Background

Cattle breeding programs in Brazil have given greater emphasis to the study and selection of reproductive traits due to their economic importance for the production system.In males,scrotal circumference traits are related to the reproductive potential of bulls,because testis size is associated with the production and quality of sperm and the production of sex hormones[1].

In general,female reproductive traits are difficult to measure and,in some cases,are strongly influenced by environmental factors.The reproductive performance of heifers depends on the age at which they calve for the first time;the ones that calve earlier have a more productive life[2].In addition to first calving,another important factor is that the cow continues producing calves regularly to maintain its productivity and diminish calving interval[3].Studies have reported that indirect selection of females based on the performance of bulls is possible,considering the favorable genetic correlations between scrotal circumference measures and age at calving[2,4,5].

Many achievements in animal breeding were obtained based on the classical approach,using information from phenotypes and pedigree.Nowadays,molecular data analyses are bringing new insights to the genetic architecture of species.Regarding reproductive traits of beef cattle,genome-wide association studies(GWAS)are useful tools for the identification of candidate genes that could be used to classify precocious or more fertile individuals[6,7].

这种象征的易象与“道”相联系，也具有形上性的特征。刘若愚在《中国文学理论》中认为:“在‘形上’的标题下，可以包括以文学为宇宙原理之显现这种概念为基础的各种理论”，“在形上理论中，宇宙原理通常称为‘道'”[12](P20)。宇宙原理仅是“道”的涵义的一部分，《周易》一书多次提到了形而上的“道”，《易传·系辞》曰：

The identification of candidate genes provides a better understanding of the distribution of genes that affect traits of economic interest,as well as a basis for further studies to identify causal mutations.Despite its potential,important observations are needed for this approach.According to Tabor et al.[8],the difficulty of replication over time or across populations in this approach might indicate that more detailed studies are needed to certify its causality.Therefore,the aim of this study was to perform a GWAS to identify genomic regions and candidate genes to uncover the genetic architecture of scrotal circumference at 210 and 420 d of age and age at first and second calving and aid in the breeding process in Canchim cattle.

Methods

Canchim breed

The Canchim is a composite breed(62.5%Charolais and 37.5%Zebu)developed in the 1940s by the Brazilian Agricultural Research Corporation(Embrapa),located in São Carlos city,SP,Brazil[9].Different crossbreeding schemes have been studied to produce Canchim cattle with different Charolais-Zebu proportions and to achieve greater genetic variability in the population[10-13].One of these schemes was used to produce animals of the“MA”genetic group,which was derived from mating Canchim-Zebu animals(F1)with Charolais animals,resulting in approximate contributions of 65.6%Charolais and 34.4%Zebu[14].

The Canchim cattle represents about 3%of the beef cattle produced in Brazil[15].Indicine breeds are much more representative as purebreds or crossbred animals,being responsible for 80%of the beef cattle industry in the country[16].

而这时要根据红豆杉植物自身喜湿、怕涝，喜阴凉，极耐阴的特点对播种的位置进行确定。因此，在进行红豆杉播种位置的选择上，都是选择土壤疏松肥沃、便于进行排灌的土地。当确定种植位置后，需要对土地进行翻耕，并对其进行基肥的施撒，从而保证土壤的营养满足幼苗成长需求。一般基肥主要以腐熟的农机有机肥为主。此外，为了降低病虫害发生的几率，在进行基肥的施加过程中，可以同时加入少量的硫酸亚铁或硫酸铜粉末进行相应的消毒。

2）自主学习与协作学习的结合情况。大部分学习者偏好自主学习，协作学习较少。调查显示，教师对于混合式远程学习的理解就是“看视频+提交作业”，协作学习开展较少。现有远程学习平台为学习者提供的协作学习方式有论坛研讨、教师工作坊。笔者以管理员的身份进入某平台中的教师工作坊中进行查看，总计47个工作坊，未开展活动的有10个，开展活动的有37个，其中活动次数≥5的只有五个，工作坊中的可用资源数量也不多，可见学习者对于开展协作学习的积极性还是不高。

The BovineHD BeadChip SNP panel(Illumina Inc.,San Diego,CA)was used to genotype 194 males and 205 females:285 animals of the Canchim breed and 114 animals of the MA genetic group,calves of 49 bulls and 355 cows.The animals were born between 1999 and 2005 and come from seven farms in the States of São Paulo and Goiás.A detailed description of these animals was previously presented by Buzanskas et al.[20]and Mokry et al.[18].Genotype quality control excluded SNPs with significant deviations(P＜10-5)from the Hardy-Weinberg equilibrium,SNPs with minor allele frequencies of less than 0.05,and call rate for SNPs and animals lower than 0.90.Only autosomal chromosomes and SNPs with known positions,according to the UMD_3.1 bovine genome assembly[21],were used for GWAS.

The estimated breeding values(EBVs)used in our study considered the following traits:scrotal circumference at weaning adjusted for 210 d of age(SC210),scrotal circumference adjusted for 420 d of age(SC420),age at first calving(AFC),and age at second calving(ASC).Linear interpolation was previously used to adjust the scrotal circumferences for 210 and 420 d of age.

在果蔬病害生物保鲜研究中发现的拮抗细菌主要是芽胞杆菌，其次是假单胞杆菌。芽胞杆菌属好氧或兼性厌氧菌，抗逆能力强，繁殖速度快，营养要求简单，容易定殖在植物表面，适合作为生防制剂。目前，应用于果蔬防腐的有枯草芽胞杆菌、多粘芽胞杆菌和蜡状芽胞杆菌等[6]。

The EBVs were obtained from the genetic evaluation carried　outby　the　Brazilian　AgriculturalResearch Corporation(Embrapa)for the Canchim breed which considered 318,307 animals in the relationship matrix and 267,002 animals with valid records.In general,the traits analyzed by Embrapa for the genetic evaluation of Canchim that could be highlighted are body weight traits measured at various ages,reproductive traits of males and females,carcass quality,navel visual score of males and females,and hair coat.The EBVs were estimated by multi-trait analysis using the REMLF90 program[17],under an animal model.For the studied traits,the fixed effects considered in the contemporary groups were year and season of birth(January to March;April to September;and October to December),farm of birth,genetic group,and feeding system.

As described by Mokry et al.[18],the genotyped animals were chosen according to the EBV for some traits(ribeye area,back fat thickness,and productive and reproductive traits),accuracy,family size,and proportion of males and females.The mean EBV values for SC210,SC420,AFC,and ASC were 1.44 mm,2.07 mm,-4.34 d,and-1.23 d,respectively.Minimum and maximum values for SC210,SC420,AFC,and ASC varied from-8.76 to 12.74,-17.41 to 21.80,-50.89 to 44.64,and-42.46 to 44.66,respectively.De-regressed proofs were not used due to limited data.Furthermore,de-regressed proofs calculated with low accuracies are expected to have a smaller genomic contribution due to Mendelian sampling and will have more noise added in de-regressed calculations[19].

Genotypes and quality control

Traits analyzed

Genome-wide association study

The Generalized Quasi-Likelihood Score(GQLS)method[22]was used for GWAS.In this method,a logistic regression was used to associate the EBVs(treated as a covariate)to the genotypes(treated as the response variable),one SNP at a time.The EBVs were represented by Xi=(X1,…,Xn)',in which Xi represents the EBVs for the ith animal.The genotype of the animals was coded as “0”,“1”or “2”and,as the genotypes were represented by Yi=(Y1,…,Yn)',in which Yi=½*(number of alleles for animal genotype i),the respective proportions would be equal to 0,½and 1.The expected SNP allele frequency is represented by μ,in which μ =(μ1,… ,μn)'=E(X|Y),thus 0＜μi＜1.

To associate μiwith Xi,the following logistic regression was defined:

我想，我们的暗恋大多无果而终，是因为自己不够优秀吧。“我不想继续开小卖部了，我要奋斗！”沐子她从小卖部撤了资，藏起浮躁的女汉子性格，不再成天把八卦和大话挂在嘴边，虔诚地皱着眉头听老师讲课，活得像一个“出家人”，灵台一片澄澈空明。

（4）部分学生过度依靠母语汉语对其的影响，甚至把听到的每一个单词和句子翻译成中文，便于理解，但是忽略了中英之间语言语法差异，心译过程造成的时间差反之增加听力理解难度。

The false discovery rate(FDR)was used for multiple testing corrections[24]to verify significant SNPs.The P-values of each SNP were sorted in ascending order and the following formula applied:

in which q is the desired level of significance,m is the total number of SNPs,and P is the P-value of the ith SNP.To account for multiple comparisons,a genomewide and chromosome-wise FDR of 5%(significant association)and 10%(suggestive association)was applied.The main reason for considering a minimum FDR of 10%was to obtain a comprehensive number of SNPs,which might assist to comprehend the genetic architecture of the studied traits.

团场学校、幼儿园通过每周一次的双语口语学习培训、民族团结主题班会、学习传统文化主题队会等系列活动415场次，把民族团结的种子播撒到了孩子幼小的心灵中。医院组成医疗队伍远赴离团场300多公里以外的青河县，为阿热勒乡达巴特村的村民进行免费义诊。元旦春节、肉孜节、古尔邦节期间，团场党员干部开展了“走亲戚庆节日”活动。通过活动的开展，加深了彼此间的了解与认识，增进了彼此间的友谊和感情。

Gene mapping and in silico functional analyses

The SNPs associated with the EBVs for SC210,SC420,AFC,and ASC traits were surveyed to their corresponding genes or to surrounding genes.The National Center for Biotechnology Information(NCBI)database[28]and Ensembl Genome Browser[29]were accessed to proceed with in silico functional analyses of the genes.If the SNP was located in the intergenic region(i.e.not assigned to any gene),we observed,through the integrated maps of the NCBI variation viewer,for the closest gene(s)and calculated the distance(s).

The PANTHER tool[30]was used to access and gather biological processes and pathway associations.The AnimalQTLdb database[31]was used to verify previous reports of quantitative trait loci(QTL)in the surroundings of significant SNPs.The main reason to use these tools was to validate our findings.

Results

A total of 672,778 SNPs and 392 animals were used for GWAS.The Manhattan plots for chromosome-wise(in blue)and genome-wide(in red)significantly associated SNPs after FDR correction for SC210,SC420,AFC,and ASC,respectively,are presented in Fig.1.The greatest number of significant SNPs was observed for SC420(435 SNPs),followed by SC210(12 SNPs),AFC(six SNPs),and ASC(four SNPs)when considering FDR 10%for all traits.

In Table 1 are presented SNPs and genes identified for SC210,AFC,and ASC.For SC420,SNPs and genes are presented in Table 2.Due to the large number of SNPs associated with SC420,full information regarding genes,pseudogenes,and non-coding RNA are presented in Additional files 1,2,and 3.For FDR 5%,a total of 249(chromosome-wise)and 50(genome-wide)SNPs were observed for SC420(Additional file 1).Considering FDR 10%,the regions observed in Table 1 presented a suggestive association;therefore,the genes identified in these regions were surveyed.

Additional file 1:Significantly associated single nucleotide polymorphism(SNP)with scrotal circumference at 420 days of age(SC420)after Bonferroni(B)and false discovery rate(FDR)correction at a chromosome-wise(CW)and genome-wide(GW)level.(DOCX 95 kb)

Error Analysis and Treatment Strategy of Ultra Short Baseline Positioning System for Submarine Cable ZHOU Jing,XIAO Bo,GONG Houcheng,GUO Qiang(138)

Fig.1　Manhattan plots for scrotal circumference at 210 d of age(a),scrotal circumference at 420 d of age(b),age at first calving(c),and age at second calving(d).Chromosomes with significant SNPs are highlighted in black.The significant SNPs,after false discovery rate correction of 10%(chromosome-wise),are highlighted in red.The significant SNPs,after false discovery rate correction of 10%(genome-wide),are highlighted in blue.On the y-axis are presented the-Log of the P-values for each SNP.On the x-axis are presented the autosomal chromosomes

For SC420,chromosome-wise and genome-wise associations were observed on chromosomes 5,9,13,14,18,and 21(Fig.1b).The SNPs were close or within a total of 64 genes,10 non-coding RNAs,13 pseudogenes,and two transfer RNAs.Full information on SNPs,position,and genes are presented in the Additional files 1,2,and 3.

Six significantly associated SNPs(P≤0.00001)for AFC(Table 1 and Fig.1c)were located on chromosomes4(NXPH1 and EXOC4 genes)and 27(ZMAT4 gene).ForASC,fourSNPsweresignificantlyassociated(P≤0.000001)and located on chromosomes 10(FMN1 gene),11(TMEM182 gene and LOC790871 pseudogene),and 15(UBQLN3 gene).Biological processes for SC210,AFC,and ASC are presented in Fig.2.

Table 1　Chromosome-wise association for scrotal circumference(SC210),age at first(AFC)and second calving(ASC)

aintergenic region bintron variant cexon variant Gene symbols,SNP reference number,and chromosomes(Chr)and positions(Pos,in megabase)were obtained from NCBI website.Distances to gene(kilobase)are presented from 5′to gene direction.If distance equals zero(0.00),the SNP is on intragenic region.P-values are presented as the minimum(Min)and maximum(Max)significance obtained from the generalized quasi-likelihood method

Trait　Genes　SNP Reference　Chr:Pos　Distance to gene　P-value(Min-Max)SC210　SMIM23　rs135355728a　20:3.48　14.22　8.96E-06 SC210　PAPD7　rs137042056a,rs136276163a　20:66.52..66.53　48.62..38.66　5.85E-06-p2.32E-05 SC210　ICE1　rs41582170b　20:67.81　0.00　2.13E-05 SC210　LOC101907249　rs136535499a　20:69.05　240.87　2.13E-05 SC210　EDARADD　rs110746860b,rs110371081b,rs109902875b,rs110870694b,rs110610232b,rs134356559b,rs210911576b 28:9.14..28:9.16　0.00　6.71E-06-5.76E-05 AFC　NXPH1　rs133411648a　4:17.46　45.77　8.26E-07 AFC　EXOC4　rs110606254a　4:98.31　0.00　1.74E-06 AFC　ZMAT4　rs134390082a,rs137553882a,rs133519327a,rs135481346a 27:35.19..35.21　48.32..29.34　1.23E-05-3.18E-05 ASC　FMN1　rs134100268b　10:29.88　0.00　2.43E-06 ASC　TMEM182　rs43661848a　11:7.69　197.15　6.29E-06 ASC　LOC790871　rs136610615a　11:22.29　69.94　2.77E-06 ASC　UBQLN3　rs43031470c　15:48.70　0.00　3.39E-06

Table 2　Highlighted genes from genome-wide(in bold)and chromosome-wise associations for scrotal circumference at 420 d of age

aintergenic regionbintron variantcdownstream variantd5′UTReupstream variant Gene symbols,SNP reference number,and chromosomes(Chr)and positions(Pos,in megabase)were obtained from NCBI website.Distances to gene(kilobase)are presented from 5′to gene and 3′to gene directions.If distance equals zero(0.00),the SNP is on intragenic region.P-values are presented as the minimum(Min)and maximum(Max)significance obtained from the generalized quasi-likelihood method

Symbol　SNP Reference Chr:Pos　Distances to gene　P-value(Min-Max)RAP1B　rs110520377a,rs133990240a,rs109547215a,rs110160018b,rs110261691c,rs109099268b,rs133124963b,rs109023687b,rs110034677b,rs110091099b,rs109950552b,rs137658592b,rs133340933b,rs134626455b,rs110001336b,rs109288126b,rs137319832b,rs109506571b,rs109248631b,rs110027103b,rs109561643b,rs109210079b,rs110160918b,rs134366426b,rs109273768b,rs109024096b,rs110798702b,rs110625630b,rs110219262b,rs134311132b,rs135497432b,rs133173059b 5:45.35..45.40　8.58..0.00　2.47E-07-7.64E-05 SRGAP1　rs110268648b,rs109748105b,rs134621421b　5:49.98..49.98　0.00　1.88E-06-6.55E-06 FOXM1　rs135705262b　5:107.39　0.00　4.00E-04 TOP1　rs134822694b,rs135287766b　13:70.39..70.40　0.00　3.86E-06 STAU2　rs137465376b,rs134711539b,rs137821036a　14:38.89..38.97　0.00..14.40　8.66E-05-5.00E-04 PEX2　rs137442228d,rs110035827a,rs41730291a　14:42.33..42.37　0.00..40.77　4.10E-04-9.80E-04 FABP5　rs109480456a,rs136045797a,rs133930486e,rs136613853b,rs137684819c,rs133054550a,rs133483556a,rs136236059a,rs136236059a,rs137344980a,rs135804214a,rs136785030a,rs135727060a,rs133436244a,rs134708967a,rs43103204a 14:46.63..46.73　15.48..0.00..83.31　1.55E-05-1.16E-03 FABP12　rs43765470b,rs43765465a,rs41730924a　14:46.89..46.92　0.00..28.04　3.48E-05-2.88E-04 MED30　rs135065691c,rs41734435b,rs135292147a　14:48.94..48.97　4.03..0.00..9.76　8.27E-04-9.48E-04 TRHR　rs133457508e　14:57.52　0.00　2.27E-04

Fig.2　Biological processes for scrotal circumference(SC210)and age at first(AFC)and second(ASC)calving

For SC420,biological processes and pathway associations are presented in Figs.3 and 4,respectively.PANTHER tool was able to account for 62 genes overrepresented among pathways when using Bos taurus as background.Most of the biological processes were involved in metabolic(32 genes),cellular(22 genes),developmental(14　genes),biologicalregulation　(13 genes),and localization(11 genes)processes.Biological processes were observed for the immune system(seven genes),multicellular organismal process(seven genes),response to stimulus(seven genes),apoptotic process(six genes),cellular component organization or biogenesis(six genes),biological adhesion(four genes),and reproduction(one gene).Pathway analysis showed that the RAP1B gene is involved in four pathways,while the IFNG,COL12A1,and SRGAP1 genes are involved in two pathways.

The average linkage disequilibrium for each trait by chromosome was equal to 0.70(AFC-BTA27),0.94(SC210-BTA20),0.89(SC210-BTA28),0.64(SC420-BTA5),0.69(SC420-BTA9),0.90(SC420-BTA13),0.56(SC420-BTA14),0.85(SC420-BTA18),and 0.99(SC420-BTA21).Some significant or suggestive regions presented r2 equal to zero.We highlighted the SC420 trait due to the higher number of regions presenting significant or suggestive associations(Additional file 4:Figs.S1,S2,S3 and S4).

Discussion

Fig.3　Biological processes for scrotal circumference at 420 d of age

The suggestive associations observed for SC210,AFC,and ASC,could provide an insight over potential regions responsible for the genetic variability of the traits.The main reason to consider FDR 10%was to study and describe,in a broader point ofview,the biological pathways to aid in the comprehension of these complex traits.Furthermore,due to the polygenic inheritance of these traits,it was expected that many loci with small effects were responsible for expressing the phenotype(or EBV).In some cases,we observed the lack of information regarding the SNP or gene identified.

We observed high average linkage disequilibrium among significantly or suggestively associated SNPs for most of the traits studied;therefore,there is evidence of direct or indirect associations that could be affecting the traits.Mokry et al.[27],when studying this Canchim population,observed that the linkage disequilibrium estimated could be used for genomic selection and GWAS(minimum r2 varying from 0.33 to 0.40 up to 2.5 kb).

Fig.4　Pathways associations for scrotal circumference at 420 d of age

For SC210,we have found that the SMIM23 gene is located inside QTL regions associated with the rate of nonreturn to estrus in Swedish Red and Swedish Holstein breed animals[32].In this same region,McClure et al.[33]found QTL for scrotal circumference in American Angus cattle.Studies reported a QTL in the PAPD7,ICE1,and LOC101907249 gene regions associated with percentage abnormal sperm in Holstein bulls[34]and calving ease in Angus breed cows[33].The EDARADD gene participates in cell differentiation and mutations in this gene can result in abnormal development of tissues and organs of ectodermal origin[35].In this region,a QTL associated with pregnancy rate trait have been reported by Boichard et al.[36].No specific biological process for SMIM23,ICE1,and LOC101907249 genes was observed in the literature.

2016年我国由《华盛顿协议》预备会员转正，成为该协议第18个正式成员，从此我国化工专业认证工作全面展开[1]。在认证过程中毕业要求的制定、分解、支撑及评价工作是专业建设持续改进的重要依据，也是专业认证中自评报告的重要组成部分。

Few suggestive associations were observed for SC210 and none of the SNPs associated with SC210 presented pleiotropic effect with SNP associated with SC420(discussed below)or other studied traits(discussed as follows),which could be due to low genetic correlation among EBVs for these traits in our dataset.

Regarding the SC420 trait,we have identified the STAU2 gene,which participates in multiple biological processes(Table 2 and Fig.3),highlighting reproduction,developmental and immune system.This gene was involved in muscle development[37]in Bos taurus indicus,Bos taurus taurus,and Bos taurus taurus x Bos taurus indicus.According to Ramayo-Caldas et al.[37],the STAU2 gene was present in two main networks of PPARGC1A and HNF4G genes,which acts as transcription factors that activate a variety of hormone receptors[38]and binding to fatty acids[39].Peddinti et al.[40]observed,through genetic network analysis,that one of the main biological networks for bovine germinal vesicle and bovine cumulus granulosa cell proteomes contain the SRGAP1 and TOP1 genes,respectively.

The RAP1B gene is involved in four pathways(Fig.4),including gonadotropin-releasing hormone receptor pathway.This gene was also observed associated with cell-tocell signaling network(integrin,ephrin receptor,and mitogen-activated protein kinase signaling network)[40].The MED30 and TRHR genes are strongly related to thyroid hormone,triggering hormonal processes associated with reproductive systems in males and females[41,42].

Genes related to fatty acid,cholesterol,and triacylglycerol,such as FABP5[43],FABP12[44],PEX2[45],and MED30[46]are critical for energy and hormone production in males and females.Cholesterol is a precursor of steroid hormone production,such as testosterone,and therefore it is involved in male growth and reproductive development.

Jin等对Nivre和Yamada方法进行改进，新的移进—归约算法采用双阶段方式进行汉语依存分析，第一阶段的归约由两部分构成，一是归约左边的依存弧，二是归约右边的体词性依存节点，第二阶段则主要是对右边的动词性依存节点进行归约。实验时，先对CTB 4.0进行转换，然后抽取转换结果中部分句子作为实验数据，依存正确率为84.52%。

Important genome-wide associations on chromosome 14 for scrotal circumference were observed by Fortes et al.[5],demonstrating that this chromosome presents regions of interest that could be explored to identify sexually precocious animals.These authors observed associations with age at first corpus luteum in the same regions as scrotal circumference and have attributed their results to the genetic correlation between these traits,thus genes and SNPs associated with puberty in heifers were likely to be relevant for puberty in bulls,and vice versa.On chromosome 14,a large number of SNPs associated with puberty were identified in both bulls and heifers.Urbinati et al.[47]found important selection signatures in Canchim cattle on chromosomes 5 and 14,which were related to pigmentation(strongly selected trait in Charolais and Canchim),productive and reproductive traits.

Reports of QTL associated with reproductive traits of interest may give support to the results found in our study;however,most of the QTL were reported for female traits.On chromosome 5,QTL associated with the concentration of follicle-stimulating hormone in Brahman and Hereford crosses[48],have been described.The QTL were observed as associated with interval to first estrus after calving[7]and dystocia in dairy cattle[49]on chromosome 13.On chromosome 14,QTL associated with gestation period[50],number of inseminations per conception[51],and ovulation rate[52]have been reported.

As favorably correlated responses between scrotal circumference measures and age at first calving traits are expected through selection,whereas the genes previously reported could be highlighted as a candidate to,directly and indirectly,improve the reproductive performance of males and females,respectively.Moreover,fat deposition in cattle could be reflected in sexual precocity and carcass finishing,traits that have become the main concern of beef cattle breeders.

Pairwise linkage disequilibrium was estimated using the r2measure[25]for the SNPs with significant and suggestive associations.The software Haploview[26]was used to estimate r2and plot the results.Due to the high linkage phase consistency between Canchim and MA genetic group(above 0.80 up to 100 kb)[27],analyses were conducted considering Canchim and MA as one population.

Few genes were observed across the positions of significant SNPs for AFC(Table 1 and Fig.1c).According to Blaschek et al.[53],an association of the SNP rs110984522(178.73 kb apart from the SNP rs133411648)was observed for sire fertility in Holstein breed.The EXOC4 gene plays a role in insulin processing,metabolism of proteins,and peptide hormone metabolism pathways.Reports of QTL associated with scrotal circumference[33]have been described in this region.The ZMAT4 gene,located on chromosome 27,participates in the apoptotic,biological,developmental,and metabolic processes.QTL associated with dystocia[49]and calving ease[54]in Holstein cattle and non-return rate[55]in Angus cattle have been described.

6月中下旬，选择温185、新新2优良核桃的半木质化新梢枝条做接穗，选取新梢枝条上部的雌花芽做为方块芽接的接芽。在选取的接芽长度3 cm、宽度1.5 cm上下左右各切一刀深达木质部，将叶柄留1 cm削除。

Significant SNPs for ASC are presented in Table 1 and Fig.1d.The FMN1 gene located on chromosome 10 participates in actin cytoskeleton organization and is of great importance for cell and muscle movements[56].QTL associated with calving ease have been observed in this region[33].

A QTL associated with scrotal circumference was observed in the region of the TMEM182 gene[33].No function or biological processes have been described for the LOC790871 and TMEM182 genes in the literature or databases consulted.In the region comprising the LOC790871 gene,QTL regions have been reported associated with the following traits:scrotal circumference[33],subcutaneous fat[57],and sperm motility[34]in Angus,Holstein,and Charolais×Holstein crossbred cattle,respectively.

Ithasbeen　verified　thattheUBQLN3geneis expressed in the testes,acts in spermatogenesis in humans and rats[58]and is conserved in mammals(Homo sapiens,Mus musculus,Rattus norvegicus,Canis lupus familiaris,and Bos taurus).This gene is involved in the protein processing in endoplasmic reticulum pathway.A QTL associated with weight and carcass traits has been described[33]in the region in which the UBQLN3 gene is located.

Conclusions

Few associations were observed for SC210,AFC,and ASC,reinforcing their polygenic inheritance and the complexity of understanding the genetic architecture of reproductive traits.Finding many associations for SC420 in various regions of the Canchim genome also reveals the difficulty of targeting specific candidate genes that could act on fertility;nonetheless,the high linkage disequilibrium between loci herein estimated could aid to overcome this issue.Therefore,all relevant information about genomic regions influencing reproductive traits may contribute to target candidate genes for further investigation of causal mutations and aid in future genomic studies in Canchim cattle to improve the breeding program.

Additional files

For SC210,chromosome-wise significantly associated SNPs(P≤0.00001)were located on chromosomes 20 and 28.Five SNPs were located in the intergenic or intragenic regions of SMIM23,PAPD7,ICE1,and EDARADD genes,and non-coding RNA LOC101907249(Table 1 and Fig.1a).On chromosome 28,seven SNPs were located in the EDARADD gene.

Additional file 2:Genome-wide(in bold)and chromosome-wise associations for scrotal circumference at 420 days of age.Gene symbols,SNP reference number,and chromosomes(Chr)and positions(Pos,in megabase)were obtained from NCBI website.Distances to gene(kilobase)are presented from 5′to gene and 3′to gene directions.If distance equals zero(0.00),the SNP is on intragenic region.P-values are presented as the minimum(Min)and maximum(Max)significance obtained from the generalized quasilikelihood method.(DOCX 34 kb)

Additional file 3:Candidate genes identified for scrotal circumference at 420 days of age.(DOCX 26 kb)

Additional file 4:Fig.S1.Linkage disequilibrium for scrotal circumference at 420 d of age on chromosome 5.Fig.S2.Linkage disequilibrium for scrotal circumference at 420 d of age on chromosome 9.Fig.S3.Linkage disequilibrium for scrotal circumference at 420 d of age on chromosome 13.Fig.S4.Linkage disequilibrium for scrotal circumference at 420 d of age on chromosome 14.Fig.S5.Linkage disequilibrium for scrotal circumference at 420 d of age on chromosome 18.Fig.S6.Linkage disequilibrium for scrotal circumference at 420 d of age on chromosome 21.(ZIP 1985 kb)

Abbreviations

Under the null hypothesis,WG follows a Chi-squared distribution with one degree of freedom[23],resulting in P-values for each SNP.This method does not account for the genetic variance explained by the marker.The GQLS provides the advantage of accounting for the population structure by means of the pedigree-based relationship among animals(A-1).

AFC:Age at first calving;ASC:Age at second calving;BTA:Bos taurus chromosome;EBV:Estimated breeding value;FDR:False discovery rate;GQLS:Generalized Quasi-Likelihood Score;GWAS:Genome-wide association study;QC:Quality control;QTL:Quantitative trait loci;r2:Linkage disequilibrium measure;SC210:Scrotal circumference at 210 days of age;SC420:Scrotal circumference at 420 days of age;SNP:Single nucleotide polymorphism

Acknowledgements

The authors would like to acknowledge the Universidade Estadual Paulista-Faculdade de Ciências Agrárias e Veterinárias(UNESP/FCAV),University of Guelph,and the Brazilian Agricultural Research Corporation(Embrapa).We also thank Dr.Mehdi Sargolzaei for assistance and providing the software used in our study.

Funding

This study was supported by the“Conselho Nacional de Desenvolvimento Científico e Tecnológico”(CNPq)-449,564/2014-2.DPM,MVGBS,LCAR,and MMA were supported by a fellowship from CNPq.NBS received a Post-Doctoral fellowship from CAPES/PNPD.TCSC and LDR were supported by the São Paulo Research Foundation(Fapesp)fellowship(2015/08939-0 and 2013/13972-0).

Availability of data and materials

The data that support the findings of this study are available upon reasonable request contacting Dr.Luciana Correia de Almeida Regitano,luciana.regitano@embrapa.br,telephone:55 16 3411-5600,Rodovia Washington Luiz,Km 234,Fazenda Canchim,CEP:13,560-970,São Carlos/SP,Brazil.

Authors’contributions

MEB:contributed to the development of this research with data analysis,interpretation,figure compositions,manuscript writing,and revision.FSS,DAG,and RVV:data analysis,manuscript discussion,and revision.SLCM,FBM,MMA,LCAR,RHH,MAM,and MVGBS:experimental design.NBS,TCSC,LDR,and DPM:data interpretation.SLCM,LCAR,MMA,and FBM:experimental design,interpretation,and manuscript revision.All authors read and approved the final manuscript.

出台了《水利建设基金筹集和使用管理办法》等4项水利非税筹资政策；通过采取建设费源信息数据库、强化宣传、履行司法程序等措施，加大非税征缴力度，做到应收尽收。2011年全省水利非税收入达49亿元，较2010年增长24.2%。2012年，全省水利建设基金、土地出让计提和水利非税收入将力争达到58.7亿元。

Ethics approval and consent to participate

This study was performed with the approval of the Embrapa Southeast Livestock Ethical Committee of Animal Use(CEUA-CPPSE)under protocol number 02/2009.

Consent for publication

针对上述问题的解决，就以我司的成本中心划分及成本核算流程为例进行详解。我司是一家专门从事向各类连锁企业提供整体展示货架的生产与销售的非标制造企业，在长期的生产实践过程中，摸索出一套适合非标生产企业的成本核算方案，能够有效平衡成本效益原则，其方案设计如下：

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

1Departamento de Zootecnia,Universidade Federal da Paraíba(UFPB),Areia,Paraíba 58397-000,Brazil.2Fast Genetics,Saskatoon,SK S7K 2K6,Canada.

3Beef Improvement Opportunities(BIO),Guelph,ON N1K 1E5,Canada.

4Department of Animal and Poultry Science,University of Guelph,Centre for Genetic Improvement of Livestock(CGIL),Guelph,ON N1G 2W1,Canada.

5Departamento de Ciências Exatas,Faculdade de Ciências Agrárias e Veterinárias,Universidade Estadual Paulista(Unesp),Jaboticabal,São Paulo 14884-900,Brazil.6Departamento de Zootecnia,Núcleo de Pesquisa e Conservação de Cervídeos,Faculdade de Ciências Agrárias e Veterinárias,Universidade Estadual Paulista(Unesp),Jaboticabal,São Paulo 14884-900,Brazil.7Department of Animal Science,Federal University of Lavras(UFLA),Lavras,Minas Gerais 37200-000,Brazil.8Department of Genetics and Evolution,Federal University of São Carlos(UFSCar),São Carlos,São Paulo 13565-905,Brazil.9Embrapa Agricultural Informatics,Campinas,São Paulo 13083-886,Brazil.10Embrapa Dairy Cattle,Juiz de Fora,Minas Gerais 36038-330,Brazil.11Embrapa Southeast Livestock,São Carlos,São Paulo 13560-970,Brazil.

References

1.　Trocóniz JF,Beltrán J,Bastidas H,Larreal H,Bastidas P.Testicular development,body weight changes,puberty and semen traits of growing Guzerat and Nellore bulls.Theriogenology.1991;35:815-26.

2.　Martin LC,Brinks JS,Bourdon RM,Cundiff LV.Genetic effects on and subsequent heifer puberty.J Anim Sci.1992;70:4006-17.

3.　Buzanskas ME,Savegnago RP,Grossi DA,Venturini GC,Queiroz SA,Silva LOC,et al.Genetic parameter estimates and principal component analysis of breeding values of reproduction and growth traits in female Canchim cattle.Reprod Fertil Dev.2013;25:775-81.

4.　Burns BM,Gazzola C,Holroyd RG,Crisp J,McGowan MR.Male reproductive traits and their relationship to reproductive traits in their female progeny:a systematic review.Reprod Domest Anim.2011;46:534-53.

5.　Fortes MRS,Lehnert SA,Bolormaa S,Reich C,Fordyce G,Corbet NJ,et al.Finding genes for economically important traits:Brahman cattle puberty.Anim Prod Sci.2012;52:143-50.

6.　Feugang JM,Kaya A,Page GP,Chen L,Mehta T,Hirani K,et al.Two-stage genome-wide association study identifies integrin beta 5 as having potential role in bull fertility.BMC Genomics.2009;10:176.

7.　Sahana G,Guldbrandtsen B,Bendixen C,Lund MS.Genome-wide association mapping for female fertility traits in Danish and Swedish Holstein cattle.Anim Genet.2010;41:579-88.

8.　Tabor HK,Risch NJ,Myers RM.Candidate-gene approaches for studying complex genetic traits:practical considerations.Nat Rev Genet.2002;3:1-7.

9.　Vianna AT,Pimentel-Gomes F,Santiago M.Formação do Gado Canchim pelo Cruzamento Charolês-Zebu.Nobel:São Paulo;1978.

10.Alencar MM.Bovino-Raça Canchim:Origem e Desenvolvimento.Brasília:Embrapa-DMU;1988.

11.Baldi F,Albuquerque LG,Alencar MM.Random regression models on Legendre polynomials to estimate genetic parameters for weights from birth to adult age in Canchim cattle.J Anim Breed Genet.2010;127:289-99.

12.Buzanskas ME,Grossi DA,Baldi F,Barrozo D,Silva LOC,Torres Júnior RAA,et al.Genetic associations between stayability and reproductive and growth traits in Canchim beef cattle.Livest Sci.2010;132:107-12.

13.Barbosa PF.O Canchim na Embrapa Pecuária Sudeste.In:Convençao Nacional da Raça Canchim,São Carlos:Embrapa Pecuária Sudeste,2000;55-69.

14.Andrade PC,Grossi DA,Paz CCP,Alencar MM,Regitano LCA,Munari DP.Association of an insulin-like growth factor 1 gene microsatellite with phenotypic variation and estimated breeding values of growth traits in Canchim cattle.Anim Genet.2008;39:480-5.

15.ABCCAN.Associação Brasileira de Criadores de Canchim 2015.http://www.abccan.com.br.Accessed 05 Nov 2015.

16.Associação Brasileira dos Criadores de Zebu-ABCZ.Estatística total Brasil RGN+RGD-período 1939 a 2012 2013.http://www.abcz.org.br.Accessed 5 Nov 2013.

17.Misztal I.Reliable computing in estimation of variance components.J Anim Breed Genet.2008;125:363-70.

18.Mokry FB,Higa RH,Mudadu MA,Lima AO,Meirelles SLC,MVG B d S,et al.Genome-wide association study for backfat thickness in Canchim beef cattle using random Forest approach.BMC Genet.2013;14:47.

19.Boddhireddy P,Kelly MJ,Northcutt S,Prayaga KC,Rumph J,DeNise S.Genomic predictions in Angus cattle:comparisons of sample size,response variables,and clustering methods for cross-validation.J Anim Sci.2014;92:485-97.

20.Buzanskas ME,Grossi DA,Ventura RV,Schenkel FS,Sargolzaei M,Meirelles SLC,et al.Genome-wide association for growth traits in canchim beef cattle.PLoS One.2014;9:e94802.

21.Zimin A V,Delcher AL,Florea L,Kelley DR,Schatz MC,Puiu D,et al.A whole-genome assembly of the domestic cow,Bos Taurus.Genome Biol.2009;10:r42.

22.Feng Z,Wong WWL,Gao X,Schenkel F.Generalized genetic association study with samples of related individuals.Ann Appl Stat.2011;5:2109-30.doi:10.1214/11-AOAS465.

23.Heyde CC.Quasi-likelihood and its application:a general approach to optimal parameter estimation.Canberra:Springer;1997.

24.Benjamini Y,Hochberg Y.Controlling the false discovery rate a practical and powerful approach to multiple testing.J R Stat Soc Ser B.1995;57:289-300.

25.Hill W,Robertson A.Linkage disequilibrium in finite populations.Theor Appl Genet.1968;38:226-31.

26.Barrett JC,Fry B,Maller J,Daly MJ.Haploview:analysis and visualization of LD and haplotype maps.Bioinformatics.2005;21:263-365.

27.Mokry FB,Buzanskas ME,Mudadu MA,Grossi DA,Higa RH,Ventura RV,et al.Linkage disequilibrium and haplotype block structure in a composite beef cattle breed.BMC Genomics.2014;15:S6.

28.National Center for Biotechnology Information.2016.http://www.ncbi.nlm.nih.gov/snp/.Accessed 11 Nov 2016.

29.Ensembl Genome Browser.2016.http://www.ensembl.org/index.html.Accessed 12 Nov 2016.

30.Mi H,Muruganujan A,Thomas PD.PANTHER in 2013:modeling the evolution of gene function,and other gene attributes,in the context of phylogenetic trees.Nucleic Acids Res.2013;41:377-86.

31.AnimalQTLdb.2016.http://www.animalgenome.org.Accessed 15 Nov 2016.

32.Holmberg M,Andersson-Eklund L.Quantitative trait loci affecting fertility and calving traits in Swedish dairy cattle.J Dairy Sci.2006;89:3664-71.

33.McClure MC,Morsci NS,Schnabel RD,Kim JW,Yao P,Rolf MM,et al.A genome scan for quantitative trait loci influencing carcass,post-natal growth and reproductive traits in commercial Angus cattle.Anim Genet.2010;41:597-607.

34.Druet T,Fritz S,Sellem E,Basso B,Gérard O,Salas-Cortes L,et al.Estimation of genetic parameters and genome scan for 15 semen characteristics traits of Holstein bulls.J Anim Breed Genet.2009;126:269-77.

35.Gargani M,Valentini A,Pariset L.A novel point mutation within the EDA gene causes an exon dropping in mature RNA in Holstein Friesian cattle breed affected by X-linked anhidrotic ectodermal dysplasia.BMC Vet Res.2011;7:1-7.

36.Boichard D,Grohs C,Bourgeois F,Cerqueira F,Faugeras R,Neau A,et al.Detection of genes influencing economic traits in three French dairy cattle breeds.Genet Sel Evol.2003;35:77-101.

37.Ramayo-Caldas Y,Fortes MRS,Hudson NJ,Porto-Neto LR,Bolormaa S,Barendse W,et al.A marker-derived gene network reveals the regulatory role of PPARGC1A,HNF4G,and FOXP3 in intramuscular fat deposition of beef cattle.J Anim Sci.2014;92:2832-45.

38.Summermatter S,Shui G,Maag D,Santos G,Wenk MR,Handschin C.PGC-1α improves glucose homeostasis in skeletal muscle in an activitydependent manner.Diabetes.2013;62:85-95.

39.Wisely GB,Miller AB,Davis RG,Thornquest AD,Johnson R,Spitzer T,et al.Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids.Structure.2002;10:1225-34.

40.Peddinti D,Memili E,Burgess SC.Proteomics-based systems biology modeling of bovine germinal vesicle stage oocyte and cumulus cell interaction.Plos One.2010;5(6):e11240.

41.Choksi NY,Jahnke GD,St Hilaire C,Shelby M.Role of thyroid hormones in human and laboratory animal reproductive health.Birth Defects Res B Dev Reprod Toxicol.2003;68:479-91.

42.Krassas GE.Thyroid disease and female reproduction.Clin Endocrinol.2007;66:309-21.

43.Michal JJ,Zhang ZW,Gaskins CT,Jiang Z.The bovine fatty acid binding protein 4 gene is significantly associated with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses.Anim Genet.2006;37:400-2.

44.Liu RZ,Li X,Godbout R.A novel fatty acid-binding protein(FABP)gene resulting from tandem gene duplication in mammals:transcription in rat retina and testis.Genomics.2008;92:436-45.

45.Van Veldhoven PP.Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism.J Lipid Res.2010;51:2863-95.

46.Wang Q,Sharma D,Ren Y,Fondell JD.A coregulatory role for the TRAP-mediator complex in androgen receptor-mediated gene expression.J Biol Chem Chem.2002;277:42852-8.

47.Urbinati I,Stafuzza NB,Oliveira MT,TCS C,Higa RH,Regitano LC d A,et al.Selection signatures in Canchim beef cattle.J Anim Sci Biotechnol.2016;7:29.

48.Casas E,Lunstra DD,Stone RT.Quantitative trait loci for male reproductive traits in beef cattle.Anim Genet.2004;35:451-3.

49.Seidenspinner T,Bennewitz J,Reinhardt F,Thaller G.Need for sharp phenotypes in QTL detection for calving traits in dairy cattle.J Anim Breed Genet.2009;126:455-62.

50.Maltecca C,Weigel KA,Khatib H,Cowan M,Bagnato A.Whole-genome scan for quantitative trait loci associated with birth weight,gestation length and passive immune transfer in a Holstein x Jersey crossbred population.Anim Genet.2009;40:27-34.

51.Schulman NF,Sahana G,Iso-Touru T,McKay SD,Schnabel RD,Lund MS,et al.Mapping of fertility traits in Finnish Ayrshire by genome-wide association analysis.Anim Genet.2011;42:263-9.

52.Gonda MG,Arias JA,Shook GE,Kirkpatrick BW.Identification of an ovulation rate QTL in cattle on BTA14 using selective DNA pooling and interval mapping.Anim Genet.2004;35:298-304.

53.Blaschek M,Kaya A,Zwald N,Memili E,Kirkpatrick BW.A whole-genome association analysis of noncompensatory fertility in Holstein bulls.J Dairy Sci.2011;94:4695-9.

54.Ashwell MS,Heyen DW,Weller JI,Ron M,Sonstegard TS,Van Tassell CP,et al.Detection of quantitative trait loci influencing conformation traits and calving ease in Holstein-Friesian cattle.J Dairy Sci.2005;88:4111-9.

55.Ron M,Feldmesser E,Golik M,Tager-Cohen I,Kliger D,Reiss V,et al.A complete genome scan of the Israeli Holstein population for quantitative trait loci by a daughter design.J Dairy Sci.2004;87:476-90.

56.Zhou F,Leder P,Martin SS.Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2.Exp Cell Res.2006;312:1119-26.

57.Gutiérrez-Gil B,Williams JL,Homer D,Burton D,Haley CS,Wiener P.Search for quantitative trait loci affecting growth and carcass traits in a cross population of beef and dairy cattle.J Anim Sci.2009;87:24-36.

58.Conklin D,Holderman S,Whitmore TE,Maurer M,Feldhaus AL.Molecular cloning,chromosome mapping and characterization of UBQLN3 a testisspecific gene that contains an ubiquitin-like domain.Gene.2000;249:91-8.