吴磊　谭光辉　李杰章　覃媛钰　张依裕　田琴

摘要：【目的】探討OCX-32基因对长顺绿壳蛋鸡蛋壳品质的遗传效应，为长顺绿壳蛋鸡的保种选育及开发利用提供参考依据。【方法】以长顺绿壳蛋鸡为材料，采用PCR产物直接测序法筛选OCX-32基因SNP多态位点，实时荧光定量PCR检测组织表达谱，运用SPSS 19.0广义线性模型（GLM）分析SNP位点基因型或双倍型与所测定性状指标的相关性。【结果】在长顺绿壳蛋鸡OCX-32基因中检测到3个SNPs位点，分别是位于内含子1上的g.22592323G>T及内含子3上的g.22597350T>C和g.22597555G>A。卡方（χ2）检测结果发现，g.22597350T>C位点的基因型分布显著偏离Hardy-Weinberg平衡（P<0.05，下同）。连锁不平衡分析结果显示，3个SNPs突变位点不存在强连锁不平衡，共发现4种单倍型和8种双倍型，单倍型H1和双倍型H1H2频率最高，分别为0.523和0.345。实时荧光定量PCR检测结果显示，OCX-32基因在长顺绿壳蛋鸡12个组织中存在不同程度的表达，表达量排序为肾脏>心脏>子宫>腹脂>小肠>脾脏>肝脏>腺胃>胸肌>胰腺>肺脏>肌胃。关联分析结果显示，g.22597350T>C位点CC基因型在蛋壳强度和蛋壳重2个品质指标上显著高于CT基因型和TT基因型;双倍型H4H4个体的蛋壳强度显著高于其他7种双倍型个体，蛋壳重显著高于H2H4个体。【结论】OCX-32基因内含子变异能影响长顺绿壳蛋鸡蛋壳品质，检测到的3个SNPs位点可作为鸡蛋壳品质选择的遗传分子标记，其中突变位点g.22597350T>C的CC基因型和双倍型H4H4是影响蛋壳强度和蛋壳重的关键基因型和双倍型，有利改善蛋壳品质。

关键词： 长顺绿壳蛋鸡;OCX-32基因;SNP;内含子;荧光定量PCR;蛋壳品质

中图分类号： S831.91                         文献标志码： A 文章编号：2095-1191（2020）08-1872-08

Effects of OCX-32 gene intron variation on eggshell quality

of Changshun green-eggshell laying hens

WU Lei， TAN Guang-hui， LI Jie-zhang， QIN Yuan-yu， ZHANG Yi-yu*， TIAN Qin

（College of Animal Science， Guizhou University/Key Laboratory of Genetics， Breeding and Reproduction of Plateau Mountain Animals， Ministry of Education/Key Laboratory of Animal Genetics， Breeding and Reproduction

of Guizhou Province， Guiyang  550025， China）

Abstract：【Objective】To explore the genetic effects of OCX-32 gene on eggshell quality of Changshun green-shell laying hens，and to provide data support for breeding，development and utilization of Changshun green-eggshell laying hens. 【Method】Using Changshun green-eggshell laying hens as material，the SNP polymorphic site of OCX-32 gene was screened by direct sequencing of PCR products，and the tissue expression profile was detected by real-time fluorescence quantitative PCR. SPSS19.0 generalized linear model（GLM） was used to analyze the correlation between SNP locus genotype or diplotype and the tested traits. 【Result】Three SNPs loci were detected in OCX-32 gene of Changshun green-eggshell laying hens，which were located in g.22592323G>T mutation in intron 1，g.22597350T>C mutation in intron 3 and g.22597555G>A mutation in intron 3，respectively. Chi-square（χ2） test showed that the genotype distribution of g.22597350T>C mutation site significantly deviated from Hardy-Weinberg equilibrium（P<0.05， the same below）. The results of linkage disequilibrium analysis showed that there was no strong linkage disequilibrium at the three SNPs mutation sites. Four haplotypes and eight diplotypes were found. Haplotype H1 and diplotype H1H2 had the highest frequencies of 0.523 and 0.345，respectively. The results of real-time fluorescence quantitative PCR showed that OCX-32 gene was expressed in different degrees in 12 tissues of Changshun green-eggshell laying hens，and the order of expression was kidney>heart>uterus>abdominal fat>small intestine>spleen>liver>glandular stomach>chest muscle>pancreas>lung>mu-scle stomach. The results of association analysis showed that the eggshell strength and eggshell weight of CC genotype at g.22597350T>C were significantly higher than those of CT and TT genotypes，and the eggshell strength of diploid H4H4 was significantly higher than that of the other seven diplotypes，and the eggshell weight of diploid H4H4 was significantly higher than that of H2H4. 【Conclusion】The intron variation of OCX-32 gene can affect the eggshell quality of Changshun green-eggshell laying hens. The three SNPs loci detected can be used as genetic markers for eggshell quality selection. The CC genotype and diplotype H4H4 of the variation site g.22597350T>C are the key genotypes and diplotypes affecting eggshell strength and eggshell weight，which are more beneficial to improve eggshell quality.

Key words： Changshun green-eggshell laying hens; OCX-32 gene; SNP; intron; fluorescence quantitative PCR;eggshell quality

Foundation item： Guizhou Science and Technology Cooperation Planning Project（Qiankehe LH〔2016〕7454）

0 引言

【研究意义】鸡蛋由59%蛋清、31%蛋黄和10%蛋壳构成（Mann and Mann，2015）。蛋壳是卵生动物的卵外壳，是抵御外界物理损伤和微生物侵染的重要屏障，构成蛋壳主要有两部分：一是由蛋白纤维所构成的基质，二是在蛋白质基质上堆积钙质的结晶物（Eddin et al.，2019）。蛋壳品质是影响家禽生产力的重要性状，直接影响蛋的保存、运输和孵化，……