulting SNP markers. This set included: a) two full-sib mapping populations, known as the quantitative trait loci (QTL) mapping population and the framework (FWK) mapping population ; b) a pr™ 2000  and DNA extracted using the NucleoSpin® Plant II DNA extraction kit , as described by Telfer et al. [® QuantIT dsDNA BR Assay kit and a Qubit® Fluorometer . A minimum of 1 μg of DNA was sent, at a minimum concentration of 20 ng/μL, to the genotyping service provider RAPiD Genomics LLC .Diploid needle tissue (100 mg) was homogenised using a Geno/Grinderr et al. . One of r et al. . Haploidr et al.  and resuP. radiata transcriptome contigs [P. taeda genome v. 1.01e [P. radiata exon templates and represented 46,342 P. taeda genomic scaffolds.Exome capture probes for the pilot 80K panel were designed by RAPiD Genomics LLC, as per the steps described by Neves et al. . RAPiD G contigs  onto thev. 1.01e . This reP. taeda reference genome v.1.01e [P. radiata transcriptome [in silico, corresponding to 18,246 gene models. An additional 42,882 probes were included in the panel to bring the total number of probes up to 80,000. These additional probes consisted of 5,211 probes from P. taeda [P. radiata exome capture probe panel designed by RAPiD Genomics LLC, therefore, consisted of 5,211 probes from P. taeda, and 74,789 probes designed from P. radiata transcriptomic sequence. Probe numbers from the pilot 80K panel were reduced to 48,914 (49K) after screening in the first 30 samples (set 1), with removal of monomorphic probes, probes that captured multiple loci, and those that were over-represented within the raw data. Inefficiencies in probe synthesis resulted in a total of 44,336 capture probes for use in the evaluation experiment (set 2 samples). Sequences for the two probe sets are included in the All possible probes (120 bp) were designed for the 449,951 predicted exon templates, yielding 1,164,086 potential probes. These were aligned to the  v.1.01e , and anycriptome , and oneP. taeda , and a fhttp://hannonlab.cshl.edu/fastx_toolkit), trimming bases with PHRED score below 20 from the 3’ end and removing reads shorter than 50 nt or containing more than 10% of bases below PHRED 20. The resulting reads were re-paired and mapped against a P. taeda reference v 1.01e [Genotyping was performed by RAPiD Genomics LLC using the exome capture genotype-by-sequencing (GBS) methods described by Neves et al , 60. DNA v 1.01e  using Mo v 1.01e  with non v 1.01e  was utilFilters were applied to the raw genotype .vcf file, in the order described in The following quality metrics were used to assess the quality of SNPs generated in the 80K pilot study: call rate per sample, read depth, call rate per SNP, polymorphic SNPs per probe, minor allele frequency (MAF), heterozygosity within haploid (megagametophyte) samples, probes that successfully capture data and probe representation within the raw data. Mendelian inheritance in trios and duos, and reproducibility between replicate samples. Departure from Hardy-Weinberg expectation and average homozygosity were not considered suitable metrics for the sample size and composition of set 1.The following quality metrics were used to assess the quality of SNPs generated in the 49K study: call rate per sample, read depth per sample, call rate per SNP, polymorphic SNPs per probe, minor allele frequency (MAF) in the unrelated 135 individuals, Mendelian segregation in the two mapping populations, linkage clustering in the one of the mapping populations, and reproducibility between replicate samples.To further examine the reliability of genotyping and the distribution of markers across the genome, we used JoinMap 4.1  to check2), used to measure composite linkage disequilibrium [A subset of SNPs were selected from those identified using the 49K probe panel for testing the efficacy of pedigree reconstruction, with the view that this subset could potentially form the basis of a smaller targeted SNP panel. The criteria for selecting SNPs for pedigree reconstruction were a SNP call rate of greater than 0.75, and an observed minor allele frequency (MAF) between 0.35 and 0.5. In addition, selected SNPs did not deviate from Hardy-Weinberg equilibrium (HWE) as assessed using a Chi-square goodness-of-fit test . SNP marilibrium , of lessAs previously described , an exclThe pedigree of individuals in the two mapping populations had been previously confirmed . To deteClone identification analysis was used to differentiate a clonal relationship between two given offspring from the other potential relationships in this study, such as full-sibs (both parents in common), half-sibs (one parent in common) or unrelated. Individuals from the two mapping populations and the 117 unrelated individuals were used in this analysis. As multiple genotypes of the four mapping population parents  were available, the relationship between these multiple samples was defined as “ramet”. The relationship between the progeny within each mapping population was defined as “full-sibs”. The relationship between parents and progeny within each population was defined as “parent-progeny”. The relationship between the 135 unrelated individuals was defined as “unrelated”. For clone identification, an exclusion analysis was conducted to evaluate whether any two plants had identical genotypes across all the SNPs used. An exclusion meant that two individuals had different genotypes for at least one SNP. The number of exclusions were calculated for all possible combinations of candidate individuals within each defined relationship group across each of the 60 panels of 110 SNPs mentioned above. The profile of the number of exclusions were compared for each of the different relationships: ramet, full-sib, parent-offspring, and unrelated. SNPs where one or both genotypes were missing from a pair-wise comparison were excluded from the analysis. Analysis results were excluded if less than 80 of the 110 SNP comparisons were available.P. radiata DNA samples in set 1 was 79,972 (99.97%). Initial SNP discovery within this small pilot study detected 284,058 SNPs , which was reduced to 49,129  after filtering  could not be assigned to a cluster at LOD 9.0, however all SNPS clustered at 2.0 < LOD < 9.0.Following the selection criteria for pedigree reconstruction, 704 SNPs were available for exclusion analysis, with MAF of the selected SNPs evenly distributed between 0.35 and 0.5 . We thenTo allow for some missing datapoints, we aimed to have a panel that contained 110 SNP markers, we generated 60 random SNP panels form the pool of 704 and assessed their discriminatory power. Almost all progeny in the QTL and FWK mapping populations were assigned to their true parents across all 60 panels . In the P. radiata and P. taeda to predict intron-exon boundaries, appears to have been highly successful, with over 99% of the original 80K probes successfully capturing genomic sequence from the prepared DNA samples. The original panel designed for P. taeda had demonstrated decreased capture efficiency associated with the presence of introns in probes [P. taeda that was used to map the P. radiata transcriptome [P. taeda genes. In addition, the linkage analysis performed in this study indicates that these gene-linked SNP markers are distributed approximately equally among the 12 linkage clusters, consistent with expectations for a species with 12 similarly sized chromosomes [The design process, which exploited the conservation in gene sequence between n probes . The refcriptome  was an icriptome . Howevercriptome , indicatomosomes .Within the numerous quality metrics we examined, the number of individual raw reads contributing to a variant being called had the greatest impact on data quality. Read depth impacted on the proportion of missing data which in turn influenced the reproducibility of genotypes between replicate samples and consistency of alleles within both parent/progeny trios and parent/megagametophyte duos. These results suggested that higher error rates were more likely in samples with low read depth due to unobserved heterozygotes being incorrectly assigned as homozygous genotypes. The decision to increase the target read depth thus allowed for improved retention of markers within the filtered pool. The impact on probe retention within the SNP pool was dramatic when the quality of individual SNPs was assessed first, and individual datapoints removed instead of entire SNPs or even whole probes. The most dramatic impact was the retention of ∼11,000 additional probes that had originally been removed due to the presence of heterozygous SNPs detected in the haploid samples. Closer examination revealed 281,391  of the putatively heterozygous SNPs had only a single read for the alternative allele. The presence of triallelic SNPs, a class we chose to exclude for further analysis, was also dramatically impacted by an initial filtering step based on read depth. Substitution error rates introduced by sequencing have been reported between 0.11% and 0.28% depending on the particular chemistry being used . The impExome capture GBS has proven very efficient at capturing SNP markers, including low frequency ones. Over 94% of the markers captured in our large scale 49K evaluation panel were present at a MAF of less than 0.03. As new populations are genotyped, we will continue to capture additional variants and grow a SNP marker resource for downstream analyses. This is an advantage of sequencing-based approaches over fixed array SNP genotyping platforms which are limited to interrogating predefined SNP loci. Non-target or adjacent SNP markers and insertions/deletions, previously undocumented in a population, can also negatively impact the quality of genotypic scores generated from SNP arrays, which generally use shorter probes of 30-50 bases. The length of the exome capture probes (120 bp) gives improved specificity in terms of single-locus binding, as well as improved tolerance for undocumented polymorphism in the binding region. The impact of a rare SNP variant resource could be beneficial for a number of downstream applications and if exome capture GBS is used operationally, it is a resource that will continue to grow.P. taeda showed that SNP markers are similarly prevalent in both genic (53%) and non-genic (42%) regions, the average allele frequency within the two classes was not discussed [Under the SNP selection criteria developed for inclusion in pedigree reconstruction (MAF 0.35-0.5), only 704 (0.05%) were suitable for this purpose. It is possible that the low percentage of SNPs with intermediate frequency alleles, present in the representative 135 putatively unrelated individuals, could relate to the nature of exome capture as a source of SNP discovery, as a higher degree of conservation would be expected for genic regions. While a recent study in iscussed . Howeveriscussed . Over 50iscussed . Once thiscussed . Therefo2 = 0.886 [All 60 test panels performed well for pedigree reconstruction using exclusion analyses, although there was a marked difference in the discriminatory power between true ramets and full-sib relationships. However, the ability to distinguish material in the more distantly related classes was not as clear, indicating that a larger pool of SNPs, such as a medium density fixed array, may be required for pedigree reconstruction where hidden relatedness is present. All genotyping platforms, particularly those based on genotype-by-sequencing, can result in incorrect genotypes being called, particularly dependent on the variant calling and filtering algorithms employed to generate a variant call file , 69. For = 0.886 . To reduP. radiata from transcriptomic data, using a related reference genome. This resource enables interrogation of SNPs at a level that has not previously been reported for this species. The final 49K probe set captures an estimated 54% of the predicted gene models in P. taeda and was able to generate a large pool of SNPs  for downstream applications, such as genomic selection, pedigree reconstruction, clone identification, association studies and linkage mapping. The majority (94%) of the SNP markers identified are present at low to extremely low minor allele frequencies (MAF < 0.03). The potential of rare variants within populations for genomic predictions will need to be explored. The linkage clustering results indicate that most SNPs behave as discrete single locus markers consistent with Mendelian expectations. Precise genomic coverage is yet to be determined, but the initial results show good co-location and approximately similar distribution across all chromosomes.We have successfully developed a high-density exome capture GBS probe panel for Through the initial 80K pilot study and subsequent evaluation process we identified a minimum read depth of 20X to accurately distinguish sequencing errors from genuine SNP markers. Improvements made to the capture protocols are now achieving an average read depth of 60X with the same initial sequencing strategy. We also developed filtering pipelines which focused on data quality prior to removing probes or individual SNPs which showed evidence of capturing multiple loci or had more than two alleles present in a population of interest. This has increased the number of gene models included within the filtered dataset from 22,740 to 27,260. The result is that a greater portion of the genome is being captured and is available for use in downstream analyses.P. radiata training populations, POP2 and POP3 were genotyped with the optimised 49K probe panel, and first proof of concept genomic estimated breeding values are presented in [We successfully identified 704 SNP markers from within the pool of SNPs which met our criteria for pedigree reconstruction and clone identification. We were able to assign parentage correctly in two large full-sib mapping populations, with as few as 70 SNP markers in most instances. The impact of missing datapoints in progeny or parents with low call rates was most likely the cause of the observed parental misassignments. Therefore, we recommend a set of 110 SNPs that can be used by our industry stakeholders for pedigree reconstruction and clone identification for radiata pine in New Zealand, to be used for the development of an operational SNP-based parentage assay using an affordable, scalable, and robust genotyping platform. Our results indicate that this set of SNPs will be robustly effective for distinguishing clonal from first-degree (i.e. parent-offspring and full sib) relationships. However, more complex pedigree reconstruction tasks  will likely require using much larger numbers of SNPs and more sophisticated methodology to account for genotyping error and rates of false positive and false negative classifications. This will most likely be achieved through design of a medium density fixed-array genotyping resource using these results. Full pedigree information will become achievable for open-pollinated and mixed-seedlot trees, provided all possible candidate parental genotypes are available for comparison with potential progeny genotypes. Generating a complete (or near-complete) database of potential parents reflective of our breeding and production populations will be a priority going forward. In addition to pedigree reconstruction, the clonal ented in .P. radiata is the wealth of additional information that is captured. Rare variants, seldom captured in fixed arrays, are available for analysis. Flanking genomic sequence is also available, which can prove beneficial in the development of low density SNP panels that are more suitable for pedigree reconstruction and routine clone identification. Well-evaluated SNPs can similarly be converted to robust fixed array medium density SNP panels, suitable for cost-effective screening of 10,000s of seedlings. These panels will enable New Zealand’s radiata pine breeding programmes to move towards the implementation of genomic selection, increasing the delivery of genetic gain through shortened generation intervals, which is increasingly important to select for resilience to new abiotic and biotic threats.The benefit of developing an exome capture GBS panel for S1 TableThe total number of progeny that were used in the analysis was 93 in the QTL mapping population and 82 in the FWK mapping population.(DOCX)Click here for additional data file.S1 FileThe sequence of probes designed for 80K pilot.(FA)Click here for additional data file.S2 FileThe sequence of probes designed for 49K pilot.(TXT)Click here for additional data file.


Zeolite synthesis is a wide area of study with increasing popularity. Several general reviews have already been published, but they did not summarize the study of imidazolium species in zeolite synthesis. Imidazolium derivatives are promising compounds in the search for new zeolites and can be used to help understand the structure-directing role. Nearly 50 different imidazolium cations have already been used, resulting in a variety of zeolitic types, but there are still many derivatives to be studied. In this context, the purpose of this short review is to help researchers starting in this area by summarizing the most important concepts related to imidazolium-based zeolite studies and by presenting a table of recent imidazolium derivatives that have been recently studied to facilitate filling in the knowledge gaps. The term zeolite (from the Greek word “boiling stone”) was first coined by A. F. Crönstedt in 1756 . New syn4 tetrahedra linked together by the O atoms that are on the vertices  tetrahedra. This principle is known as the Loewenstein rule decyl)-1H-imidazol-3-ium. TON zeolites were often synthesized from the same imidazolium derivatives as the MFI zeolite, such as with 1,3-diethylimidazolium or 1-(propan-2-yl)-3-(6-[1-(propan-2-yl)-1H-imidazol-3-ium-3-yl] hexyl)-1H-imidazol-3-ium. Therefore, it may be possible to obtain the MFI type in cases where the TON zeolite was already achieved and vice versa. The length of the terminal alkyl chains has also been an object of study, such as in the article by Wen et al.  tetradecyl)-1H-imidazol-3-ium.Another two types of zeolites seemed to follow a similar rule: BEA* and FAU. Both have been synthesized using imidazolium derivatives with voluminous lateral groups, such as 1,3-diisobutyl-imidazolium or 1,3-bis(cyclohexyl)imidazolium. Comparing the cell parameters or framework densities, the two zeolite types appear to be very different at first sight . Howeverfor STW) . In addiIt is also important to note that imidazolium derivatives have certain control of the flexibility around the imidazolium ring; that is, the substituent can be chosen based on their conformational possibilities. An example of this fact is the work published by Variani et al. . The catSecond, the imidazolium derivatives can also exhibit a templating effect in which their directing influence is not only established by their geometry but also by their electronic configuration. Imidazolium cations are able to form π-π interactions with each other through their imidazolium rings , which mH-imidazol-3-ium), but further studies and tests of different conditions would probably lead to other zeolite types. Therefore, in this sense, it is interesting to continue studying different conditions with different imidazolium derivatives because to date, there is not enough proof to state that any imidazolium derivative is a pure structure-directing agent for only one zeolite type in the presence of different heteroatoms. In this sense, studying the hydrophobicity of the OSDAs can help explaining their role. Kubota et al. [The last role of OSDAs that was described in a et al.  already a et al. . The derFinally, the continued study of imidazolium derivatives in zeolite synthesis is important, as they proved to be interesting in their ability to form new types of zeolites ,25,66 foIn this article, the state of the art techniques relevant to the synthesis of zeolites with imidazolium derivatives were summarized, and a brief introduction on zeolites and zeotypes was presented. Several questions related to the synthesis and structure of these materials were addressed based on analysis of the literature. It was shown that imidazolium derivatives can act as space-fillers, templates and structure-directing agents. Finally, a list of ionic liquids employed in zeolite and zeotype syntheses was presented in relation to the obtained zeolitic framework in an attempt to help researchers with experimental planning and with the discussion of results in an area that it is important for zeolitic research and is difficult to keep up to date.


Citrus trifoliata and Citrus clementina, we performed genome resequencing on the two citrus species. Compared with the citrus reference genome, a total of 9,449,204 single-nucleotide polymorphisms (SNPs) and 846,615 insertion/deletion polymorphisms (InDels) were identified in the two citrus species, while 1,868,115 (19.77%) of the SNPs and 190,199 (22.47%) of the InDels from the two citrus species were located in the genic regions. Meanwhile, a total of 8,091,407 specific SNPs and 692,654 specific InDels were identified in the two citrus genotypes, yielding an average of 27.32 SNPs/kb and 2.34 InDels/kb. We identified and characterized the patterns of gene exchanges in the grafted citrus plants by using specific genetic variation from genome resequencing. A total of 4396 transporting genes across graft junctions was identified. Some specific genetic variation and mobile genes was also confirmed by Sanger sequencing. Furthermore, these mobile genes could move directionally or bidirectionally between the scions and the rootstocks. In addition, a total of 1581 and 2577 differentially expressed genes were found in the scions and the rootstocks after grafting compared with the control, respectively. These genetic variations provide fundamental information on the genetic basis of important traits between C. trifoliata and C. clementina, as the transport of genes would be applicable to horticulture crops.To identify the genetic variation between  Grafting has been used in a number of plant species, including fruits, vegetables, and ornamentals, for over 2000 years of history to improve plant production, water-use efficiency, and other fruit quality traits, as well as deal with abiotic stress ,2. GraftRecently, many transcriptome and proteome studies have investigated the rootstock–scion interaction in various plants that occur at the early phase in response to grafting ,8,9. ForCitrus grandis (Linnai), and found that a large number of genes were involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction. However, the underlying molecular and genetic mechanisms for how the graft partners interact with each other to produce a successful graft remain largely unknown. The recent release of the citrus genome dramatically enhanced the efficiency of grafting research in citrus [Citrus trifoliata) and Clementine mandarin (Citrus clementina), and a large number of putative genetic variations were identified based on the citrus reference genome. The distribution and predicted function of these genetic variations were analyzed in detail. To evaluate this genetic variation dataset, some genetic variations were amplified and sequenced using the Sanger method. Using specific genetic variations derived from Citrus trifoliata and C. clementina as markers, we identified the patterns of genome-wide gene exchanges between C. trifoliata and C. clementina, and provided insights into how genome-wide gene exchanges between scions and rootstocks may contribute to the genetic success of grafted plants.Citrus is one of the most important fruit trees in the world. To affect citrus trees’ vigor, size, precocity, fruit quality, taste, and harvestable yield, the use of rootstocks is very popular in citrus ,5. To obn citrus . Here, wC. trifoliata was the rootstock and C. clementina was the scions, was used to identify the genes that moved across the graft union. All of the plants were grown in the greenhouse of the National Citrus Breeding Center at Huazhong Agricultural University  at a temperature of 25 ± 1 °C with 60–70% relative humidity. The juvenile material from C. trifoliata was obtained from the seeds of the adult mother plants. Since the embryo originates from a nuclear cell, the seedlings have the same genetic background as the mother plants. Seeds of C. trifoliata were sown in 20-cm pots containing commercial potting mix and perlite . Following germination, the seedlings were watered regularly with a nutrient solution. The rootstock trial contained three replicates of grafts involving three scions and three rootstocks grown in the greenhouse. The scions were grafted to six-month-old rootstocks. In this study, the scions from C. clementina were grafted to six-month-old C. trifoliata as an experimental grafting system  . For resC. trifoliata and C. clementina were aligned to the citrus genome  [http://bio-bwa.sourceforge.net/) software with default parameters [http://www.broadinstitute.org) was used to realign reads [The DNA sequencing reads from mentina)  separaterameters . Furtherrameters . ‘Mark dgn reads . Finallygn reads .http://circos.ca/) [From the variant call format (VCF) files obtained through filtering, SNPs and InDels were annotated based on genomic location and classified by the likely effects of the variations, including functional categories using SnpEff . The Snpcos.ca/) .http://hannonlab.cshl.edu/fastx_toolkit/) [http://www.stargroup.uwaterloo.ca/) [Total RNA was extracted using TRIzol reagent , and then treated with RNase-free DNase I, according to the manufacturer’s instructions. The total RNA was sent to Beijing Genomics Institute , where the libraries were produced and sequenced using Illumina’s Genome Analyzer (Solexa). Raw sequence reads were filtered for quality using the FASTX-Toolkit (oolkit/)  with defoolkit/)  using STloo.ca/) . Differeloo.ca/) . In thisloo.ca/) . Gene anloo.ca/) . The datTo detected SNPs in RNA-seq, we merged the three biological replicates to increase the coverage of RNA-seq. Following alignment, the coverage of each genomic position by base A, G, C, and T was calculated based on the mpileup file generated by SAMtools . Only loβ-actin. Three biologic repeats were assayed in this study.The differentially expressed genes were confirmed by real-time with SYBR green I (SG) chemistry , as described previously . The priC. trifoliata and C. clementina, two resequencing libraries from the two genotypes of citrus were constructed. A total of 121 and 125 million reads were generated for C. trifoliata and C. clementina, respectively. In C. trifoliata, 110.4 million (90.94%) reads were mapped to the reference genome, representing 84.40% of the citrus reference genome. In C. clementina, 118 million (94.36%) reads were mapped to the reference genome, representing 97.57% of the citrus reference genome, and indicating that the generated dataset was highly relevant with the reference genome.To identify the genome-wide genetic variation between C. trifoliata and C. clementina were identified by comparing with the reference sequence, respectively. After combining the data, a total of 7,744,203 and 3,062,798 SNPs were detected in C. trifoliata and C. clementina compared to the citrus reference genome, respectively. The detected SNPs from two citrus species were classified into two groups on the basis of nucleotide substitutions: transitions (A/G and C/T) and transversions . In C. trifoliata, the identified SNPs comprised 8,243,171 transitions and 4,770,130 transversions. Among these detected SNPs, 2,475,105 (31.96%) were heterozygous, and 5,269,098 (68.04%) were homozygous  were heterozygous, and only 52,120 (1.70%) were homozygous  insertions and 396,912 (52.97%) deletions; C. clementina had 115,624 (46.33%) insertions and 137,055 (53.67%) deletions (C. trifoliata) and 1174 bases (C. clementina).The genome-wide SNPs and InDels of mozygous A. The obmozygous A. Consideletions B. The apC. trifoliata and C. clementina were widely distributed to each chromosome. However, there was a uniform distribution of SNPs and InDels in the nine chromosomes of both citrus species (C. trifoliata were identified throughout nine chromosomes. Chromosome 3 (chr 3) showed the highest number of SNPs , and very few SNPs  were detected on chromosome 7 , was used for analyzing the chromosomal distributions of the identified SNPs and InDels . The SNP species . A totalmosome 7 C. The deementina D. The frribution E. For C.in chr 7 F. The reC. trifoliata and C. clementina, we compared the SNPs and InDels from two citrus species. A total of 9,449,204 SNPs were identified in the two citrus species, of which 1,357,797 were identified to be common, and 8,091,407  were specific in the two genotypes , 521,094 (33.30%) were located in intron regions , and 661,317 (42.24%) were located in CDS regions . The number of non-synonymous SNPs was 280,502 (52.83%) and 71,705 (55.01%) in CDS regions of C. trifoliata and C. clementina, respectively. Meanwhile, 5636 (1.06%) and 1656 (1.27%) other SNPs gave rise to variants in the start or stop codons of coding genes in C. trifoliata and C. clementina, respectively  were specific in the two citrus species  were specific to C. clementina and distributed in 9250 genes , 80,948 (53.06%) were located in intron regions , and 12,656 (8.29%) were located in CDS regions . Within the gene body, the maximum number of specific SNPs (C. trifoliata: 41.82% and C. clementina: 44.12%), and only a small proportion of specific InDels (C. trifoliata: 7.63% and C. clementina: 12.72%) from genic regions were present in the CDS regions , and 366,991 deletions (one to five bp) were observed. The frequency of different types of InDels varied, and was negatively correlated with the number of nucleotides. Mononucleotide InDels  were the most frequent InDel in C. trifoliata and C. clementina, following by dinucleotide  and trinucleotide InDels . The specific SNPs and InDels from C. trifoliata and C. clementina were also visualized in the citrus reference chromosome using Circos  and 3,679,148 (98.10%) SNPs were classified as sequence modifiers (introns or affecting non-coding genes), and a moderate effect accounted for 279,357 (1.88%) and 71,394 (1.90%) of the SNP in C. trifoliata and C. clementina, respectively  and 2133 (0.06%) in C. trifoliata and C. clementina, respectively  missense SNPs and 246,325 (46.37%) silent SNPs, with a 1.14 missense/silent ratio. In C. clementina, 1285 SNPs (0.99%) were assigned as high effect and comprised 71,705 (55.01%) missense SNPs and 57,359 (44.01%) silent SNPs, with a missense/silent ratio of 1.25  and C. clementina 664 (0.26%). The remaining high-effect variants accounted for 5472 (0.32%) and 1483 (0.58%) in total in C. trifoliata and C. clementina, respectively  were selected randomly and verified in two citrus species . Of thesTo detect the gene exchange between scions and rootstocks, we devised a strategy based on grafting two different citrus species displaying a high frequency of genomic sequence SNPs. The species-specific SNPs that were present in long RNA molecules allowed us to identify their origin—and thus, their mobility—in grafted plants . Two setC. trifoliata and C. clementina  term analysis may provide valuable hints regarding their functions during the grafting process. For the categories based on biological processes, a GO term analysis of the 2450 transmitting genes from the scions to the rootstocks C and 241nd stems . Nine ovTo analyze the transcriptome changes of scions and rootstocks during the grafting process, RNA-seq was performed on the stems and leaves after grafting and before grafting. In this study, 1510 genes were DEGs after the grafting of leaves compared with the before-grafting leaves, of which 1085 genes were up-regulated and 425 were down-regulated based on a probability ≥0.8 and an absolute log2 fold change value ≥1 A. MeanwhWe performed GO term analysis on the 1549 and 2470 DEGs from scions and rootstocks, respectively. The analysis revealed that these DEGs were involved in 12 and seven main biological processes in scions and rootstocks, respectively. The three largest groups were the single-organism process, oxidation–reduction process, and carbohydrate metabolic process. A comparative analysis of the biological processes from scions and rootstocks indicated that the cell wall organization or biogenesis, response to oxidative stress, and movement of cell or subcellular components were enriched in scions, and photosynthesis was enriched in rootstocks. An overview of biotic stresses was performed with MapMan software to visualize the stress pathways in which DEGs were involved . In the C. trifoliata and C. clementina. Genome resequencing is an effective strategy to discover a large number of genetic variants in plant genomes. Therefore, genome analysis based on resequencing these two citrus species was used for the comprehensive identification of SNPs and InDels. A total of 9,449,204 SNPs and 846,616 InDels were identified between C. trifoliata and C. clementina. These detected genetic variants from this study will expand the genomic resources that are available for citrus, and can be used to develop genotyping platforms to perform a marker–trait association study. In addition, we identified and characterized the patterns of genome-wide gene exchanges across graft junctions in grafted citrus using diagnostic SNPs derived from species-specific SNPs. The large genomic-scale exchanges of genes between scions and rootstocks could be the key genetic basis of the superior performance of grafted plants.Gene-based molecular markers are increasingly used in crop-breeding programs for marker-assisted selection. However, the identification of genetic variants that are associated with important agronomic traits has remained a difficult task in citrus . One of C. clementina and C. trifoliata. Interestingly, the number of specific genetic variations (including SNPs and InDels) from C. clementina was less than that of C. trifoliata in this study, which indicates that C. clementina is more closely related to the reference genome than C. trifoliata. Among the SNPs of the CDS regions, the frequency of non-synonymous substitutions was much higher than that of synonymous substitutions. Similar mutagenic effects were also observed in other citrus species [C. trifoliata and C. clementina. Therefore, the high frequency of non-synonymous changes and frameshift InDels could help explain the differences in agronomic traits between C. trifoliata and C. clementina.There has been concerted interest in the last few years in the analysis of genetic variation in some important horticultural plants ,41. Furt species . Non-syn species . MeanwhiArabidopsis that was previously reported [Successful grafting may improve the quality and production of the horticultural plants by enhancing nutrient absorption and conferring resistance to many biotic and abiotic stresses in the graft units . The lonreported .Arabidopsis into the parasitic Cuscuta in the Cuscuta–Arabidopsis study [Comparisons of the abundance, movement directions, and patterns of transmitting genes in the stem and leaves revealed an important fact: while thousands of genes could transmit their mRNAs between scions and rootstocks, only a small number of them might reach certain tissues to become biologically relevant (approximately 13.83% of the transmitting genes were shared between the stems and leaves). For example, a high number of mRNAs were found to be transported from is study . Howeveris study ,47. Baseis study ,19,48. Iis study ,46. ThesCitrus grandis L. Osbeck) and (C. trifoliata and fragrant citrus) rootstocks, and a total of 1950 DEGs were identified. The KEGG pathway enrichment analysis revealed that these genes were involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction. Liu et al. [A larger signaling molecules exchange between scions and rootstocks through the graft union, and small differences in signal concentration or in their receptors/targets, can alter gene expression. Therefore, we analyzed the transcriptomes of the leaves and stems of grafting plants during the grafting process. A total of 1549 and 2470 DEGs were identified in scions and rootstocks, respectively. For the categories based on biological processes, the analysis revealed that these DEGs were associated with the oxidation-reduction process, carbohydrate metabolic process, and abiotic stimulus responses. He et al.  studied u et al.  performeu et al. . The anaC. trifoliata and C. clementina. A total of 9,449,204 SNPs and 846,615 InDels were identified in the two citrus species. Meanwhile, a total of 8,091,407 specific SNPs and 692,654 specific InDels were identified in the two citrus genotypes. These genetic variations provided a foundation for the further exploration of citrus diversity and gene–phenotype relationships, and for future research on molecular breeding to improve citrus and related species. Using these diagnostic SNPs derived from high throughput genome sequencing, we identified and characterized the patterns of genome-wide mRNA exchanges across graft junctions between scions and rootstocks. More than 4000 genes transporting mRNAs were identified across graft junctions. These genes were involved in diverse biological processes such as metabolism, hormone signal transduction, and transcriptional regulation. In addition, a total of 3326 DEGs were also found in the scions and the rootstocks after grafting was compared with the control. The results that were obtained in this study further improve our knowledge of the molecular and metabolic changes that are involved in this interaction between the rootstocks and the scions.Here, we performed whole genome resequencing to reveal the comprehensive genetic variation between


Olea europaea L. subsp. europaea var. europaea), the most important oil tree crop of the Mediterranean basin, has been poorly evaluated up to now. A deep sequence analysis of fragments of four genes, OeACP1, OeACP2, OeLUS and OeSUT1, in 90 cultivars, revealed a wide range of polymorphisms along all recognized allele forms and unexpected allele frequencies and genotype combinations. High linkage values among most polymorphisms were recorded within each gene fragment. The great sequence variability corresponded to a low number of alleles and, surprisingly, to a small fraction of genotype combinations. The distribution, frequency, and combination of the different alleles at each locus is possibly due to natural and human pressures, such as selection, ancestrality, or fitness. Phylogenetic analyses of allele sequences showed distant and complex patterns of relationships among cultivated olives, intermixed with other related forms, highlighting an evolutionary connection between olive cultivars and the O. europaea subspecies cuspidata and cerasiformis. This study demonstrates how a detailed and complete sequence analysis of a few gene portions and a thorough genotyping on a representative set of cultivars can clarify important issues related to sequence polymorphisms, reconstructing the phylogeny of alleles, as well as the genotype combinations. The identification of regions representing blocks of recombination could reveal polymorphisms that represent putatively functional markers. Indeed, specific mutations found on the analyzed OeACP1 and OeACP2 fragments seem to be correlated to the fruit weight.Gene sequence variation in cultivated olive ( Olea europaea L. subsp. europaea var. europaea) is the most important oil tree crop of the Mediterranean basin. The species is characterized by an unusually rich germplasm, represented by a high number of traditional varieties, vegetatively propagated over 1000s of years , followed by empirical local selection, probably associated to episodic varietal substitution, leading to the current varietal framework  genes (SUT1) (LUS)  synthesis and desaturation, as well as in other metabolic pathways  genes , a gene s (SUT1)  and anot1) (LUS)  were chopathways . The funmulation , meanwhiic acids . The SUThe plant . Differehe plant . In olivcterized . The LUS cyclase , is invo cyclase . Up to n cyclase .Polymorphic fragments of these genes were deeply analyzed within a large set of olive varieties, with the aims to: (i) understand the level of genomic variation within the chosen fragments; (ii) assign the alleles to each olive varieties; (iii) understand evolutionary and phylogenetic relationships among haplotypes, and (iv) verify potential correlations between detected polymorphisms and phenotypic traits.O. europaea subsp. cuspidata from Nepal and subsp. cerasiformis from the Canary Islands were used to clarify the ancestry and the phylogenetic relationships of the cultivar allele groups, whereas one sample of the related species Olea paniculata, belonging to the subgenus Paniculatae .In order to obtain polymorphic E. coli XL1 blue strain. DNA from 10 colonies for each genotype was amplified using the ExTaq (Takara) following the manufacturer instructions for the reaction and PCR amplification. Direct sequencing in both directions of the PCR products was performed on an ABI 3130 Genetic Analyzer  using the ABI Prism BigDye Terminator v.3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems). To evaluate the presence of polymorphisms, sequences were aligned by BioEdit 7.1.7 (BioEdit1) and by Geneious 6.1.6 (Geneious2). Afterward, new allele-specific primers were designed and applied to amplify distinct alleles at each locus in all 90 cultivars . Amplifications were performed on a thermal cycler PCR System 9600 , using the following cycling conditions: initial denaturation at 95°C for 5 min, followed by 50 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 90 s, with a final elongation at 72°C for 10 min. Amplicons of different lengths were obtained. Considering that different fragment lengths prevent direct sequencing, in order to identify and distinguish the two alleles at each of the 20 diploid genotypes, amplicons of each locus were cloned, by using pGEM-T Easy Vector (Promega) and In order to verify whether the obtained alleles effectively belong to a single locus, a co-segregation analysis was performed by genotyping 94 individuals of a F1 intervarietal cross progeny (Leccino × Dolce Agogia), through length and SNP markers at each locus heterozygous in at least one parental variety (data not shown).O. europaea subspecies cuspidata and cerasiformis and the related species O. paniculata.The same cloning strategy was also applied to obtain at least one allele for each sample of the 34O. europaea subsp. europaea var. sylvestris5,6, in order to find the position of these genes on the chromosome.The obtained sequences were used as queries in a BLAST search on the genome of the cultivars Farga 3 and LeccAt each locus, the number of SNPs, indels, SSRs and repeats was calculated. The frequency of polymorphisms was obtained by the ratio between number of polymorphisms and total fragment length. Linkage analysis among polymorphisms was evaluated through MapMaker/EXP Version 3.0b .2 parameters and computing a non-linear regression line. The 180 fragments sequenced for each gene were analyzed, as well as the haplotypes inferred by the software. Due to the nature of the analyzed fragments, in this manuscript the terms ‘allele’ and ‘haplotype’ have been used as synonymous. The same software was used to detect the degree of intragenic recombination (IR) in real and inferred haplotypes at the four loci using genotypic data with the r2 parameter. The same software was used for haplotype reconstruction which was implemented by PHASE with 1,000 iterations, thinning intervals equal to 10 and 1,000 burn-in iterations, and either forcing or not the program to use the naive Gibbs sampler (GS options)  among polymorphisms at each locus. The degree of LD was estimated by calculating roptions) . The twoO. paniculata sample, used as an out-group, and one allele for each of the two subspecies cuspidata and cerasiformis.Median joining phylogenetic networks based on SNP and length markers for each allele were obtained by Network 4.6.11 software , includiOeACP1–OeACP2 for 56 genotypes, were analyzed by following the same parameters through the same software.The evolutionary history was inferred using the Neighbor-Joining method by MEGA7 software . The optTo assess whether signatures of positive selection or demographic processes were present in each of the four candidate loci, three neutrality tests were separately performed   on exonsOeACP genes, a subset of 56 varieties from the Olive Varietal Collection established in Zagaria  was phenotyped for FA composition and fruit weight. Fruit samples were collected from two plants per cultivar, for 2 years and two dates/year. Methods of analysis refer to 7.Following the neutrality test results, to verify the presence of any genotype/phenotype correlation for Furthermore, Box and Whisker plots were chosen to graphically show the correlations between genotype and phenotypic traits . For eacOeACP1 and OeACP2 genes, separately. This software uses a Bayesian model that accounts for the linkage present between polymorphic sites within aligned sequences. For each gene, BAPS carried out a genetic mixture analysis to determine the best partition of the data into K groups. The mixture analysis was run with 100 iterations, the input number of reference genotypes from each group was 10; the input number of iterations for reference genotype was 100, the minimum size of a group was 10. Subsequently, BAPS carried out a genetic admixture analysis to estimate admixture proportions within each genotype, taking into consideration the most likely number of K groups identified previously. TASSEL genotype alignments were first filtered using minimum counts of 56 sequences, with a minimum SNP frequency of 0.05 , including all sites for each fragment.Finally, TASSEL was used to establish genotype/phenotype relationships by using the association analyses with general linear model (GLM) and mixed lineage model (MLM), the latter only for significant correlations by using kinship matrix obtained by the same software. A Q matrix was built by using the Bayesian model-based approach and the “Clustering with linked loci” module, as implemented in BAPS 6.0 , inferriOeSUT1, and eight SSRs, the latter exclusively present in the OeACP2 fragment.Combined approaches, including cloning and direct sequencing performed by means of locus- and allele-specific primers, allowed us to distinguish all alleles at the four loci on 90 olive cultivars. Nucleotide diversity in the gene fragments is summarized in Table OeACP gene fragments, as detailed below.Single nucleotide polymorphism frequency was 7.9 times higher than length polymorphisms . The variation density was one polymorphism every 19.5 bp (only one every 21.9 bp for SNPs), one every 45.9 bp on exons and one every 17 bp on introns. The 17 SNPs detected in coding regions included 11 transitions and six transversions, leading to amino acid changes only in the OeACP1 allele fragments, with a maximum length of 990 bp, revealed 29 SNPs and four indels, giving a frequency of one polymorphism every 30 bp. A high degree of linkage was observed: on the 33 polymorphisms, only two SNPs at positions 407 and 503 and one indel at position 537 were unlinked, whereas the remaining (90.9%) were grouped in three linkage groups.The analysis of the OeACP2 allele fragments, with a maximum length of 1,363 bp, showed higher variability than that found in the other loci. The 127 polymorphisms included 112 SNPs, seven indels and eight SSRs, with a frequency of one polymorphism every 10.7 bp. The OeACP2 fragments carried insertions and various microsatellite regions, made up of mono-, di-, and tri-nucleotide motifs. As for OeACP1, a high linkage degree was observed: 104 polymorphisms (81.9%) were linked in five linkage groups.The In this gene fragment (555 bp), ranging from the seventh exon to a portion of the ninth exon, only 10 nucleotide mutations were detected, with a frequency of one every 55.5 bp. Four SNPs were unlinked, whereas the remaining were grouped into two linkage groups.OeSUT1 allele fragments (maximum length of 914 bp) revealed 23 SNPs, two indels of 1 and 19 bp, respectively, and a motif of 166 bp that in some cultivars was repeated twice in a head-tail arrangement, generating a tandem repeated sequence. The alignment of the isolated gene fragments with the SUT1 sequence of other plant species – i.e., Populus trichocarpa (HM749897.1), Spinacia oleracea (X67125.1), Vitis vinifera (HQ323258.1), or Verbascum phoeniceum (FJ797307.1) – showed that the first exon of the olive gene was divided into two portions by a tandem repeated sequence made up of a 166 bp motif. By aligning this sequence with the collection of repetitive DNA elements of Dfam database (Dfam8), it was not identified as a part of a transposable element. The frequency of polymorphisms was one every 35.2 bp. Fifty percent of polymorphisms were linked into two linkage groups. No linked SNPs were found among the four gene fragments.Overall, the sequence analysis of the OeACP1, 12.2% in OeACP2, 38.9% in OeLUS, and 24.4% in OeSUT1). The co-segregation analysis of alleles confirmed that all four genes effectively belong to four distinct loci (data not shown).The list of alleles found at each locus, along with the corresponding GenBank accession numbers, is reported in Supplementary Table OeACP1. The allele named 1A was clearly distinct from the others, due to two deletions at positions 306 and 802  and a 12 bp insertion at position 485, joined with 23 linked SNPs. Three out of the four indels were linked. The other alleles belonged to the same group called 3 , and were distinguished among each other by a few polymorphisms. Three out of four polymorphisms found in exon two generated three amino acid changes  were found in our sample set. Out of these, the most frequent genotype was 2D-4A (18.9%), derived from the combination of the most frequent alleles , and only 12 of the possible 28 genotypes were observed, the most common being C–C, shared by the 35% of cultivars Figure .OeSUT1 alleles were divided into three groups: long (L), medium (M), and small (S). Length differences between S and M alleles were due to a 19 bp indel, whereas L alleles contained a duplication of a 166 bp motif in respect to M alleles. SNPs in the exon sequences did not produce any amino acid change. The M group represented 60% of total alleles and, within this group, M2 showed the highest frequency. The S alleles represented 27% of the total, and the two L alleles only the remaining 13%. No random combinations of OeSUT1 alleles were observed, in fact, only 22 out of the possible 120 (18.3%) genotypes were identified. The most frequent genotypes were M2-S4 and M2-M2. Taken together, they represented more than 44% of the observed ones  , each representing a ‘haplotype block.’ In OeACP2, the IR for calculated haplotypes was 0.252, while the IR obtained with real haplotypes was 0.023, corresponding to a calculated minimum number of 19 recombination events versus the true five ones. The latter were located at 140–147, 147–151, 318–348, 348–470, and 851–1,167 bp. Interestingly, one of them included the last part of the second exon (from 318 to 337 bp). All the other OeACP2 portions represented haplotype blocks. In OeSUT1, the IR value was four times higher in calculated versus true haplotypes, and the only RE was located at 259–566 bp, in the intron region containing the 166 bp repeated motif , whose separation from the other alleles of group 2 was sustained by a bootstrap value of 100% , linking the sequences at each locus, are shown in Figure OeACP1-1A, OeACP2-4A, OeLUS-C, OeSUT1-M2)  and for OeACP2 SNP 869  with fruit weight  percentage  and OeACP2 (p = 0.047) .When the functional contribution of polymorphisms at each OeACP1 and OeACP2 sequences of 56 cultivars, in order to verify if the cultivars that grouped in the same genetic cluster showed also a relationship with the fruit fresh weight. Almost all cultivars carrying both or only one of the two alleles OeACP1-3D and OeACP2-4F are included in three clusters  and showed a medium-high fruit weight  and single nucleotide polymorphisms (one every 21.9 bp) was high compared to that found in other plant species, in which SNP frequency was detected by the same sequencing method. The values detected in olive were higher than that observed not only in perennial woody species, such as  species , but witultivars . Interesultivars . This diultivars .The frequency of indels and microsatellites along the introns of the analyzed fragments of the four loci resulted quite low compared to total polymorphisms . Due to the high SNP frequency and the low indel frequency, their ratio resulted higher than in other plant species: 12.4 against 6.4 in apple , 3.6 in OeSUT1 gene sequences respect to other species. The first exon was interrupted by an intron, not recognized as a part of a transposable element . Several hypotheses can be conceived to explain this evidence: (i) a different level of ancestrality may have affected the chance of combination; (ii) the complex varietal diversification process  could haO. paniculata species, was probably due to the existence of unsampled or extinct ancestral sequences. The high affinity between the allele clusters showed that the different allele forms derived from each other through single or multiple nucleotide mutations. Moreover, the relationships established in our material may be considered as an evidence of introgression of alleles in cultivated olives from other subspecies. Interesting is the case of OeACP1-1A allele, highly widespread among cultivars of different geographical origin, that appeared before all other alleles, including those carried by different subspecies, and for which a different introgression process could be hypothesized. In OeACP2, a close affinity with subsp. cuspidata can be deduced for alleles of group 4, whereas group 2 alleles derived from subsp. cerasiformis. A similar frame can be seen in OeLUS, in which the two subspecies are distinctly linked to different allele groups. It is interesting to highlight that cerasiformis subspecies is characteristic to the extreme western side of Mediterranean, while cuspidata subspecies spreads from north-east and south of Africa to the south of Asia, two completely different ways of diffusion.The use of single-copy genes to reconstruct phylogenies of the olive complex, has been limited by the high allele diversity, coupled to numerous ancient admixture events, because very different topologies may be supported by different loci . In thisAs interpreted for the distribution of lupeol synthase alleles in wild Mediterranean olives , the recOeACP2-2L, which is more present in the cultivars of the West-Central Mediterranean and characterizes a large number of Spanish cultivars. The same result was obtained by specific Neighbor-Joining analysis.This also explains why in the set of cultivars analyzed, which included the main commercial ones, it was not possible to group the varieties according to their geographical origin. Despite this, some alleles or allelic associations have been reasonably regionalized, see the French cultivars and the case of OeACP2) were specific to single varieties , thus representing powerful markers, exploitable not only for genotyping purposes but also for olive oil DNA testing, parentage analysis and association mapping studies.Due to the strong association, only 21% on total polymorphisms resulted informative, but these were able to discriminate a vast majority of the analyzed cultivars. Some polymorphisms , and other six double-purpose (oil and table olives) varieties . Box plot graphs highlighted also three SNPs in OeACP2 sequences as significantly related to the percentage of palmitoleic acid. When each relationship was tested by TASSEL, only the correlation between fruit weight and both SNPs in 4F and 3D alleles remained within the significance range, when using GLM analysis. Box and Whisker plots, produced on the basis of the allele estimates output provided by TASSEL, underlined that when the estimate values were >0.5 the effect of allele’s genotypic class clearly emerged. In particular, homozygous genotypes were associated with lower phenotypic values.This low recombination level, together with a very high and significant values of Tajima test, encouraged us to investigate the occurrence of any genotypic/phenotypic correlation. Box and Whisker plots showed a good correlation between fruit weight and two specific polymorphisms, the ACP genes are characterized, in the upstream and downstream sequences, by regulatory sequences that could play a major role in affecting the trait and, in an oil crop as olive, genes involved in oil accumulation may have undergone to a strong selective pressure, shaping the distribution, frequency and combination of the different alleles. Further functional genomic studies are needed to validate these SNPs as effective putative causal polymorphisms.The lack of strong marker/phenotype correlation may be due to numerous factors. Fruit weight and FAs content are quantitative traits presumably controlled by multiple loci and the effect of one over the others is still totally unknown. Acyl carrier protein is a cofactor and a structural protein that serves for the plastidial condensation of the acyl chains C16, but it represents a member of the FAs synthesis chain that involves different actors . ACP genThis research showed how an in-depth gene sequence analysis may help to clarify important issues related to allele variation and apportioning of variability in a perennial cultivated species.The polymorphisms found could be applied for the discrimination and evaluation of the olive genetic resources, in studies on the evolution and domestication of the crop, or as a starting point for further functional genomic experiments.NC, LB, MC, RM, and VS conceived and designed the experiments. NC, VS, FA, RM, SM, and CR performed the experiments. RM, LL, NC, LB, and FA analyzed the data. LB and MC contributed reagents, materials, and analysis tools. LB, NC, RM, LL, SM, FA, and MC wrote the manuscript. All the authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Up to the present time, seafood, and in particular, fish oil-derived products, have been the richest sources of n-3 LC-PUFA. The human diet generally contains insufficient amounts of these essential FA due largely to the low consumption of seafood. This issue provides opportunities to enrich the content of n-3 PUFA in other common food groups. Milk and milk products have traditionally been a major component of human diets, but are also among some of the poorest sources of n-3 PUFA. Consideration of the high consumption of milk and its processed products worldwide and the human health benefits has led to a large number of studies targeting the enhancement of n-3 PUFA content in dairy products. The main objective of this review was to evaluate the major strategies that have been employed to enhance n-3 PUFA content in dairy products and to unravel potential knowledge gaps for further research on this topic. Nutritional manipulation to date has been the main approach for altering milk fatty acids (FA) in ruminants. However, the main challenge is ruminal biohydrogenation in which dietary PUFA are hydrogenated into monounsaturated FA and/or ultimately, saturated FA, due to rumen microbial activities. The inclusion of oil seed and vegetable oil in dairy animal diets significantly elevates ALA content, while the addition of rumen-protected marine-derived supplements is the most effective way to increase the concentration of EPA, DHA, and DPA in dairy products. In our view, the mechanisms of n-3 LC-PUFA biosynthesis pathway from ALA and the biohydrogenation of individual n-3 LC-PUFA in ruminants need to be better elucidated. Identified knowledge gaps regarding the activities of candidate genes regulating the concentrations of n-3 PUFA and the responses of ruminants to specific lipid supplementation regimes are also critical to a greater understanding of nutrition-genetics interactions driving lipid metabolism.Omega-3 polyunsaturated fatty acids (n-3 PUFA) are termed essential fatty acids because they cannot be synthesized de novo by humans due to the lack of delta-12 and delta-15 desaturase enzymes and must therefore be acquired from the diet. n-3 PUFA include α-linolenic acid , eicosapentaenoic , docosahexaenoic , and the less recognized docosapentaenoic acid . The three long-chain (≥C The common types of n-3 PUFA are: Shorter chain SC, ≤C8 n-3 PUF-chain ≥C0 n-3 PUF SDA, 18:n-3, and  EPA, 20:n-3; doco DHA, 22:n-3; and 3) acids ,8 diseas3) acids ,10,11. P3) acids . The unaChronic or non-communicable diseases have remained as the most leading cause of death worldwide, with 41 million deaths accounting for 71% of reported global deaths (57 million) . This reDietary intake recommendations of n-3 LC-PUFA from different organizations vary largely and also depend on many factors including age, gender, and consumption purposes of consumers ,22. AdheDue to the lack of delta-12 and delta-15 desaturase enzymes, mammals (including humans) cannot synthesize n-3 PUFA de novo, thus these essential FA must be acquired via foods or nutritional supplements . The firThere are two recognised biosynthesis pathways for n-3 LC-PUFA , includiDue to the limitation of n-3 LC-PUFA biosynthesis in the human body from ALA, the best way of acquiring these essential FA is from dietary sources . Fish anThe biological functions of n-3 LC-PUFA are firstly represented by their occurrence in all cellular membranes in all tissues of the body, and in particular, at high content levels in the retina, brain, and myocardium ,49. For Cardiovascular diseases refer to a collective term for heart and/or blood vessels related diseases that are by far, the most leading cause of mortality worldwide with 17.9 million deaths reported in 2018 . TherefoApart from CVD and cancer, large studies have recognised the role of n-3 LC-PUFA in regards to brain related cognitive treatments and other common chronic diseases such as rheumatoid arthritis, type-2 diabetes and obesity. Relating to brain issues in humans, bioactivities of n-3 LC-PUFA, particularly DHA, play an important role in neural membrane structure, neurotransmission, and signal transduction , and posThe controversies regarding the role of n-3 PUFA in chronic diseases may be explained by many factors such as dose, duration, baseline intake , specifiSince all of the long-chain FA in milk fat are derived from the absorption of fatty acids from the small intestine and body fat reserves that have both originated from dietary FA ,87, maniDietary lipid sources for ruminants are mainly from forages, supplements or concentrates including cereal grains, oilseeds and animal fats. Lipids derived from forages contain largely glycolipids and phospholipids, while triglycerides are found primarily in supplements . Once diUnder the activity of rumen microbes, unsaturated fatty acids (UFA) including PUFA are hydrogenated to monounsaturated FA (MUFA) and ultimately, saturated FA (SFA) through the addition of a double bond of two hydrogen atoms. The principal role of this process is to maintain a stable rumen environment by reducing the toxic effects of free UFA on bacterial growth in the rumen . Due to Apart from ruminal BH, given the relatively low absorption rate from the small intestine into the mammary gland at 49% for ALA, and ranging from 14% to 33% for EPA, and from 13% to 25% for DHA , it is nUp to the present time, the nutritional manipulation of feeding regimes and supplementation with lipid sources containing high amounts of n-3 PUFA ,96 are tPrevious studies had demonstrated that feeding regime, particularly changes in forage sources and feeding systems, had significant effects on shorter chain n-3 PUFA, but minor effects on n-3 LC-PUFA concentrations in both dairy ewes and cows . This isB. proteoclasticus has been implemented with limited success due to many factors. For more details, see comprehensive coverage by Lourenco et al. [The transfer of n-3 PUFA from forage into milk and milk products is also influenced by forage species . Grazingo et al. .Lipid supplementation has been used as an effective tool to improve animal performance due to its significant energy contribution , and it Plant-derived fat is the most common fat source in ruminant supplements, and includes both oilseeds and extracted vegetable oils. This is because these materials not only contain a high concentration of PUFA , proteinOil infusion is also considered an effective form of providing plant oil supplements that increases the escape rate of UFA from the BH of rumen microbes, thus enhancing the availability of n-3 PUFA for absorption . Khas etFeeding dairy animals with marine oil resulted in the highest n-3 LC-PUFA concentration in milk and milk products  among alRecent focus on achieving quantitatively significant amounts of n-3 PUFA per standard serve of milk and milk products has occurred ,46. ThisAttempts at understanding and estimating genetic parameters influencing milk FA content that may be beneficial for human health had been made a decade ago ,147. Up Since first being reported some four decades ago, the number of studies confirming the beneficial effects of n-3 PUFA on reducing the risk of chronic diseases including cardiovascular disease, cancer, mental illnesses, age-related cognitive decline, and inflammatory diseases were over and above those that found neutral or adverse effects. Specific mechanisms with respect to the independent and/or supplementary influences of n-3 PUFA on human health, however, remain controversial and need further investigations. Consumption of seafood as the major source of n-3 LC-PUFA is still generally insufficient, resulting in the need for enhancing the concentration of these beneficial n-3 LC-PUFA in other common dietary sources for human consumption. Attempts to increase the content of n-3 LC-PUFA in milk and milk products, which are important components of the human diet, has therefore been a major research focus for the last two decades. Current studies generally demonstrate that the processes of lipolysis combined with dietary lipid BH in the rumen are the main obstacles in modulating n-3 LC-PUFA content of dairy products. In addition, the biosynthetic pathway of these FA from the precursor ALA, is limited, with only minor conversion occurring. Thus, supplementing ruminants with marine-derived sources in a rumen-protected form is at this time, one of the best strategies to increase n-3 LC-PUFA content in milk and its processed products. Yet, there seems to be a lack of evaluation regarding the economic efficiency of different supplemented ingredients utilized in the current research. We propose that further studies are required to better elucidate the n-3 LC-PUFA biosynthesis pathway from ALA, together with the BH mechanism of individual n-3 LC-PUFA. A more comprehensive knowledge of these aspects would be a guiding principle for further research on increasing the escape rate of dietary PUFA from the rumen and their absorption rate from the small intestine into milk. Genetically modifying oilseeds as an alternative and sustainable source of dietary n-3 LC-PUFA for ruminants should be taken into account when future supplies of fish oil may not be sufficient. Finally, activities of regulated genes on the concentration of n-3 PUFA and their reaction to non-genetic factors such as lipid supplementation for specific dairy species also needs to be better defined. Identified knowledge gaps regarding the activities of candidate genes regulating the concentrations of n-3 PUFA and the responses of ruminants to specific lipid supplementation regimes are also critical to a greater understanding of nutrition-genetics interactions driving lipid metabolism.