Evaluation of the causality of the low‐density lipoprotein receptor gene (LDLR) for serum lipids in pigs

A significant quantitative trait locus (QTL) for low‐density lipoprotein cholesterol (LDL‐C) and total cholesterol (TC) was identified around the LDLR gene on chromosome 2 (SSC2) in a White Duroc × Erhualian F₂ resource population and Sutai pigs in our previous study. However, in previous reports, the causality of LDLR with serum lipids is controversial in pigs. To systematically assess the causality of LDLR with serum lipids, association analyses were successively performed in three populations: Sutai pigs, a White Duroc × Erhualian F₂ resource population and a Duroc × (Landrace × Large White) population. We first performed a haplotype‐based association study with 60K SNP genotyping data and evidenced the significant association with LDL‐C and TC around the LDLR gene region. We also found that there is more than one QTL for LDL‐C and TC on SSC2. Then, we evaluated the causalities of two missense mutations, c.1812C>T and c.1520A>G, with LDL‐C and TC. We revealed that the c.1812C>T SNP showed the strongest association with LDL‐C (P = 5.40 × 10^?11) and TC (P = 3.64 × 10^?8) and explained all the QTL effect in Sutai pigs. Haplotype analysis found that two missense SNPs locate within a 1.93‐Mb haplotype block. One major haplotype showed the strongest significant association with LDL‐C (P = 4.62 × 10^?18) and TC (P = 1.06 × 10^?9). However, the c.1812C>T SNP was not identified in the White Duroc × Erhualian intercross, and the association of c.1520A>G with both LDL‐C and TC did not achieve significance in this F₂ population, suggesting population heterogeneity. Both missense mutations were identified in the Duroc × (Landrace × Large White) population and showed significant associations with LDL‐C and TC. Our data give evidence that the LDLR gene should be a candidate causative gene for LDL‐C and TC in pigs, but heterogeneity exists in different populations.     

Identification of genes related to intramuscular fat independent of backfat thickness in Duroc pigs using single‐step genome‐wide association

Intramuscular fat (IMF) is an important meat‐quality trait of pigs, which influences pork’s shearing force, hydraulics, tenderness and juicy flavor. However, to achieve a higher percentage of lean meat, pigs with lower backfat thickness (BF) are intensively selected for, which may lead to a reduction in pork quality. Therefore, the objective of this study was to locate loci that affect IMF without changing BF. A single‐step GWAS was performed on 950 Duroc pigs genotyped by a 50K SNP chip in order to detect genomic variants relevant to IMF and BF. The significant SNPs detected were afterwards divided into a BF subset (seven SNPs), an IMF subset (11 SNPs) and a subset of both traits (12 SNPs), according to their P‐value and LD. After SNP and QTL annotation, our results indicated that SSC1: 167938652, 166363826, 164829874 and 167171587 might be associated with IMF without changing BF. In the subset of both traits, we found that the combined effect of ALGA0006602 (SSC1: 159538854) and 12784636 (SSC1: 160773437) might improve the IMF without changing BF. Our gene annotation result showed that TLE3, ITGA11, SMAD6, PAQR5 and [RNF152^A/G × MC4R^A/A] genes might affect IMF independently of BF. We believe that the SNPs and genes identified in this study will be valuable for the future molecular breeding of IMF in Duroc pigs.     

Genome‐wide detection of genetic loci and candidate genes for teat number and body conformation traits at birth in Chinese Sushan pigs

Body conformation at birth and teat number are economically important traits in the pig industry, as these traits are usually explored to evaluate the growth and reproductive potential of piglets. To detect genetic loci and candidate genes for these traits, we performed a GWAS on 269 pigs from a recently developed Chinese breed (Sushan) using 38  128 informative SNPs on the Affymetrix Porcine SNP 55K Array. In total, we detected one genome‐wide significant (P = 1.31e‐6) SNP for teat number on chromosome X and 15 chromosome‐wide significant SNPs for teat number, body weight, body length, chest circumference and cannon circumference at birth on chromosomes 1, 3, 4, 6, 7, 9, 10, 13, 14, 15, 17 and 18. The most significant SNP had an additive effect of 0.74 × total teat number, explaining 20% of phenotypic variance. Five significant SNPs resided in the previously reported quantitative trait loci for these traits and seven significant SNPs had a pleiotropic effect on multiple traits. Intriguingly, 12 of the genes nearest to the significant SNPs are functionally related to body conformation and teat number traits, including SPRED2, MKX, TMSB4X and ESR1. GO analysis revealed that candidate genes proximal to the significant SNPs were enriched in the G‐protein coupled receptor and steroid hormone‐mediated signaling pathway. Our findings shed light on the genetic basis of the measured traits and provide molecular markers especially for the genetic improvement of teat number in Sushan and related pigs.     

The combined effect of 1‐methylcyclopropene and citral suppressed postharvest grey mould of tomato fruit by inhibiting the growth of Botrytis cinerea

1‐Methylcyclopropene (1‐MCP, 1 μl/L) and 1 × minimum fungicidal concentration (MFC) citral alone and in combination were used to treat on postharvest tomato fruits to investigate their influence on disease incidence and postharvest quality during fruit storage, which were stored at 90%–95% relative humidity and 25 ± 2°C. Weight loss, pH, hue angle (Hue^°), total soluble solid (TSS), ascorbic acid content, firmness and antioxidant enzyme activities were evaluated after each storage period. 1 μl/L 1‐MCP or 1 × MFC citral reduced weight loss, retarded peel colour changes and retained postharvest fruit quality. 1 μl/L 1‐MCP + 1 × MFC citral could better maintain firmness and ascorbic acid content and increase antioxidant enzyme activities, compared to other treatments. Disease incidence of tomato fruit was significantly decreased, and spore germination and mycelia growth of Botrytis cinerea were suppressed by the combined treatment with 1 μl/L 1‐MCP and 1 × MFC citral. These results indicate that the combined treatment could effectively delay postharvest tomato fruits senescence and inhibit postharvest pathogens in vitro.     

Diet of farm‐escaped sturgeon in the Yangtze River

The diet of farm‐escaped sturgeon accidentally introduced into the Yangtze River ecosystem was investigated from samples collected over 10 days. The fish had consumed adequate quantities of natural food, with prey type related to fish body size. The fish were categorized according to their primary dietary component: demersal fish were most common prey of kaluga Huso dauricus and H. dauricus × Acipenser schrenckii hybrids of total length 120.3–188.7 cm. Shrimp Macrobrachium nipponensis made up the major portion of the diet of H. dauricus × A. schrenckii and Acipenser baerii × A. schrenckii of total length 91.1–106.8 cm. Gammarid amphipods were consumed by A. baerii × A. schrenckii of total length 35.0–81.2 cm. The body length of prey was proportional to the fork length of the sturgeon, Y = 0.19X‐9.46, R² = 0.997. Escaped sturgeon had travelled at least 898 km downstream from the point of escape in the Yangtze River and were feeding on native macrobenthos and demersal fish.     

Identification of new single nucleotide polymorphisms affecting total number born and candidate genes related to ovulation rate in Chinese Erhualian pigs

The Chinese Erhualian pig has the highest record for litter size in the world. However, the genetic mechanism of its high prolificacy remains poorly understood. In our study, large phenotypic variations in litter size were found among Erhualian sows. Significant differences in total number born (TNB) and corpora lutea numbers were observed between sows with high and low estimated breeding values (EBVs) for TNB. To identify single nucleotide polymorphisms (SNPs) associated with TNB, a selective genomic scan was conducted on 18 sows representing the top 10% and 18 sows representing the bottom 10% of EBVs of 177 sows using Illumina Porcine SNP60 genotype data. Genome‐wide fixation coefficient (F_ST) values were calculated for each SNP between the high‐ and low‐EBV groups. A total of 154 SNPs were significantly differentiated loci between the two groups. Of the top 10 highest F_ST SNPs, rs81399474, rs81400131 and rs81405013 on SSC8 and rs81434499 and rs81434489 on SSC 12 corresponded to previously reported QTL for litter size. The other five SNPs, rs81367039 on SSC2, rs80891106 on SSC7, rs81477883 on SSC12 and rs80938898 and rs80971725 on SSC14, appeared to be novel QTL for TNB. Significant associations between rs81399474 on SSC8 and TNB were confirmed in 313 Erhualian sows. Forty genes were identified around the top 10 highest F_ST SNPs, of which UCHL1, adjacent to rs81399474, and RPS6KB1 and CLTC, adjacent to rs81434499, have been reported to affect the ovulation rate in pig. The findings can advance understanding of the genetic variations in litter size of pigs.     

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis,Polyscias balfouriana and Bougainvillea spectabilis in Southern China

Species of Lasiodiplodia are important pathogens of a wide variety of plants covering a wide geographical distribution. These fungi can be associated with different symptoms such as stem cankers, shoot blights, fruit rots, dieback and gummosis. Diseases caused by Lasiodiplodia were surveyed on Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in a nursery in southern China. Based on morphology characteristics and phylogenetic analyses of ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, four species of Lasiodiplodia were identified. Lasiodiplodia theobromae was identified from E. urophylla × grandis, P. balfouriana and B. spectabilis. L. hormozganensis, L. iraniensis and L. pseudotheobromae were identified from B. spectabilis. To our knowledge, with the exception of L. theobromae on E. urophylla × grandis, this study represents the first report of these fungi on the host plants. Pathogenicity tests showed that all Lasiodiplodia spp. obtained in this study are virulent to E. urophylla × grandis and B. spectabilis, and L. theobromae was virulent to P. balfouriana.     

Red and blond Mangalitza pigs display a signature of divergent directional selection in the SLC45A2 gene

The Mangalitza lard‐type pig breed is well known for its fat appearance and curly hair, and it is mainly distributed in Eastern Europe. Four main lines were created in the nineteenth century by artificial selection: Blond Mangalitza, Black Mangalitza, Swallow‐Belly Mangalitza and Red Mangalitza. The Swallow‐Belly line has a black coat combined with yellow‐blond throat and underbelly. In the current work, we aimed to investigate if the colourations of Mangalitza pigs are genetically determined by one or a few loci whose frequencies have been modified by artificial selection. The results of selection scans, with Hap FLK and BayeScan , and of a GWAS for coat colour highlighted the existence of one region on SSC16 (18–20 Mb) with potential effects on hair pigmentation (Red vs. Blond contrast). The analysis of the gene content of this region allowed us to detect the solute carrier family 45 member 2 (SLC45A2) locus as a candidate gene for this trait. The polymorphism of the SLC45A2 locus has been associated with reduced levels or the absence of melanin in several mammalian species. The genotyping of four missense polymorphisms evidenced that rs341599992:G > A and rs693695020:G > A SNPs are strongly but not fully associated with the red and blond coat colours of Mangalitza pigs, a result that was confirmed by performing a haplotype association test. The near fixation of alternative SLC45A2 genotypes in Red and Blond Mangalitza pigs provides a compelling example of the consequences of a divergent directional selection for coat colour in a domestic species.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Genotyping by sequencing reveals a new locus for pig teat number

Teat number is an extremely valuable trait for sow reproduction performance and piglet survival. Here, we used genotyping‐by‐sequencing and a general liner model to carry out genome‐wide analysis of sow total teat number in an Erhualian population. The results indicated that eight SNPs on chromosomes 4, 5, 9 and 10 were significant genome wide (Bonferroni method, P < 2.85E‐7) for sow total teat number. Validation analyses were performed in 298 Erhualian and 904 Large White sows using these significant SNPs and the general liner model procedure in sas . Finally, only the SNP on chromosome 5 was found to be significantly associated with sow teat number in both populations. The GG genotype individuals had 2.23 and 0.82 more teat numbers respectively than did the AA genotype individuals in these two populations (P < 0.05). According to the expression and annotation analyses, we inferred the presence of a gene or lincRNA that could affect teat number by regulating other genes and ultimately affecting the mammogenesis of pigs. Further studies using methods such as Cas9 editing and gene silencing analysis are necessary for additional function analysis.     

Analysis of non‐synonymous SNPs of the porcine SERPINA6 gene as potential causal variants for a QTL affecting plasma cortisol levels on SSC7

Recently, the SERPINA6 gene encoding corticosteroid‐binding globulin (CBG) has been proposed as a candidate gene for a quantitative trait locus (QTL) affecting cortisol level on pig chromosome 7. The QTL was repeatedly detected in different lines, including a Piétrain × (German Landrace × German Large White) cross (PiF1) and purebred German Landrace (LR). In this study, we investigated whether the known non‐synonymous polymorphisms c.44G>T, c.622C>T, c.770C>T, c.793G>A, c.832G>A and c.919G>A of SERPINA6 are sufficient to explain the QTL in these two populations. Our investigations revealed that SNPs c.44G>T, c.622C>T, c.793G>A and c.919G>A are associated with cortisol level in PiF1 (P < 0.01). Haplotype analysis showed that these associations are largely attributable to differences between a major haplotype carrying SNPs c.793G>A and c.919G>A and a haplotype carrying SNPs c.44G>T and c.622C>T. Furthermore, some SNPs, particularly c.44G>T and c.622C>T and the carrier haplotype, showed association with meat quality traits including pH and conductivity (P < 0.05). In LR, the non‐synonymous SNPs segregate at very low frequency (<5%) and/or show only weak association with cortisol level (SNPs c.832G>A and c.919G>A; P < 0.05). These findings suggest that the non‐synonymous SNPs are not sufficient to explain the QTL across different breeds. Therefore, we examined whether the expression of SERPINA6 is affected by cis‐regulatory polymorphisms in liver, the major organ for CBG production. We found allelic expression imbalance of SERPINA6, which suggests that its expression is indeed affected by genetic variation in cis‐acting elements. This represents candidate causal variation for future studies of the molecular background of the QTL.     

Biomass yields,cytogenetics, fertility,and compositional analyses of novel bioenergy grass hybrids (Tripidium spp.)

High biomass yields have been documented for Tripidium spp. (Erianthus spp., Saccharum spp.), but targeted breeding for bioenergy applications has been limited. Advanced, interspecific hybrids between Tripidium ravennae and T. arundinaceum were planted in replicated field plots in 2016. Comparative feedstock evaluations examined biomass yields, cytogenetics, plant fertility, and compositional analyses relative to Miscanthus × giganteus. Dry biomass yields varied as a function of year and accession and increased each year ranging from 3.4 to 10.6, 8.6 to 37.3, and 23.7 to 60.6 Mg/ha for Tripidium hybrids compared to 2.3, 16.2 and 27.9 Mg/ha for M. × giganteus in 2016, 2017, and 2018, respectively. Cytology and cytometry confirmed that Tripidium hybrids were tetraploid with 2n = 4x = 40 (2C genome size = 5.06 pg) and intermediate between T. ravennae with 2n = 2x = 20 (2C genome size = 2.55 pg) and T. arundinaceum with 2n = 6x = 60 (2C genome size = 7.61 pg). Plant fertility characteristics varied considerably with some accessions producing no viable seeds or fewer than that observed for M. × giganteus. Accessions varied significantly for flowering culm number and height and dates of peak anthesis ranging from 14 September to 2 October. Variations in yield and compositional analyses contributed to variations in theoretical ethanol yields ranging from 10,181 to 27,546 L/ha for Tripidium accessions compared to 13,095 L/ha for M. × giganteus. Relative feed value (RFV) indices for winter‐harvested Tripidium accessions varied from 52.8 to 60.0 compared to M. × giganteus with 45.4. RFV for summer‐harvested Tripidium accessions varied from 71.6 to 80.5 compared to M. × giganteus with 61.0. These initial findings for Tripidium hybrids, including high biomass yields, cold hardiness, and desirable traits for multiple markets (e.g., forage, bioenergy, bioproducts), are promising and warrant further development of Tripidium as a temperate bioenergy feedstock.     

Development of an integrated 200K SNP genotyping array and application for genetic mapping,genome assembly improvement and genome wide association studies in pear (Pyrus)

Pear (Pyrus; 2n = 34), the third most important temperate fruit crop, has great nutritional and economic value. Despite the availability of many genomic resources in pear, it is challenging to genotype novel germplasm resources and breeding progeny in a timely and cost‐effective manner. Genotyping arrays can provide fast, efficient and high‐throughput genetic characterization of diverse germplasm, genetic mapping and breeding populations. We present here 200K AXIOM^® PyrSNP, a large‐scale single nucleotide polymorphism (SNP) genotyping array to facilitate genotyping of Pyrus species. A diverse panel of 113 re‐sequenced pear genotypes was used to discover SNPs to promote increased adoption of the array. A set of 188 diverse accessions and an F₁ population of 98 individuals from ‘Cuiguan’ × ‘Starkrimson’ was genotyped with the array to assess its effectiveness. A large majority of SNPs (166 335 or 83%) are of high quality. The high density and uniform distribution of the array SNPs facilitated prediction of centromeric regions on 17 pear chromosomes, and significantly improved the genome assembly from 75.5% to 81.4% based on genetic mapping. Identification of a gene associated with flowering time and candidate genes linked to size of fruit core via genome wide association studies showed the usefulness of the array in pear genetic research. The newly developed high‐density SNP array presents an important tool for rapid and high‐throughput genotyping in pear for genetic map construction, QTL identification and genomic selection.     

Genome‐wide association study identifies variants in the CAPN9 gene associated with umbilical hernia in pigs

Pig umbilical hernia (UH) affects pig welfare and brings considerable economic loss to the pig industry. To date, the molecular mechanisms underlying pig UH are still poorly understood. To identify potential loci for susceptibility to this disease, we performed a genome‐wide association study in an Erhualian × Shaziling F₂ intercross population. A total of 45 animals were genotyped using Illumina Porcine SNP60 BeadChips. We observed a SNP (rs80993347) located in the calpain‐9 (CAPN9) gene on Sus scrofa chromosome 14 that was significantly associated with UH (P = 1.97 × 10^?10). Then, we identified a synonymous mutation rs321865883 (g.20164T>C) in exon 10 of the CAPN9 gene that distinguished two affected individuals (CC) from their normal full‐sibs (TC). Finally, quantitative polymerase chain reaction was explored to investigate the mRNA expression profile of the CAPN9 gene in 12 tissues in Yorkshire pigs at different developmental stages (3, 90 and 180 days). CAPN9 showed high expression levels in the gastrointestinal tract at these three growth stages. The results of this study indicate that the CAPN9 gene might be implicated in UH. Further studies are required to establish a role of CAPN9 in pig UH.