高通量测序技术在鉴别影响猪产仔数QTL及候选基因中的应用

产仔数是反映猪场生产水平和经济效应的一个重要的指标,作为多基因控制、遗传力低的复杂经济性状,鉴别影响产仔数的基因位点,利用分子选育标记来提高猪的产仔性能倍受重视。随着重测序成本的不断降低,基因组学研究不断深入,以全基因组重测序、简化基因组测序和基于高通量测序技术获得的芯片技术已得到广泛应用。本文综述了通过高通量测序技术以及基于高通量测序的芯片技术研究影响猪产仔数性能的数量性状座位(quantitative trait loci,QTL)及候选基因的研究进展,为后期鉴别影响产仔数变异主效基因提供参考,为生产采用分子育种进行产仔选育提升提供分子标记信息。     

Three SNPs within exons of INHA and ACVR2B genes are significantly associated with litter size in Dazu black goats

The aim of this study was to analyse the association between single-nucleotide polymorphisms within INHA and ACVR2B and litter size in Dazu black goats. In total, twenty-two SNPs were genotyped in 190 individuals by SNaPshot and resequencing. The results showed that three SNPs (SNP_1, SNP_12 and SNP_13 in this study) were detected to have significant additive genetic effect on the recorded goat litter size (p < .05). The SNP_1 (NC_030809.1), a non-synonymous substitution of G for T at chr2-g. 28314990 in the exon 2 of INHA gene (NM_001285606.1), resulted in homozygote 2 (HOM2) contributed 0.25 and heterozygote (HET) contributed 0.12 larger litter than homozygote 1 (HOM1). Meanwhile, SNP_12 (Chr22-g. 11721225 A > T) and SNP_13 (Chr22-g. 11721227 A > C) (NC_030829.1) simultaneously mutated at the first and third position of a triplet AAA (lysine, K) in the exon 4 of ACVR2B gene (XM_018066623.1) had estimated genetic effects of HOM1 (0.00) and HOM2 (0.03) larger than HET (−0.12). In conclusion, one SNPs (chr2-g. 28314990 T > G) within the exon 2 of INHA and two SNPs (Chr22-g. 11721225 A > T and Chr22-g. 11721227 A > C) i n the exon 4 of ACVR2B gene were highly recommended as candidate markers of litter size in Dazu black goats. A large-scale association study to assess the impact of these variants on litter size is still necessary.     

Polymorphism of GDF9 and BMPR1B genes and their association with litter size in Markhoz goats

The main objective of this study was to investigate the polymorphism of GDF9 and BMPR1B genes and their relationship with litter size in Markhoz goats. The polymorphism of GDF9 and BMPR1B genes as well‐documented genes regarding fecundity in sheep and goat was investigated using RFLP‐PCR and a tetra‐primer amplification refractory mutation system‐PCR (T‐ARMS‐PCR) in Markhoz goats. The 164 blood samples were collected from the raised goats in Sanandaj Markhoz goat Performance Testing Station. The DNA extraction was carried out by salting‐out procedure, and then, PCR was performed using four and two pairs of primers to detect polymorphism in GDF9 and BMPR1B genes, respectively. To disclose GDF9 loci polymorphism, PCR products were digested with SspI (G3288A), PvuII (G423A), MvaI (A959C) and MspI (G1189A) restriction enzymes. The results showed that these mutations are available in tested animals. Parity had no significant effect on litter size. Also, the effects of different genotypes of GDF9 and BMPR1B had no significant effect on litter size. Further studies with a high number of animals with minimum relatedness for testing the association of these SNPs and others in the fecundity genes with reproductive traits may be worthwhile.     

日粮纤维的营养学功能及对母猪产仔数的影响

母猪繁殖性状主要包括总产仔数、产活仔数、初生个体均重及初生窝重、泌乳力、断奶仔猪数、断奶个体均重及断奶窝重,其中,产仔数是最重要的繁殖力评定指标。日粮纤维对家畜的营养物质的消化率存在负面影响,但是近半个世纪以来由于日粮纤维独特的性质在母猪的营养研究中备受关注。文中将从日粮纤维的定义及分析方法、理化特性着手,阐述日粮纤维添加到母猪妊娠日粮中对产仔数的影响并分析其可能原因。     

影响山羊产羔数lncRNA的筛选及功能分析

旨在分析高、低繁殖力川中黑山羊卵巢组织中长链非编码RNA（long non-coding RNA,lncRNA）的表达谱,探讨lncRNA与山羊生殖调控的关系,为解释lncRNA在山羊繁殖调控中的分子机制提供理论依据。本研究选择3胎以上的10头健康川中母羊（3.5~4.5岁）,将其分为高繁组和低繁组。高繁组为每胎产羔数在2头以上的母羊（n=6）,低繁组为每胎产羔数为1头的母羊（n=4）。同期发情后,采集高繁和低繁山羊的卵巢样品,并从中提取总RNA构建测序文库,通过HigSeq X ten平台进行测序,筛选出差异lncRNAs,通过对差异显著的lncRNAs与mRNAs位置关系以及结合能的判定预测顺式作用的靶基因,并对其进行GO和KEGG通路分析。最后,随机抽取6个差异lncRNAs进行实时荧光定量验证。结果表明,本研究共鉴定出168个差异表达lncRNAs,其中,163个lncRNAs在高繁组卵巢显著上调,5个lncRNAs在低繁组卵巢显著上调。筛选出的差异表达lncRNAs可能为调控山羊繁殖力的候选lncRNAs,包括ENSCHIG00000001479、ENSCHIG00000002617和ENSCHIG00000002622等。这些候选lncRNAs的靶基因（MYC、CDH2、FGF9、PPARGC1A等）主要富集于Jak-STAT、细胞黏附分子及AMPK等信号通路,ENSCHIG00000001479的靶基因MYC参与了Jak-STAT、TGF-β和MAPK通路;ENSCHIG00000002617和ENSCHIG00000002622的靶基因CDH2参与了细胞黏附分子通路。通过qRT-PCR验证发现,定量结果与测序结果基本一致。研究结果推断,ENSCHIG00000001479、ENSCHIG00000002617和ENSCHIG00000002622等lncRNAs可能对山羊生殖发育过程中的卵巢发育及排卵等进程具有重要的调控作用,认为鉴定出的差异表达lncRNAs与山羊产羔数有关。本研究利用RNA-Seq技术筛选高、低繁殖力山羊的差异表达lncRNAs,并对其进行GO和KEGG功能分析,为探讨山羊产羔数的繁殖机制提供更完善的转录组数据。     

Polymorphisms of the melatonin receptor 1A gene that affects the reproductive seasonality and litter size in Small Tail Han sheep

Previous researches have shown that MTNR1A plays an essential role in sheep reproduction. However, most researches focused more on the reproductive seasonality of sheep, and few scientists had studied the association of polymorphisms of the MTNR1A gene with ovine litter size and reproductive seasonality. Therefore, we chose MTNR1A gene to detect its novel sequence polymorphisms and population genetics and analyse their association with seasonal reproduction and litter size in ewes. The mRNA expression level in hypothalamus, pituitary and ovary was also detected. In this study, five polymorphisms (g.15118664G > T, g.15118683C > T, g.15118756C > T, g.15118774C > T and g.15118951G > A) were identified in exon 2. Most importantly, the g.15118683C > T and g.15118951G > A were significant difference between year‐round oestrous sheep and seasonal oestrous sheep (p < .01), and g.15118756C > T had a great effect on litter size of Small Tail Han sheep (p < .05). In addition, the mRNA expression level of MTNR1A in the hypothalamus of polytocous Small Tail Han sheep was significantly higher than that in monotocous Small Tail Han sheep (p < .01) and the expression of MTNR1A in the hypothalamus of year‐round oestrous sheep was significantly higher than that in seasonal oestrous sheep (p < .01). Polymorphisms in exon 2 may regulate the reproductive seasonality and litter size of ewes by influencing gene expression to regulate the reproductive seasonality and litter size of ewes. Our studies provided useful guidance in marker‐assisted selection of the litter size in Small Tail Han sheep.     

BMPR-IB基因多态性与河北肉用绵羊产羔数及羔羊生长发育的相关性研究

为了研究BMPR-IB基因单核苷酸多态性及其对绵羊产羔数和羔羊生长发育的影响,试验采用PCR-RFLP方法对小尾寒羊、杜泊、德克赛尔及河北肉用绵羊的BMPR-IB基因单核苷酸多态性进行了测定,并对BMPR-IB基因多态性与河北肉用绵羊产羔数和生长发育的关系进行了研究。结果表明:基因型频率在各品种间分布极不平衡,BB基因型河北肉用绵羊群体的总体平均产羔数显著高于B+基因型(P<0.05),极显著高于++基因型群体(P<0.01);90日龄时,++基因型河北肉用绵羊的胸宽和腰角宽显著高于B+基因型群体(P<0.05)。说明BMPR-IB基因的基因型对河北肉用绵羊羔羊的生长发育有一定影响。     

DNA polymorphism of 5' flanking region of prolactin gene and its association with litter size in sheep

A single nucleotide polymorphism of 5' flanking region of the prolactin gene was investigated in both high prolificacy breeds (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset and Suffolk sheep) using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). The results indicated that two genotypes (AA and AB) were detected in Small Tail Han sheep (n   =   239), only one genotype (AA) was detected in Hu (n   =   40), Dorset (n   =   50) and Suffolk sheep (n   =   39). The mutant homozygous genotype (BB) was not detected in four sheep breeds. In Small Tail Han sheep (n   =   239), the frequency of genotypes AA and AB was 0.91 and 0.09, the frequency of the A and B alleles was 0.95 and 0.05, respectively. The fitness tests showed that the Small Tail Han sheep population was in Hardy–Weinberg equilibrium. Sequencing revealed a mutation (G→T) at the position 63 bp of the 5' flanking region of prolactin gene in AB genotype compared with AA genotype in Small Tail Han sheep. The Small Tail Han ewes with AB genotype had 0.83 (p < 0.05) lambs more than those with AA genotype. These results preliminarily showed that the prolactin locus is either a major gene that influences the high prolificacy in Small Tail Han sheep or is in close linkage with such a gene.     

Parity and litter size effects on maternal behavior of Small Tail Han sheep in China

The effects of parity and litter size on maternal behavior of Small Tail Han sheep was investigated at Linyi University, China. Sixty‐eight ewes were observed from parturition to weaning. Continuous focal animal sampling was used to quantify the duration of maternal behaviors. Ewe feces were collected every 2 days and estradiol concentration was measured with an enzyme immunoassay kit. All lambs were weighed 24 h after parturition and again at 35 days of age. Parity increased sucking, following, grooming, low‐pitched bleat, head‐up and udder‐refusal behavior and decreased aggressive behavior (P < 0.01, P < 0.01, P < 0.05, P < 0.05, P < 0.05, P < 0.05, P < 0.01, respectively), and litter size showed significant effect on sucking, following and low‐pitched bleat behavior (P < 0.05, P < 0.01, P < 0.05, respectively). The lambs of multiparous ewes were significantly heavier than primiparous ewes at birth (P < 0.01) and were significantly heavier at weaning age (P < 0.01). Similar results were founded for birth weight and weaning weight gain in litter size (P < 0.01, P < 0.01, respectively). Estradiol concentration in feces was higher in multiparous ewes than primiparous ewes. Parity and litter size may have effects on maternal behavior during lactation. Ewes that have 2–3 lambs may be more suitable for production of Small Tail Han sheep in China.     

Components of heterosis for growth traits and litter size in line crosses of mice after long-term selection

Strains selected for 54 generations for large 8-week weight (N8, S8), for small weight (N6, S6), for high 3–5-week gain and low 8-week weight (N1) as well as unselected randomly mated control strains (N9, S9) were crossed, and F ₁, F ₂ and both back crosses created. N strains are derived from a NMRI base, S strains from a synthetic four-way cross among inbred lines. Heterosis for 8-week weight (8-ww) was some 6%, but 30% for 3–5-week gain and it was on average negative (22%) for 5–8-week gain. When epistatic effects were taken into account, it appeared that heterosis for 8-ww was largely due to beneficial effects of non-parental gene combinations, i.e. additive × additive epistatic effects were negative. These non-parental combinations neutralized the mostly negative dominance effects. The latter were mostly positive for early gain but in all 9 line crosses negative for late gain. The digenic effects of the non-parental origin were not large enough to balance the negative dominance effects for late gain, so that heterosis was negative. It appears therefore that early and late gain are affected by different sets of genes. Maternal additive effects on 8-ww and early gain are smaller than direct additive effects but mostly of similar sign. For late gain there appears to be no connection with direct additive effects. Direct litter heterosis was almost 50% but much of this was due to positively acting recombinational gene combinations, while direct litter dominance was mostly negative. Maternal additive effects on litter size differed depending on whether they were estimated from dams with crossbred or with purebred litter. Heterosis was small in crosses between control strains. Heterosis caused by recombinant gene pairs should be amenable to improvement by selection.     

The contribution of dominance and inbreeding depression in estimating variance components for litter size in Pannon White rabbits

In a synthetic closed population of Pannon White rabbits, additive (V_A), dominance (V_D) and permanent environmental (V_Pe) variance components as well as doe (b_Fd) and litter (b_Fl) inbreeding depression were estimated for the number of kits born alive (NBA), number of kits born dead (NBD) and total number of kits born (TNB). The data set consisted of 18,398 kindling records of 3883 does collected from 1992 to 2009. Six models were used to estimate dominance and inbreeding effects. The most complete model estimated V_A and V_D to contribute 5.5 ± 1.1% and 4.8 ± 2.4%, respectively, to total phenotypic variance (V_P) for NBA; the corresponding values for NBD were 1.9 ± 0.6% and 5.3 ± 2.4%, for TNB, 6.2 ± 1.0% and 8.1 ± 3.2% respectively. These results indicate the presence of considerable V_D. Including dominance in the model generally reduced V_A and V_Pe estimates, and had only a very small effect on inbreeding depression estimates. Including inbreeding covariates did not affect estimates of any variance component. A 10% increase in doe inbreeding significantly increased NBD (b_Fd = 0.18 ± 0.07), while a 10% increase in litter inbreeding significantly reduced NBA (b_Fl = ?0.41 ± 0.11) and TNB (b_Fl = ?0.34 ± 0.10). These findings argue for including dominance effects in models of litter size traits in populations that exhibit significant dominance relationships.     

Genetic parameters for litter size,piglet growth and sow's early growth and body composition in the Chinese–European line Tai Zumu

Genetics of piglet growth in association with sow's early growth and body composition were estimated in the Tai Zumu line. Piglet and sow's litter growth traits were calculated from individual weights collected at birth and at 3 weeks of age. Sow's litter traits included the number of piglets born alive (NBA), the mean piglet weight (MW) and the standard deviation of weights within the litter (SDW). Sow's early growth was measured by the age at 100 kg (A₁₀₀), and body composition included backfat thickness (BF₁₀₀). A main objective of this study was to estimate separately the direct genetic effect (d) and the maternal genetic effect (m) on piglet weight and daily weight gain during lactation. Variance components were estimated using the restricted maximum likelihood methodology based on animal models. The heritability estimates were 0.19 for NBA, 0.15 and 0.26 for SDW and MW at 3 weeks and 0.42 and 0.70 for A₁₀₀ and BF₁₀₀. The NBA was almost independent from SDW. Conversely, the A₁₀₀ and BF₁₀₀ were correlated unfavourably with SDW (r_g<−0.24, SE<0.12). A stronger selection for litter size should have little effect on litter homogeneity in weights. Selection for lean growth rate tends to favour heterogeneity in weights. The direct effect on piglet weight at birth and daily weight gain accounted for 12% (h² (d) = 0.02) and 50% (h² (d) = 0.11) of the genetic variance, respectively. The association between d and m for piglet weight was not different from zero at birth (r_g = 0.19, SE = 0.27), but a strong antagonism between d and m for daily weight gain from birth to 3 weeks was found (r_g = −0.41, SE = 0.17). Substantial direct and maternal genetic effects influenced piglet growth until weaning in opposite way.     

Relationships among steroid hormone levels in newborn piglets,birth weight,placental weight,vitality of offspring and litter size

Blood estrone sulfate (E₁S), estrone (E₁), estradiol (E₂) and progesterone (P₄) in newborn piglets were measured to clarify the relationships among birth and placental weight, vitality of offspring and litter size. First, the association between vital status (normal, weak and stillborn) from 165 newborn piglets of 18 litters and steroid concentrations; second, steroid concentrations from 152 newborn normal piglets and litter size; and third, steroid content in fetal placenta from 50 newborn normal piglets of six litters and litter size, were investigated. In the normal group, the birth and placental weight were significantly higher than those in the other groups. Blood E₁S levels in the stillborn group were significantly lower, whereas E₁, E₂ and P₄ were significantly higher compared to the normal group. Blood and placental E₁S levels in the small litter group were significantly higher than those in the other groups. However, there was no significant difference among the three litter size groups in the levels of steroid hormones in maternal blood. These results indicate that vitality of newborn piglets is related to E₁S concentration of neonate, to birth weight and placental weight. However, steroid hormone concentrations of newborn piglets were greatly affected by the number of littermates.     

Comparison of genetic improvement for litter size at birth by direct and indirect selection in swine herd

Responses to selection for number of piglets born alive (NBA) by the total number of piglets born (TNB), the NBA, and the NBA plus number of piglets born dead (NBD) were compared using the accuracy of selection and expected genetic gain calculated from the selection index with family information and the real response to selection, using data generated by Monte Carlo computer simulation. The accuracy of selection for NBA selected by TNB was higher than that by NBA only if the genetic correlation between TNB and NBA was close to 1.0, or the value of heritability for the TNB was much larger than that for the NBA. The accuracy of selection for the NBA selected by the combination of the TNB and the NBA was generally highest in the three selection methods in each family structure. Selection by the TNB resulted in the greatest expected genetic gain for the TNB among the selection methods. In the best linear unbiased prediction (BLUP) selection, the genetic gain for the NBA accumulated by the NBA tended to be similar to that accumulated by the combination of the NBA and the NBD, and both genetic gains at generation 10 were significantly larger than that by the TNB (P < 0.001). The accumulated responses selected by the two‐trait animal model BLUP estimated from genetic parameters with errors were similar to those estimated from the true parameters, and there was no significant difference between them. These results indicate that selection by the NBA or by the NBA and the NBD gives more genetic improvement in the NBA than that by the TNB.     

紫茎泽兰对建昌黑山羊肌肉中氨基酸和脂肪酸含量的影响

为研究紫茎泽兰(Ageratina adenophora)对建昌黑山羊肌肉中氨基酸和脂肪酸含量的影响,本研究采用氨基酸自动分析仪和氯仿-甲醇抽提法,对自然放牧采食紫茎泽兰的建昌黑山羊背最长肌中氨基酸、脂肪酸含量进行测定分析.结果显示,与对照组比较,建昌黑山羊肌肉必需氨基酸(essential amino-acid,EA...     

不同品种绵羊和山羊主要先天性免疫指标的比较

对4个山羊品种和4个绵羊品种的7项主要先天性免疫学指标进行了检测并比较。对动物进行颈静脉采血,制备血清,用ELISA试剂盒测定IL-1、IL-2、IL-6、IL-18、IFN-γ、NK细胞、血清溶菌酶。利用生物统计学的方法和原理,以品种和品种来源地为应变量对检测结果进行比较分析。结果表明,山羊当地品种的NK和LYS高于引进品种（P〈0.05）;绵羊引进品种的IL-1、IL-2、IL-6、IL-18、IFN-γ、NK、LYS均明显高于地方品种（P〈0.01）。在不同品种之间,莱芜黑山羊NK和LYS水平高于崂山奶山羊、波尔山羊和鲁波山羊,萨福克羊的IL-1、IL-6、IL-18、IFN-γ、LYS水平最高且差异极显著（P〈0.01）。小尾寒羊IL-18、IFN-γ水平最低且差异极显著（P〈0.01）。说明不同种群绵羊、山羊的先天性免疫水平存在显著差异,为以后抗病育种提供合理的试验数据。     

应用灰色关联度法对肉兔产仔数与相关性状关联度的探讨

本文结合肉兔产业基地生产,获取了肉兔开产1～3胎的平均产仔数与相关性状,通过灰色关联度分析,初步看出与产仔数关联度高的性状依次是体长〉体重〉耳长〉耳宽,说明在开产早期选择体长、发育好的母兔个体对提高产仔数有实际意义。     

高谷物日粮促进山羊瘤胃上皮单羧酸转运蛋白1及钠钾ATP酶mRNA的表达

本试验旨在研究高谷物日粮对山羊瘤胃上皮形态结构及单羧酸转运蛋白(monocarboxylate transporter, MCT)和钠钾ATP酶mRNA表达的影响。将10头装有永久性瘤胃瘘管的健康阉割公山羊随机分为饲喂全粗料日粮的对照组(Hay,0%谷物,n=5)和饲喂高谷物日粮的处理组(HG,65%谷物,n=5),试验期为7周。试验开始后,于每周晨饲后的0、2、3、4、6、8和12 h连续采集瘤胃液监测瘤胃pH值的变化,收集其中第0、3、6和12 h的瘤胃液待测挥发性脂肪酸(volatile fatty acid, VFA)浓度。试验的第50天,屠宰采集瘤胃上皮用于形态学及基因定量分析。研究结果显示:与全粗料组山羊相比,高谷物组山羊瘤胃pH值、乙酸浓度及乙丙比都显著下降(P<0.001),而瘤胃丙酸浓度、丁酸浓度及其他VFA浓度都显著升高(P<0.001);高谷物日粮组的瘤胃乳头长度显著高于对照组(P=0.001),瘤胃乳头宽度显著低于对照组(P<0.001),但是两组间的瘤胃乳头表面积并无显著差异;透射电镜结果显示,长期饲喂高谷物日粮导致瘤胃上皮细胞线粒体发生降解;实时定量PCR结果表明,与对照组相比,高谷物日粮显著升高了MCT1(P<0.001)和钠钾ATP酶(P=0.001)的mRNA表达量,显著降低了MCT4的mRNA表达量(P=0.041),但对MCT2的表达没有显著影响(P=0.305);进一步分析这些基因的mRNA表达量与pH值和VFA浓度之间的相关性,结果显示,MCT1和钠钾ATP酶的mRNA表达量与瘤胃pH值和乙酸浓度呈显著负相关,与总VFA、丙酸、丁酸的含量呈显著正相关,而MCT4的mRNA表达量与pH值呈显著正相关,与总VFA、丙酸、丁酸的含量呈显著负相关。以上结果提示:高精料引起的瘤胃pH值下降和VFA的变化可能与瘤胃上皮MCT和钠钾ATP酶表达量的变化相关。研究结果对深入认识高谷物饲喂引发的瘤胃功能紊乱具有重要意义。     

Identification of polymorphisms in the oocyte-derived growth differentiation growth factor 9 (GDF9) gene associated with litter size in New Zealand sheep (Ovis aries) breeds

Having the ability to control litter size is important for sheep farmers and breeders worldwide. However, making genetic gain in key livestock traits like reproductive performance needs typically a lot of time, and both the fecundity and fertility traits have a great economic importance. Attention has therefore turned to better understanding the genes that control reproductive performance. Of these genes, research has focussed on the growth differentiation growth factor 9 (GDF9) gene (GDF9). In this study, a PCR-single strand conformation polymorphism (PCR-SSCP) approach was used to investigate variation in this gene in separate groups of purebred Finnish Landrace sheep, Finnish Landrace × Texel-cross sheep and composite sheep of undefined breed background, but based on New Zealand Romney-type genetics. Three GDF9 variants (named A, B and C) were found, and upon DNA sequencing, the nucleotide substitutions c.978A>G, c.994G>A and c.1111G>A were revealed. The frequency of variant A (containing nucleotides c.978A, c.994G and c.1111G) in the Finnish Landrace, Finnish Landrace × Texel-cross and composite sheep was 0.86, 0.78 and 0.76, respectively. In these three sheep groups, the frequency of B (defined by the presence of nucleotides c.978G and c.994A) was 0.01, 0.03 and 0.23 and for C (containing c.1111A) was 0.13, 0.18 and 0.01, respectively. An animal model was used to estimate the additive effect of fertility data for Finnish Landrace × Texel-cross sheep and revealed an association between litter size and the c.1111G>A variation (p = .036), but this was not observed for the Finnish Landrace sheep (p = .27) or the composite sheep (p = .17). When all the sheep were analysed together, the presence of c.1111A was associated (p < .05) with increased litter size, when compared to ewes that had c.1111G. Litter size did not differ between sheep with and without c.994A in all three groups of sheep investigated. This study suggests that c.1111A could be a useful genetic marker for improving fecundity in New Zealand sheep breeds and that it could be introgressed into other breeds, but analysis of more sheep will be required to confirm the associations that have been observed here.