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Abstract ［Objectives］ This study was conducted to speed up the process of improving the quality of lamb breeds in China, and to solve the problems that restrict the development of lamb breeds in China, such as low quality of lamb breeds, low meat production rate, and poor meat quality.
［Methods］ By studying molecular markers of fat content in sheep muscle, the CAPN gene was used to find polymorphic sites related to fat content in sheep muscle, and the correlation between two polymorphic sites C724A and A601G on CAPN1 and CAPN2 and fat content in sheep muscle was analyzed.
［Results］ The two polymorphic sites, C724A on CAPN1 and A601G on CAPN2 were significantly related to intramuscular fat in mutton.
［Conclusions］ These two molecular markers can be used as a molecular marker reference when breeding high-quality meat or hair and meat using German Merino sheep breeds.
Key words Intramuscular fat (IMF); Meat sheep; Molecular markers; Breed
Received: February 13, 2020Accepted: April 8, 2020
Supported by Mutton Sheep Research System (CARS-38).
Guizhi TONG (1967-), female, P. R. China, researcher, master, devoted to research about animal breeding and reproduction.
*Corresponding author.
German Merino sheep is a collective name for fine wool sheep breeds. After years of extensive breeding around the world, many breeds of wool, hair and meat, and meat and hair have been obtained. The German Merino sheep has been an excellent meat and wool combined breed introduced in China since the 1950s. It has good performance in growth cycle, reproductive performance, adaptability and meat production performance. Scholars have tried to cross fine-hair hybrid sheep and native sheep to further improve the breeds. In view of the poor meat quality of German Merino sheep, the use of molecular markers to efficiently screen excellent succulent individuals for reproduction and breeding is an efficient way to further improve German merino sheep breeds［1］.
Intramuscular fat (IMF) is one of the main indicators of the flavor of lamb muscle［2］. In the normal range, there is a direct positive correlation between the intramuscular fat content and the tenderness, taste and succulence of mutton［3-5］. When a certain amount of fat is deposited between muscle fibers, the marble pattern increases, the meat is fresh and tender, and the juiciness is improved［6］. Studies have shown that there is a negative correlation between intramuscular fat content and lean meat percentage. Therefore, it is theoretically feasible to breed sheep breeds with a high lean meat content and a high internal fat content ( i.e. , good meat texture). However, the screening of molecular markers for intramuscular fat content in sheep, especially sheep of specific breeds, is currently underway. Intramuscular fat content and fatty acid composition have an impact on sheep meat quality［7-10］. The firmness of fat is affected by intramuscular fat and fatty acid composition, which in turn affects the nutritional value, flavor and edible quality of fat, while fatty acid composition is affected by animal species, breeds, gender and other factors［11］. The content of fat in sheep muscle is an important factor affecting the quality of lamb meat, and its regulatory mechanism is very complex. It is mainly affected by many aspects such as nutrition, environment and genetics. Among them, gene regulation is one of the fundamental ways to improve sheep muscle fat deposition［12］. Discovery of CAPN1 and CAPN2 (Calpain 1 and Calpain 2) Polymorphism Sites in German Merino Sheep
CAPN is a thiol-alanine endonuclease that is ubiquitous in cells. Existing studies have shown that CAPN , especially CAPN1 , is related to meat tenderization in certain cattle and sheep species. Based on this, the polymorphisms of CAPN in Germany Merino sheep and their correlation with intramuscular fat were studied.
Subjects: 355 healthy, adult purebred German Merino sheep were selected from sheep farms. Jugular blood (5 ml) was collected from each sheep before slaughter, added to an anti-coagulant centrifuge tube and stored at -20 ℃. The longissimus dorsi was removed after slaughter and stored at -80 ℃.
Determination of intramuscular fat content: The Soxhlet extraction method was used to detect the intramuscular fat content in the longissimus dorsi of sheep. At first, 3-5 g of sample was ground in liquid nitrogen to a fine powder, which was put into a weighed dry filter paper bag. The filter paper bag containing the wet sample was weighed, and after being dried for 2 h, the filter paper bag containing the dry sample was weighed again. A Soxhlet extractor was used to extract the sample at room temperature with ether for 6 h. After the ether was volatilized, the filter paper bag was dried for 2 h and weighed. The percentage of intramuscular fat content was calculated according to: Weight of the filter paper bag containing the dry sample-Weight of the filter paper bag after extraction)/(Weight of the filter paper bag containing the wet sample-Weight of the filter paper bag).
The molecular biology experiment was entrusted to Beijing Weiyue Gene Technology Co., Ltd.
DNA was extracted from blood samples by phenol-chloroform method, and the DNA was detected by agarose gel electrophoresis for its concentration and stored at -20 ℃.
According to the sequence information of sheep CAPN 1 and CAPN 2 on Genbank (NM_001127267 and NM_001112817, see SEQ ID NO.1-4 in the present application), primers were designed using Primer Premier 5.1 and PCR-SSCP analysis was performed. Two polymorphic loci, C724A on CAPN 1 (Leu-Met, non-polar amino acids) and A601G on CAPN 2 (Thr-Ala, from polar amino acids to non-polar amino acids) were found to be associated with intramuscular fat.
Genotype Frequency and Allele Frequency of Polymorphic Loci The genotype frequencies and allele frequencies of C724A on CAPN 1 and A601G on CAPN 2 in the experimental German Merino flock (355) are shown in Table 1.
After Chi-square test, the German Merino flock was in Hardy-Weinberg equilibrium at two polymorphic sites ( P >0.05) . C724A on CAPN 1 is a low polymorphic site, and A601G on CAPN 2 is a moderate polymorphic site.
Correlation Between Different Genotypes and Intramuscular Fat Content
The method described in Table 1 was used to detect the intramuscular fat content of each meat-like substance, and the correlation between each genotype and intramuscular fat content is shown in Table 2.
As can be seen from the data in the table above, the relationship between the least squares mean of the intramuscular fat content in the longissimus dorsi is CC<CA <AA and AA<AG<GG at the two sites, respectively. At the CAPN 1 724 position, the differences between AA and CC and between CA and CC were extremely significant ( P <0.01). The difference between GG and AA at the CAPN 2 601 position was extremely significant ( P <0.01) , while the difference between AG and AA was not significant ( P =0.166). Both polymorphic loci have good potential as molecular markers for breeding.
Polymorphic Molecular Markers Used in Breeding Practice of German Merino Sheep
After identifying the polymorphic loci, the sheep jugular vein blood samples were sent to Beijing Weiyue Gene Technology Co., Ltd. The primers of SEQ ID NO.5-8 were amplified and sequenced to detect genotypes at C724A on CAPN 1 and A601G on CAPN 2. Based on the genotypes, 3 breeding rams and 25 ewes with genotype AA at the CAPN 1 724 position and genotype GG at the CAPN 2 601 position, and 4 breeding rams with genotype CA at the CAPN 1 724 position and genotype GG at the CAPN 2 601 position, were selected and used for natural mating and breeding. The statistics of intramuscular fat in longissimus dorsi and gluteus maximus in 8 samples at the age of 6 months were 9.67%±0.31% and 5.75%±0.25%, respectively. From experience, the values exceeded the levels of Germany Merino sheep in the same period raised in the laboratory before by about 1.6 and 0.7 percentage points, respectively, and the taste was improved to some extent. Meanwhile, there was no significant change in the body weight, wool and other traits. Although data on intramuscular fat in adulthood has not been obtained, the practical effects of molecular marker-assisted selection in this study have also been relatively clear. Conclusions
The two polymorphic sites, C724A on CAPN 1 and A601G on CAPN 2, are related to intramuscular fat in mutton, which can be used as a reference for molecular markers in breeding new breeds of mutton.
References
［1］ CHEN QX. Research on molecular breeding technology and biological breeding of Zhongyuan meat sheep［J］. Feed and Husbandry, 2019(4): 46-49. (in Chinese)
［2］ GAN ML, DU JJ, YANG Q. Research progress of intramuscular fat affecting meat quality and its molecular mechanism［J］. Modern Journal of Animal Husbandry and Veterinary Medicine, 2017(10): 51-57. (in Chinese)
［3］ XU ZY, WU WC, WANG YZ. Research progress of molecular mechanism regulating intramuscular fat deposition［J］. Chinese Journal of Animal Science, 2018(5): 1-5. (in Chinese)
［4］ Gerbens F, et al. Assoeiations of heart and adipoeyte fatty acid-binding protein gene expression with intramuscular fat content in pigs［J］. AnimSei, 2001(79): 347-354.
［5］ MA Y, LUO HL, WANG YP. Effects of intramuscular fat content and fatty acid composition on quality traits of sheep meat［J］. Modern Journal of Animal Husbandry and Veterinary Medicine, 2016(9): 9-11. (in Chinese)
［6］ LI HQ, LUO HQ. Research progress on the regulation of intramuscular fat and fatty acid in ruminants［J］. Chinese Journal of Animal Science, 2019(8): 1-5. (in Chinese)
［7］ LUAN ZJ, QU XX, HE JN. Advances in research on regulation of intramuscular fat in sheep［J］.Journal of Domestic Animal Ecology, 2014(9): 8-13. (in Chinese)
［8］ GUO B.Bioinformatics analysis on the relationship of beef cattle intramuscular fat and related functional gene set［D］. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
［9］ LIU JY, HAO CL, LI WH. Association of the MC4R gene expression with intramuscular fat content in Hu sheep muscles［D］. Nanjing: Nanjing Agricultural University, 2011. (in Chinese)
［10］ TIAN J. Effects of MyoG, H-FABP and LPL genes on meat production and meat quality of two local sheep breeds in Xinjiang［D］. Urumchi: Xinjiang Agricultural University, 2014. (in Chinese)
［11］ WANG YP, XU CC, LUO HL. Research progress of candidate genes related to animal intramuscular fat deposition［J］. Acta Zoonutrimenta Sinica, 2017(5): 1475-1480. (in Chinese)
［12］ LI B, LI W, WANG T. Research progress in intramuscular fat cell growth and regulation factors［J］. Science and Technology of Food Industry, 2013(22): 363-366. (in Chinese)
Editor: Yingzhi GUANGProofreader: Xinxiu ZHU
［Methods］ By studying molecular markers of fat content in sheep muscle, the CAPN gene was used to find polymorphic sites related to fat content in sheep muscle, and the correlation between two polymorphic sites C724A and A601G on CAPN1 and CAPN2 and fat content in sheep muscle was analyzed.
［Results］ The two polymorphic sites, C724A on CAPN1 and A601G on CAPN2 were significantly related to intramuscular fat in mutton.
［Conclusions］ These two molecular markers can be used as a molecular marker reference when breeding high-quality meat or hair and meat using German Merino sheep breeds.
Key words Intramuscular fat (IMF); Meat sheep; Molecular markers; Breed
Received: February 13, 2020Accepted: April 8, 2020
Supported by Mutton Sheep Research System (CARS-38).
Guizhi TONG (1967-), female, P. R. China, researcher, master, devoted to research about animal breeding and reproduction.
*Corresponding author.
German Merino sheep is a collective name for fine wool sheep breeds. After years of extensive breeding around the world, many breeds of wool, hair and meat, and meat and hair have been obtained. The German Merino sheep has been an excellent meat and wool combined breed introduced in China since the 1950s. It has good performance in growth cycle, reproductive performance, adaptability and meat production performance. Scholars have tried to cross fine-hair hybrid sheep and native sheep to further improve the breeds. In view of the poor meat quality of German Merino sheep, the use of molecular markers to efficiently screen excellent succulent individuals for reproduction and breeding is an efficient way to further improve German merino sheep breeds［1］.
Intramuscular fat (IMF) is one of the main indicators of the flavor of lamb muscle［2］. In the normal range, there is a direct positive correlation between the intramuscular fat content and the tenderness, taste and succulence of mutton［3-5］. When a certain amount of fat is deposited between muscle fibers, the marble pattern increases, the meat is fresh and tender, and the juiciness is improved［6］. Studies have shown that there is a negative correlation between intramuscular fat content and lean meat percentage. Therefore, it is theoretically feasible to breed sheep breeds with a high lean meat content and a high internal fat content ( i.e. , good meat texture). However, the screening of molecular markers for intramuscular fat content in sheep, especially sheep of specific breeds, is currently underway. Intramuscular fat content and fatty acid composition have an impact on sheep meat quality［7-10］. The firmness of fat is affected by intramuscular fat and fatty acid composition, which in turn affects the nutritional value, flavor and edible quality of fat, while fatty acid composition is affected by animal species, breeds, gender and other factors［11］. The content of fat in sheep muscle is an important factor affecting the quality of lamb meat, and its regulatory mechanism is very complex. It is mainly affected by many aspects such as nutrition, environment and genetics. Among them, gene regulation is one of the fundamental ways to improve sheep muscle fat deposition［12］. Discovery of CAPN1 and CAPN2 (Calpain 1 and Calpain 2) Polymorphism Sites in German Merino Sheep
CAPN is a thiol-alanine endonuclease that is ubiquitous in cells. Existing studies have shown that CAPN , especially CAPN1 , is related to meat tenderization in certain cattle and sheep species. Based on this, the polymorphisms of CAPN in Germany Merino sheep and their correlation with intramuscular fat were studied.
Subjects: 355 healthy, adult purebred German Merino sheep were selected from sheep farms. Jugular blood (5 ml) was collected from each sheep before slaughter, added to an anti-coagulant centrifuge tube and stored at -20 ℃. The longissimus dorsi was removed after slaughter and stored at -80 ℃.
Determination of intramuscular fat content: The Soxhlet extraction method was used to detect the intramuscular fat content in the longissimus dorsi of sheep. At first, 3-5 g of sample was ground in liquid nitrogen to a fine powder, which was put into a weighed dry filter paper bag. The filter paper bag containing the wet sample was weighed, and after being dried for 2 h, the filter paper bag containing the dry sample was weighed again. A Soxhlet extractor was used to extract the sample at room temperature with ether for 6 h. After the ether was volatilized, the filter paper bag was dried for 2 h and weighed. The percentage of intramuscular fat content was calculated according to: Weight of the filter paper bag containing the dry sample-Weight of the filter paper bag after extraction)/(Weight of the filter paper bag containing the wet sample-Weight of the filter paper bag).
The molecular biology experiment was entrusted to Beijing Weiyue Gene Technology Co., Ltd.
DNA was extracted from blood samples by phenol-chloroform method, and the DNA was detected by agarose gel electrophoresis for its concentration and stored at -20 ℃.
According to the sequence information of sheep CAPN 1 and CAPN 2 on Genbank (NM_001127267 and NM_001112817, see SEQ ID NO.1-4 in the present application), primers were designed using Primer Premier 5.1 and PCR-SSCP analysis was performed. Two polymorphic loci, C724A on CAPN 1 (Leu-Met, non-polar amino acids) and A601G on CAPN 2 (Thr-Ala, from polar amino acids to non-polar amino acids) were found to be associated with intramuscular fat.
Genotype Frequency and Allele Frequency of Polymorphic Loci The genotype frequencies and allele frequencies of C724A on CAPN 1 and A601G on CAPN 2 in the experimental German Merino flock (355) are shown in Table 1.
After Chi-square test, the German Merino flock was in Hardy-Weinberg equilibrium at two polymorphic sites ( P >0.05) . C724A on CAPN 1 is a low polymorphic site, and A601G on CAPN 2 is a moderate polymorphic site.
Correlation Between Different Genotypes and Intramuscular Fat Content
The method described in Table 1 was used to detect the intramuscular fat content of each meat-like substance, and the correlation between each genotype and intramuscular fat content is shown in Table 2.
As can be seen from the data in the table above, the relationship between the least squares mean of the intramuscular fat content in the longissimus dorsi is CC<CA <AA and AA<AG<GG at the two sites, respectively. At the CAPN 1 724 position, the differences between AA and CC and between CA and CC were extremely significant ( P <0.01). The difference between GG and AA at the CAPN 2 601 position was extremely significant ( P <0.01) , while the difference between AG and AA was not significant ( P =0.166). Both polymorphic loci have good potential as molecular markers for breeding.
Polymorphic Molecular Markers Used in Breeding Practice of German Merino Sheep
After identifying the polymorphic loci, the sheep jugular vein blood samples were sent to Beijing Weiyue Gene Technology Co., Ltd. The primers of SEQ ID NO.5-8 were amplified and sequenced to detect genotypes at C724A on CAPN 1 and A601G on CAPN 2. Based on the genotypes, 3 breeding rams and 25 ewes with genotype AA at the CAPN 1 724 position and genotype GG at the CAPN 2 601 position, and 4 breeding rams with genotype CA at the CAPN 1 724 position and genotype GG at the CAPN 2 601 position, were selected and used for natural mating and breeding. The statistics of intramuscular fat in longissimus dorsi and gluteus maximus in 8 samples at the age of 6 months were 9.67%±0.31% and 5.75%±0.25%, respectively. From experience, the values exceeded the levels of Germany Merino sheep in the same period raised in the laboratory before by about 1.6 and 0.7 percentage points, respectively, and the taste was improved to some extent. Meanwhile, there was no significant change in the body weight, wool and other traits. Although data on intramuscular fat in adulthood has not been obtained, the practical effects of molecular marker-assisted selection in this study have also been relatively clear. Conclusions
The two polymorphic sites, C724A on CAPN 1 and A601G on CAPN 2, are related to intramuscular fat in mutton, which can be used as a reference for molecular markers in breeding new breeds of mutton.
References
［1］ CHEN QX. Research on molecular breeding technology and biological breeding of Zhongyuan meat sheep［J］. Feed and Husbandry, 2019(4): 46-49. (in Chinese)
［2］ GAN ML, DU JJ, YANG Q. Research progress of intramuscular fat affecting meat quality and its molecular mechanism［J］. Modern Journal of Animal Husbandry and Veterinary Medicine, 2017(10): 51-57. (in Chinese)
［3］ XU ZY, WU WC, WANG YZ. Research progress of molecular mechanism regulating intramuscular fat deposition［J］. Chinese Journal of Animal Science, 2018(5): 1-5. (in Chinese)
［4］ Gerbens F, et al. Assoeiations of heart and adipoeyte fatty acid-binding protein gene expression with intramuscular fat content in pigs［J］. AnimSei, 2001(79): 347-354.
［5］ MA Y, LUO HL, WANG YP. Effects of intramuscular fat content and fatty acid composition on quality traits of sheep meat［J］. Modern Journal of Animal Husbandry and Veterinary Medicine, 2016(9): 9-11. (in Chinese)
［6］ LI HQ, LUO HQ. Research progress on the regulation of intramuscular fat and fatty acid in ruminants［J］. Chinese Journal of Animal Science, 2019(8): 1-5. (in Chinese)
［7］ LUAN ZJ, QU XX, HE JN. Advances in research on regulation of intramuscular fat in sheep［J］.Journal of Domestic Animal Ecology, 2014(9): 8-13. (in Chinese)
［8］ GUO B.Bioinformatics analysis on the relationship of beef cattle intramuscular fat and related functional gene set［D］. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
［9］ LIU JY, HAO CL, LI WH. Association of the MC4R gene expression with intramuscular fat content in Hu sheep muscles［D］. Nanjing: Nanjing Agricultural University, 2011. (in Chinese)
［10］ TIAN J. Effects of MyoG, H-FABP and LPL genes on meat production and meat quality of two local sheep breeds in Xinjiang［D］. Urumchi: Xinjiang Agricultural University, 2014. (in Chinese)
［11］ WANG YP, XU CC, LUO HL. Research progress of candidate genes related to animal intramuscular fat deposition［J］. Acta Zoonutrimenta Sinica, 2017(5): 1475-1480. (in Chinese)
［12］ LI B, LI W, WANG T. Research progress in intramuscular fat cell growth and regulation factors［J］. Science and Technology of Food Industry, 2013(22): 363-366. (in Chinese)
Editor: Yingzhi GUANGProofreader: Xinxiu ZHU