略阳乌鸡褐壳蛋壳颜色量化体系的建立和遗传基础分析

Establishment of Quantization Method and Genetic Basis Analysis of Brown Eggshell Color in the Lüeyang Black-Boned Chicken

【背景】褐壳蛋壳颜色与蛋壳强度、蛋内抗菌肽含量、血斑肉斑和孵化率有密切联系,是影响蛋品质和销售的一个重要的指标。然而,一些地方鸡因选育程度低,存在蛋色偏浅、均匀度差等不足,给鸡蛋的销售和品牌的打造造成不利影响。【目的】探讨以略阳乌鸡为对象,建立能灵敏度量蛋壳颜色变异的量化体系,估计品种特异性蛋壳颜色的遗传力,筛选参与蛋壳颜色调控的候选基因,为略阳乌鸡褐壳蛋壳颜色选育奠定理论基础。【方法】在略阳乌鸡蛋用系62个半同胞家系中选取841只母鸡,每只鸡收集3枚蛋,用L*a*b色度体系量化蛋壳颜色。在贝叶斯框架下,用马尔可夫链蒙特卡罗(Markov Chain Monte Carlo,MCMC)算法计算各色度指标的后验遗传力。用逆γ分布指定育种值和残差方差的先验分布。执行130000迭代,弃去前30000次迭代结果,按间隔100存储迭代结果,获得各方差组分的后验分布和各指标的后验遗传力。选取产浅褐壳、褐壳、浅绿壳的母鸡各8只,每只鸡收集3枚蛋用于蛋壳色素含量的测定。用原卟啉和胆绿素标准品建立浓度与吸光度值的回归关系,分别在670 nm和412 nm处测定胆绿素和原卟啉的吸光度,用回归法求得待测样本两种蛋壳色素的浓度。采集褐壳(n=8)和浅褐壳(n=8)母鸡的蛋壳腺,用qPCR法检测ALAS1和CPOX在蛋壳腺中的表达量。【结果】在3个色度指标中,a值递减的变化规律与褐色向绿色调过渡的视觉观感吻合度最高。L值和b值能够反映褐壳颜色深浅的变化,但在区分褐色和绿色调上存在较大误差。L值和b值分布呈现出较高的集中度,50%的样本的L值和b值集中分布于73.3—79.1和13.1—18.2之间,而a值分布更为分散,50%的样本分布于1.4—7.1之间。与上述分布特征保持一致,a值的变异系数88.2%要高于L值5.7%和b值24.0%。遗传力估计结果显示,a值主要受遗传效应调控(h2=0.77),而L值(h2=0.46)和b值(h2=0.37)受环境效应影响更大。在蛋壳色素和蛋壳颜色之间,原卟啉浓度与3个色度指标均存在强相关关系(L:r=-0.86,a:r=0.73,b:r=0.88),但胆绿素浓度仅与a值存在强负相关(r=-0.73)。ALAS1和CPOX是两个催化原卟啉前体物合成的酶。表达结果显示,CPOX在褐壳鸡中的表达水平是浅褐壳的1.5倍(P<0.05),但ALAS1的表达量无显著差异(P>0.05)。【结论】原卟啉是影响略阳乌鸡蛋壳颜色的关键色素,而CPOX是与蛋壳原卟啉含量和褐壳颜色显著关联的候选基因。a值是量化略阳乌鸡蛋壳颜色准确度和灵敏度最高的指标,且具有较高的遗传力。对a值向上选择,有望在提高褐壳颜色深度和均匀度方面取得较大选择反应。

【Background】Brown eggshell color is closely relevant to eggshell strength,concentrations of egg white antimicrobial protein and yolk carotenoid,blood and meat spots,and hatchability,and is an important index affecting quality and sale of eggs.However,due to absence of selection for eggshell color,color of brown eggs that some indigenous breeds lay is light and highly variable,which has an adverse effect on sale of eggs and creation of egg brands.【Objective】The aims of this study are to establish a quantization method that could accurately and sensitively capture the variation of eggshell color,to estimate breed-specific heritability of eggshell color and to identify candidate genes associated with eggshell color in the Lüeyang black-boned chicken(LBC),so as to provide a theoretic basis for genetic improvement of LBC eggshell color.【Method】841 hens from 62 half-sibling families of LBC breeding population were selected.Three eggs were collected from each hen,and then the eggshell color was quantified using the L*a*b color space,and posterior heritabilities of L,a,and b values were estimated in a Bayesian framework using the Markov Chain Monte Carlo(MCMC)algorithm.The prior distributions of breeding value and residual variances were set using an inverse-Gamma distribution.MCMC performed 130000 iterations,dropped 30000 iterations at the beginning and stored one every 100 iterations to obtain posterior distributions of variance components and posterior heritability estimates of color indexes.Hens that laid light brown(n=8),brown(n=8)and light blue(n=8)eggs were selected from LBC population.Three eggs were collected from each hen for the measurement of eggshell pigment,and the absorbance values were measured at 670 nm for biliverdin and at 412 nm for protoporphyrin.The concentrations of protoporphyrin and biliverdin were calculated by regression equations,which were fitted using absorbances and concentrations of standard samples.Shell glands of brown-(n=8)and light brown-shelled(n=8)hens were collected.Expression levels of ALAS1 and CPOX in shell glands were detected using qPCR.【Result】Among three color indexes,a*value kept a high consistency with the change of eggshell color based on subjective perception as a*value generally decreased with transition of egg color from brown to green hues.L and b values could accurately reflect the change of egg color from light to dark brown,but L and b values were unable to discriminate between brown and green hues reliably.L and b values showed low variation as L and b values of 50%of samples were distributed between 73.3-79.1 and 13.1-18.2.In contrast,a*values were evenly distributed among the samples as a*values of 50%of samples ranged from 1.4 to 7.1.In line with the distribution characteristics,the coefficient of variation(88.2%)of a value was higher than ones of L(5.7%)and b(24.0%)values.Estimation results of heritability showed that a*value(h2=0.77)was predominately affected by genetic factors.In contrast,L(h2=0.46)and b(h2=0.37)values were controlled by environmental effect to larger extents.For the relationship of eggshell pigment and color,protoporphyrin concentration had a strong correlation with all of three color indexes(L:r=-0.86,a:r=0.73,b:r=0.88).But biliverdin concentration showed a strong(r=-0.73)negative correlation with a*value alone.ALAS1 and CPOX were two key enzymes that catalyze the biosynthensis of protoporphyrin precursors.Expression results showed that expression levels of CPOX in shell glands of brown-shelled chickens were 1.5-fold higher(P<0.05)than that in shell glands of light brown-shelled chickens.ALAS1 had no significant(P>0.05)difference between brown-and light brown-shelled chickens.【Conclusion】These results indicated that protoporphyrin was the key pigment affecting LBC eggshell color,and CPOX was a candidate associated with protoporphyrin concentration and color of brown eggs.The a*value was an optimal index quantifying eggshell color of LBC with high accuracy and sensitivity.In view of high heritability estimate of a*value,it was possible to increase brown hue and uniformity of LBC eggshell color via positive selection of a*value.