Quantitative trait loci mapping for fatty acid contents in the backfat on porcine chromosomes 1, 13, and 18

A partial genome scan using microsatellite markers was conducted in order to detect quantitative trait loci (QTLs) for 10 fatty acid contents of the backfat in a pig reference population. Two QTLs were found by studying SSC1, SSC13, and SSC18, where QTLs had already been identified for backfat thickness. A QTL was located between marker loci S0113 and SW974 on chromosome 1; this QTL was only significantly detected (P < 0.05) for linoleic acid. The other QTL was discovered between markers S0062 and S0120 on chromosome 18, and its significance only showed (P < 0.05) for myristic acid. The two QTLs mapped to the same location as the backfat thickness QTL. A third of the phenotypic variation was explained for linoleic acid by the QTL on chromosome 1, and a quarter for myristic acid by the QTL on chromosome 18. Further studies on fine mapping and positional comparative candidate gene analyses will be the next step toward a better understanding of the genetic architecture of fatty acid contents.     

Quantitative trait loci mapping for fatty acid composition traits in perirenal and back fat using a Japanese wild boar x Large White intercross

Here, we analysed quantitative trait loci (QTL) for fatty acid composition, one of the factors affecting fat quality, in a Japanese wild boar x Large White cross. We found 25 significant effects for 17 traits at 13 positions at the 5% genome-wise level, of which 16 effects for 12 traits at 10 positions were significant at the 1% level. QTL for saturated fatty acids (SFA) in back fat were mapped to swine (Sus scrofa) chromosomes (SSC) 1p, 9 and 15. QTL for unsaturated fatty acids in back fat were mapped to SSC1p, 1q, 4, 5, 9, 15 and 17. Using a regression model that fits back fat thickness as a covariate, two of the QTL for linoleic acid content on SSC4 and SSC17 were not significant, but one QTL for total SFA composition was detected on SSC5 with correction for back fat thickness. Wild boar alleles at six of seven QTL tended to increase SFAs and to decrease unsaturated fatty acids. QTL for fatty acid composition in perirenal fat were mapped on SSC2, 3, 4, 5, 6, 14, 16 and X. QTL for melting point (in back fat samples) were mapped on SSC1, 2 and 15. Wild boar alleles in QTL on SSC1 and SSC15 were associated with elevated melting points whereas those on SSC2 were associated with lower melting point measurements.     

Interval mapping of carcass and meat quality traits in a divergent swine cross.

An autosomal scan of the swine genome with 119 polymorphic microsatellite (ms) markers and data from 116 F2 barrows of the University of Illinois Meishan x Yorkshire Swine Resource Families identified genomic regions with effects on variance in carcass composition and meat quality at nominal significance (p-value <0.05). Marker intervals on chromosomes 1, 6, 7, 8 and 12 (SSC1, SSC6, SSC7, SSC8, SSC12) with phenotypic effects on carcass length, 10th rib backfat thickness, average backfat thickness, leaf fat, loin eye area and intramuscular fat content confirm QTL effects identified previously based on genome wide significance (p-value <0.05). Several marker intervals included nominally significant (p-value <0.05) dominance effects on leaf fat, 10th rib backfat thickness, loin eye area, muscle pH and intramuscular fat content.     

A genome scan reveals QTL for growth, fatness, leanness and meat quality in a Duroc-Pietrain resource population

We performed a genome-wide QTL scan for production traits in a line cross between Duroc and Pietrain breeds of pigs, which included 585 F(2) progeny produced from 31 full-sib families genotyped with 106 informative microsatellites. A linkage map covering all 18 autosomes and spanning 1987 Kosambi cM was constructed. Thirty-five phenotypic traits including body weight, growth, carcass composition and meat quality traits were analysed using least square regression interval mapping. Twenty-four QTL exceeded the genome-wide significance threshold, while 47 QTL reached the suggestive threshold. These QTL were located at 28 genomic regions on 16 autosomal chromosomes and QTL in 11 regions were significant at the genome-wide level. A QTL affecting pH value in loin was detected on SSC1 between marker-interval S0312-S0113 with strong statistical support (P < 3.0 x 10(-14)); this QTL was also associated with meat colour and conductivity. QTL for carcass composition and average daily gain was also found on SSC1, suggesting multiple QTL. Seventeen genomic segments had only a single QTL that reached at least suggestive significance. Forty QTL exhibited additive inheritance whereas 31 QTL showed (over-) dominance effects. Two QTL for trait backfat thickness were detected on SSC2; a significant paternal effect was found for a QTL in the IGF2 region while another QTL in the middle of SSC2 showed Mendelian expression.     

Quantitative trait locus analysis of unsaturated fatty acids in a recombinant inbred population of soybean

Soybean [Glycine max (L.) Merr.] is an important oilseed crop which produces about 30 % of the world’s edible vegetable oil. The quality of soybean oil is determined by its fatty acid composition. Soybean oil high in oleic and low in linolenic fatty acids is desirable for human consumption and other uses. The objectives of this study were to identify quantitative trait loci (QTLs) for unsaturated fatty acids and to evaluate the genetic effects of single QTL and QTL combinations in soybean. A population of recombinant inbred lines derived from the cross of SD02-4-59 × A02-381100 was evaluated for fatty acid content in seven environments. In total, 516 polymorphic single nucleotide polymorphism markers, 477 polymorphic simple sequence repeat markers and three GmFAD3 genes were used to genotype the mapping population. By using the composite interval mapping and/or the interval mapping method, a total of 15 QTLs for the three unsaturated fatty acids were detected in more than two environments. Two QTLs for oleic acid on linkage groups G [chromosome (Chr) 18] (qOLE-G) and J (Chr 16) (qOLE-J), three QTLs for linoleic acid on linkage groups A1 (Chr 5) (qLLE-A1) and G (Chr 18) (qLLE-G-1 and qLLE-G-2), and five QTLs for linolenic acid on linkage groups C2 (Chr 6), D1a (Chr 1), D1b (Chr 2), F (Chr 13) and G (Chr 18) were consistently detected in at least three individual environments and the average data over all environments. Significant QTL × QTL interactions were not detected. However, significant QTL × environment interactions were detected for all the QTLs which were repeatedly detected. Some QTLs reported previously were confirmed, and seven new QTLs (two for oleic acid, two for linoleic acid and three for linolenic acid) were identified in this study. Comparisons of two-locus and three-locus combinations indicated that cumulative effects of QTLs were significant for all the three unsaturated fatty acids. QTL pyramiding by molecular marker-assisted breeding would be an appropriate strategy for the improvement of unsaturated fatty acids in soybean.     

A quantitative trait locus for oleic fatty acid content on Sus scrofa chromosome 7

A partial genome scan using microsatellite markers was conducted to detect quantitative trait loci (QTLs) for 10 fatty acid contents of backfat on 15 chromosomes in a porcine resource population. Two QTLs were discovered on Sus scrofa chromosome 4 (SSC4) and SSC7. The QTL on SSC4 was located between marker loci sw1336 and sw512, and this QTL was detected (P < 0.05) only for linoleic acid. Its position was in proximity of those mapped for linoleic acid content in previous studies. The QTL on SSC7 was mapped between markers swr1343 and sw2155, and it was significant (P < 0.05) only for oleic acid. A novelty of the QTL for oleic acid was suggested because the QTL was located far from any other QTLs previously mapped for fatness traits. The QTL on SSC7 explained 19% of phenotypic variation for oleic acid content. Further studies on fine mapping and positional comparative candidate gene analysis would be the next step toward better understanding of the genetic architecture of fatty acid contents.     

Detection of quantitative trait loci for growth- and fatness-related traits in a large-scale White Duroc × Erhualian intercross pig population

Growth and fatness are economically important traits in pigs. In this study, a genome scan was performed to detect quantitative trait loci (QTL) for 14 growth and fatness traits related to body weight, backfat thickness and fat weight in a large-scale White Duroc × Erhualian F(2) intercross. A total of 76 genome-wide significant QTL were mapped to 16 chromosomes. The most significant QTL was found on pig chromosome (SSC) 7 for fatness with unexpectedly small confidence intervals of ～2 cM, providing an excellent starting point to identify causal variants. Common QTL for both fatness and growth traits were found on SSC4, 5, 7 and 8, and shared QTL for fat deposition were detected on SSC1, 2 and X. Time-series analysis of QTL for body weight at six growth stages revealed the continuously significant effects of the QTL on SSC4 at the fattening period and the temporal-specific expression of the QTL on SSC7 at the foetus and fattening stages. For fatness traits, Chinese Erhualian alleles were associated with increased fat deposition except that at the major QTL on SSC7. For growth traits, most of White Duroc alleles enhanced growth rates except for those at three significant QTL on SSC6, 7 and 9. The results confirmed many previously reported QTL and also detected novel QTL, revealing the complexity of the genetic basis of growth and fatness in pigs.     

Mapping Quantitative Trait Loci for Omega-3 Fatty Acids in Asian Seabass

Omega-3 fatty acids are essential fatty acids for human health. Therefore, increasing both percentage of omega-3 and a better fatty acid profile in fish fillets is one of the breeding goals in aquaculture. However, it is difficult to increase the omega-3 content in fish fillets, as the phenotypic selection of these traits is not easily feasible. To facilitate the genetic improvement of the Asian seabass for optimal fatty acid profiles, a genome-wide scan for quantitative trait loci (QTL) affecting fatty acid level in the flesh of the Asian seabass was performed on an F₂ family containing 314 offspring. All family members were genotyped using 123 informative microsatellites and 22 SNPs. High percentages of n-3 polyunsaturated fatty acids (PUFA), especially C22:6 (DHA 16.48?±?3.09 %) and C20:5 (EPA 7.19?±?0.86 %) were detected in the flesh. One significant and 54 suggestive QTL for different fatty acids and a water content trait were detected on the whole genome. QTL for C18:0b was located on linkage groups (LG) 5. QTL for total n-3 PUFA content in flesh were mapped onto LG6 and LG23 with the phenotypic variance explained ranging from 3.8 to 6.3 %. Four QTL for C22:6 were detected on LG6, LG23, and LG24, explaining 3.9 to 4.9 % of the phenotypic variance, respectively. Mapping of QTL for contents of different fatty acids is the first step towards improving the omega-3 content in the fillets of fish by using marker-assisted selection and is important for understanding the biology of fatty acid deposition.     

QTL mapping for growth and carcass traits in an Iberian by Landrace pig intercross: additive,dominant and epistatic effects

Results from a QTL experiment on growth and carcass traits in an experimental F2 cross between Iberian and Landrace pigs are reported. Phenotypic data for growth, length of carcass and muscle mass, fat deposition and carcass composition traits from 321 individuals corresponding to 58 families were recorded. Animals were genotyped for 92 markers covering the 18 porcine autosomes (SSC). The results from the genomic scan show genomewide significant QTL in SSC2 (longissimus muscle area and backfat thickness), SSC4 (length of carcass, backfat thickness, loin, shoulder and belly bacon weights) and SSC6 (longissimus muscle area, backfat thickness, loin, shoulder and belly bacon weights). Suggestive QTL were also found on SSC1, SSC5, SSC7, SSC8, SSC9, SSC13, SCC14, SSC16 and SSC17. A bidimensional genomic scan every 10 cM was performed to detect interaction between QTL. The joint action of two suggestive QTL in SSC2 and SSC17 led to a genome-wide significant effect in live weight. The results of the bidimensional genomic scan showed that the genetic architecture was mainly additive or the experimental set-up did not have enough power to detect epistatic interactions.     

Quantitative trait loci for fatty acid composition in longissimus dorsi and abdominal fat: results from a White Duroc × Erhualian intercross F2 population

A whole-genome scan was performed on 660 F₂ animals including 250 barrows and 410 gilts in a White Duroc × Erhualian intercross population to detect quantitative trait loci (QTL) for fatty acid composition in the longissimus dorsi muscle and abdominal fat. A total of 153 QTL including 63 genome-wide significant QTL and 90 suggestive effects were identified for the traits measured. Significant effects were mainly evident on pig chromosomes (SSC) 4, 7, 8 and X. No association was detected on SSC3 and 11. In general, the QTL detected in this study showed distinct effects on fatty acid composition in the longissimus muscle and abdominal fat. The QTL for fatty acid composition in abdominal fat did not correspond to those identified previously in backfat and the majority of QTL for the muscle fatty acid composition were mapped to chromosomal regions different from previous studies. Two regions on SSC4 and SSC7 showed significant pleiotropic effects on monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA) in both longissimus muscle and abdominal fat. Another two QTL with significant multi-faceted effects on MUFA and PUFA in the longissimus muscle were found each on SSC8 and SSCX. Chinese Erhualian alleles were associated with increased ratios of MUFA to saturated fatty acid at most of the QTL detected, showing beneficial effect in terms of human health.     

QTL detection on porcine chromosome 12 for fatty-acid composition and association analyses of the fatty acid synthase, gastric inhibitory polypeptide and acetyl-coenzyme A carboxylase alpha genes

Refinement of previous QTL on porcine chromosome 12 for fatty-acid composition and a candidate gene association analysis were conducted using an Iberian × Landrace cross. The concentrations of ten fatty acids were assayed in backfat tissue from which four metabolic ratios were calculated for 403 F₂ animals. Linkage analysis identified two significant QTL. The first QTL was associated with the average chain length ratio and the percentages of myristic, palmitic and gadoleic acids. The second QTL was associated with percentages of palmitoleic, stearic and vaccenic acids. Based upon its position on SSC12, fatty acid synthase was tested as a candidate gene for the first QTL and no significant effects were found. Similarly, gastric inhibitory polypeptide ( GIP ) and acetyl-coenzyme A carboxylase alpha ( ACACA ) were tested as candidate genes for the second QTL using three SNPs in GIP and 15 synonymous SNPs in ACACA cDNA sequences. Two missense SNPs in GIP showed significant effects with palmitoleic and stearic fatty-acid concentration. Highly significant associations were found for two SNPs in ACACA with stearic, palmitoleic and vaccenic fatty-acid concentrations. These associations could be due to linkage disequilibrium with the causal mutations.     

Genetic parameters of backfat fatty acids and carcass traits in Large White pigs

Subcutaneous fat thickness and fatty acid composition (FAC) play an important role on seasoning loss and organoleptic characteristics of seasoned hams. Dry-cured ham industry prefers meats with low contents of polyunsaturated fatty acids (PUFA) because these negatively affect fat firmness and ham quality, whereas consumers require higher contents in those fatty acids (FA) for their positive effect on human health. A population of 950 Italian Large White pigs from the Italian National Sib Test Selection Programme was investigated with the aim to estimate heritabilities, genetic and phenotypic correlations of backfat FAC, Semimembranosus muscle intramuscular fat (IMF) content and other carcass traits. The pigs were reared in controlled environmental condition at the same central testing station and were slaughtered at reaching 150 kg live weight. Backfat samples were collected to analyze FAC by gas chromatography. Carcass traits showed heritability levels from 0.087 for estimated carcass lean percentage to 0.361 for hot carcass weight. Heritability values of FA classes were low-to-moderate, all in the range 0.245 for n-3 PUFA to 0.264 for monounsaturated FA (MUFA). Polyunsaturated fatty acids showed a significant genetic correlation with loin thickness (0.128), backfat thickness (−0.124 for backfat measured by Fat-O-Meat’er and −0.175 for backfat measured by calibre) and IMF (−0.102). Obviously, C18:2(n-6) shows similar genetic correlations with the same traits (0.211 with loin thickness, −0.206 with backfat measured by Fat-O-Meat’er, −0.291 with backfat measured by calibre and −0.171 with IMF). Monounsaturated FA, except with the backfat measured by calibre (0.068; P<0.01), do not show genetic correlations with carcass characteristics, whereas a negative genetic correlation was found between MUFA and saturated FA (SFA; −0.339; P<0.001). These results suggest that MUFA/SFA ratio could be increased without interfering with carcass traits. The level of genetic correlations between FA and carcass traits should be taken into account in dealing with the development of selection schemes addressed to modify carcass composition and/or backfat FAC.     

A quantitative trait locus for live weight maps to bovine Chromosome 23

A multiple-marker mapping approach was used to search for quantitative trait loci (QTLs) affecting production, health, and fertility traits in Finnish Ayrshire dairy cattle. As part of a whole-genome scan, altogether 469 bulls were genotyped for six microsatellite loci in 12 families on Chromosome (Chr) 23. Both multiple-marker interval mapping with regression and maximum-likelihood methods were applied with a granddaughter design. Eighteen traits, belonging to 11 trait groups, were included in the analysis. One QTL exceeded experiment level and one QTL genome level significance thresholds. Across-families analysis provided strong evidence (P_experiment= 0.0314) for a QTL affecting live weight. The QTL for live weight maps between markers BM1258 and BoLA DRBP1. A QTL significant at genome level (P_genome= 0.0087) was mapped for veterinary treatment, and the putative QTL probably affects susceptibility to milk fever or ketosis. In addition, three traits exceeded the chromosome 5% significance threshold: protein percentage of milk, calf mortality (sire), and milking speed. In within-family analyses, protein percentage was associated with markers in one family (LOD score = 4.5). Received: 14 December 1998 / Accepted: 28 March 1998     

Detection of quantitative trait loci for backfat thickness and intramuscular fat content in pigs (Sus scrofa).

In an experimental cross between Meishan and Dutch Large White and Landrace lines, 619 F(2) animals and their parents were typed for molecular markers covering the entire porcine genome. Associations were studied between these markers and two fatness traits: intramuscular fat content and backfat thickness. Association analyses were performed using interval mapping by regression under two genetic models: (1) an outbred line-cross model where the founder lines were assumed to be fixed for different QTL alleles; and (2) a half-sib model where a unique allele substitution effect was fitted within each of the 19 half-sib families. Both approaches revealed for backfat thickness a highly significant QTL on chromosome 7 and suggestive evidence for a QTL at chromosome 2. Furthermore, suggestive QTL affecting backfat thickness were detected on chromosomes 1 and 6 under the line-cross model. For intramuscular fat content the line-cross approach showed suggestive evidence for QTL on chromosomes 2, 4, and 6, whereas the half-sib analysis showed suggestive linkage for chromosomes 4 and 7. The nature of the QTL effects and assumptions underlying both models could explain discrepancies between the findings under the two models. It is concluded that both approaches can complement each other in the analysis of data from outbred line crosses.     

New insight into the SSC8 genetic determination of fatty acid composition in pigs

Background

Fat content and fatty acid composition in swine are becoming increasingly studied because of their effect on sensory and nutritional quality of meat. A QTL (quantitative trait locus) for fatty acid composition in backfat was previously detected on porcine chromosome 8 (SSC8) in an Iberian x Landrace F₂ intercross. More recently, a genome-wide association study detected the same genomic region for muscle fatty acid composition in an Iberian x Landrace backcross population. ELOVL6, a strong positional candidate gene for this QTL, contains a polymorphism in its promoter region (ELOVL6:c.-533C < T), which is associated with percentage of palmitic and palmitoleic acids in muscle and adipose tissues. Here, a combination of single-marker association and the haplotype-based approach was used to analyze backfat fatty acid composition in 470 animals of an Iberian x Landrace F₂ intercross genotyped with 144 SNPs (single nucleotide polymorphisms) distributed along SSC8.

Results

Two trait-associated SNP regions were identified at 93 Mb and 119 Mb on SSC8. The strongest statistical signals of both regions were observed for palmitoleic acid (C16:1(n-7)) content and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) elongation ratios. MAML3 and SETD7 are positional candidate genes in the 93 Mb region and two novel microsatellites in MAML3 and nine SNPs in SETD7 were identified. No significant association for the MAML3 microsatellite genotypes was detected. The SETD7:c.700G > T SNP, although statistically significant, was not the strongest signal in this region. In addition, the expression of MAML3 and SETD7 in liver and adipose tissue varied among animals, but no association was detected with the polymorphisms in these genes. In the 119 Mb region, the ELOVL6:c.-533C > T polymorphism showed a strong association with percentage of palmitic and palmitoleic fatty acids and elongation ratios in backfat.

Conclusions

Our results suggest that the polymorphisms studied in MAML3 and SETD7 are not the causal mutations for the QTL in the 93 Mb region. However, the results for ELOVL6 support the hypothesis that the ELOVL6:c.-533C > T polymorphism has a pleiotropic effect on backfat and intramuscular fatty acid composition and that it has a role in the determination of the QTL in the 119 Mb region.     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998     

Disentangling Two QTL on Porcine Chromosome 12 for Backfat Fatty Acid Composition

A previous study allowed the identification of two QTL regions at positions 11–34 cM (QTL1) and 68–76 cM (QTL2) on porcine chromosome SSC12 affecting several backfat fatty acids in an Iberian x Landrace F2 intercross. In the current study, different approaches were performed in order to better delimit the quoted QTL regions and analyze candidate genes. A new chromosome scan, using 81 SNPs selected from the Porcine 60KBeadChip and six previously genotyped microsatellites have refined the QTL positions. Three new functional candidate genes (ACOX1, ACLY, and SREBF1) have been characterized. Moreover, two putative promoters of porcine ACACA gene have also been investigated. New isoforms and 24 SNPs were detected in the four candidate genes, 19 of which were genotyped in the population. ACOX1 and ACLY SNPs failed to explain the effects of QTL1 on palmitic and gadoleic fatty acids. QTL2, affecting palmitoleic, stearic, and vaccenic fatty acids, maps close to the ACACA gene location. The most significant associations have been detected between one intronic (g.53840T > C) and one synonymous (c.5634T > C) ACACA SNPs and these fatty acids. Complementary analyses including ACACA gene expression quantification and association studies in other porcine genetic types do not support the expected causal effect of ACACA SNPs.     

A primary screen of the bovine genome for quantitative trait loci affecting twinning rate

An autosomal genome scan for quantitative trait loci (QTL) affecting twinning rate was carried out in the Norwegian Cattle population. Suggestive QTL were detected on Chromosomes (Chr) 5, 7, 12, and 23. Among these, the QTL positions on both Chr 5 and Chr 23 are strongly supported by literature in the field. Our results also confirm previous mapping of a QTL for twinning to Chr 7, but definitely suggest a different location of the QTL on this chromosome. The most convincing QTL peak was observed for a region in the middle part of Chr 5 close to the insulin-like growth factor 1 (IGF1) gene. Since IGF1 plays an important role in the regulation of folliculogenesis, a mutation search was performed by sequencing more than 3.5 kb of the gene in actual families. The sequencing revealed three polymorphisms in noncoding regions of the gene that will be important in fine structure mapping and characterization of the QTL. Received: 14 December 1999 / Accepted: 25 May 2000     

Quantitative trait loci associated with blood pressure of metabolic syndrome in the progeny of NZO/HILtJ × C3H/HeJ intercrosses

In a previous study in 15 inbred mouse strains, we found highest and lowest systolic blood pressures in NZO/HILtJ mice (metabolic syndrome) and C3H/HeJ mice (common lean strain), respectively. To identify the loci involved in hypertension in metabolic syndrome, we performed quantitative trait locus (QTL) analysis for blood pressure with direction of cross as a covariate in segregating F₂ males derived from NZO/HILtJ and C3H/HeJ mice. We detected three suggestive main-effect QTLs affecting systolic and diastolic blood pressures (SBP and DBP). We analyzed the first principle component (PC1) generated from SBP and DBP to investigate blood pressure. In addition to all the suggestive QTLs (Chrs 1, 3, and 8) in SBP and DBP, one suggestive QTL on Chr 4 was found in PC1 in the main scan. Simultaneous search identified two significant epistatic locus pairs (Chrs 1 and 4, Chrs 4 and 8) for PC1. Multiple regression analysis revealed three blood pressure QTLs (Bpq10, 100 cM on Chr 1; Bpq11, 6 cM on Chr 4; Bpq12, 29 cM on Chr 8) accounting for 29.4% of blood pressure variance. These were epistatic interaction QTLs constructing a small network centered on Chr 4, suggesting the importance of genetic interaction for development of hypertension. The blood pressure QTLs on Chrs 1, 4, and 8 were detected repeatedly in multiple studies using common inbred nonobese mouse strains, implying substantial QTL independent of development of obesity and insulin resistance. These results enhance our understanding of complicated genetic factors of hypertension in metabolic diseases. Eri Nishihara, Shirng-Wern Tsaih, Chieko Tsukahara and Sarah Langley contributed equally to this work.