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Abstract 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. By studying molecular markers of fat content in sheep muscles， we searched for polymorphic loci related to fat content in sheep muscles on the ANGPTL4 gene. Among the 9 polymorphic loci retrieved， ANGPTL4 was found. Two polymorphic sites， C724A and A601G， were significantly associated with intramuscular fat in mutton. In summary， 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; Inosine acid
Intramuscular fat （IMF） is one of the main indicators of flavour of lamb muscle[1]. In the normal range， the content of intramuscular fat has a direct positive correlation with the tenderness， taste and juicyness of lamb. When a certain amount of fat is deposited between muscle fibers， the marble pattern increases， and the tenderness and juicyness of the meat are improved[2]. Previous 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 percentage and a high internal fat content （that is， good meat texture）. Inosinic acid （IMP）， also known as hypoxanthine nucleotide， has a variety of sources. It can be synthesized from scratch in animals or formed by ATP after slaughter. It is an important flavor substance that mainly provides umami taste in chickens， ducks， fish， cattle and sheep （the first International Symposium on umami taste in 1985 listed the taste provided by inosinic acid as umami）[3-4]， but few studies have been conducted on the screening of molecular markers for intramuscular fat and inosinic acid content in cattle and sheep， especially sheep of specific breeds.
Discovery of ANGPTL4 （Angiopoietin-like Protein 4） Polymorphism Site in German Merino Sheep
ANGPTL4 has a Chinese name： angiopoietin-like protein 4. Existing research shows that it is widely present in mammals and has been linked to lipid metabolism， glucose metabolism and even tumors. The ANGPTL4 polymorphism in German Merino sheep and its association with intramuscular fat and inosinic acid have been studied. Specifically， 355 healthy， adult purebred German Merino sheep were selected in the test ranch. Jugular blood was collected from the jugular vein before slaughter. 5 ml of blood was added to a centrifuge tube containing anticoagulant and stored at -20 ℃. The longest back muscles were stored at -80 ℃ or directly used for the determination of inosinic acid in fresh meat.
Determination of the intramuscular fat content： Soxhlet extraction method was used to detect the intramuscular fat content of the longest muscle in sheeps back. 3-5 g of the sample was ground to fine powder in liquid nitrogen， and put into a weighed dry filter paper bag， which was then weighed again. The bag containing the wet sample was dried for 2 h， and then， the filter paper bag containing the dry sample was weighed. A Soxhlet extractor was used to extract the sample with ether for 6 h at room temperature. 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 dry filter paper bag after extraction）/（Weight of the filter paper bag containing the wet sample-Weight of the filter paper bag）.
Determination of the inosinic acid content in the longest muscle of sheeps back： The inosinic acid content in the longest muscle of sheeps back was determined by referring to the prior art using HPLC. 2 g of meat sample was cut and added into 10 ml of 5% perchloric acid， followed by homogenization. The homogenate was centrifuged at 4 000 r/min for 5 min， obtaining the supernatant， which was filtered. 5 ml of 5% perchloric acid was added to the precipitation， followed by centrifugation at 4 000 r/min for 5 min， giving the supernatant， which was merged with the previous supernatant. The pH of the mixed supernatant was adjusted to 6.5， and diluted to 25 ml with purified water， followed by mixing and HPCL detection. An Agilent 1200HPLC instrument equipped with C18 column was used. The detection was performed using a mobile phase of 5% methanol and 95% phosphoric acid-triethylamine solution at a rate of 0.7 ml/min with a temperature of 25 ℃ and a detection wavelength of 254 nm. A standard curve was established using an inosinic acid standard， for the calculation of inosinic acid content （mg/g fresh meat）.
The molecular biology experiment was entrusted to Beijing Weiyue Gene Technology Co.， Ltd. Specifically， DNA was extracted from blood samples using phenol-chloroform method， and the DNA concentration was detected by agarose gel electrophoresis and stored at -20 ℃. According to the sequence information of sheep ANGPTL4 on Genbank （KF873613， CDS： 237-1469， the present application： SEQ ID NO.1-2）， primers were designed using Primer Premier 5.1 for amplification and PCR-SSCP analysis. Two polymorphic sites， G303A （based on KF873613 full-length mRNA sequence， Ala-Thr， changed from non-polar amino acid to polar amino acid） and G396T （based on KF873613 full-length mRNA sequence， Gly-Trp， changed from non-polar amino acid to critical polar amino acid） related to intramuscular fat and inosinic acid content， were found. Genotype Frequency and Allele Frequency of Polymorphic Loci
The genotype frequencies and allele frequencies of G303A and G396T on ANGPTL4 in the experimental German Merino flock （355） are shown in Table 1：
It can be seen from the data in Table 3 that the relationships between the least squares mean of the intramuscular fat content of the longest muscles at the two sites were GG<GA<AA and GG<GT<TT， respectively. The differences between GG and GA and between GA and AA at 303 were both significant （P<0.05）， and the differences between GG and GT and between GT and TT at position 396 were also significant （P<0.05）， while the difference between AG and AA was not significant （P=0.166）. Both polymorphic loci have good potential as molecular markers for breeding.
As can be seen from the data in the table above， the relationships between the least squares mean of the longest muscle inosine content at the two sites were GG<GA<AA and GG<GT<TT， respectively. The differences between GG and GA and between GA and AA at 303 were both significant （P<0.05）， and the difference between GT and TT was significant at position 396 （P<0.05）， while the difference between GG and GT was not significant （P=0.135）. 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， a blood sample from the jugular vein of the sheep was sent to Beijing Weiyue Gene Technology Co.， Ltd. The primers of SEQ ID NO.5-8 were amplified and sequenced to detect genotypes at G303A and G396T on ANGPTL4. Based on the genotype， one ram and five ewes with genotype AA at position 303 and genotype TT at position 396， one ram and 14 ewes with genotype AA at position 303 and genotype GT at position 396， and 2 rams and 6 ewes with genotype GA at 303 and TT at 396， were selected. The above rams and ewes were used for natural mating and breeding. Eight samples at the age of 6 months were investigated to have the average intramuscular fat contents in the longest back muscles and gluteus maximus muscles of 9.31% ± 0.28% and 5.82% ± 0.18%， respectively and the inosinic acid content of 1.45 mg/g fresh meat. From experience， the values exceeded the German Merino sheep raised in this laboratory by about 1.3， 0.8 percentage points， and 0.12 mg/g of fresh meat over the same period， respectively; the taste was improved to a certain extent; and there were no significant changes in body weight， wool and other characteristics.
References
[1] 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）
[2] GERBENS F， et al. Assoeiations of heart and adipoeyte fatty acid-binding protein gene expression with intramuscular fat content in pigs[J]. Anim Sei， 2001（79）： 347-354
[3] YUKIKO M， et al. Multiple taste functions of the umami substances in muscle extracts of yellowtail and bastard halibut[J]. Fish Science， 2010（76）： 521-528.
[4] QU GJ， MENG KY， YU D. The correlation analysis about different hybridized combinative beef cattle intramuscular fat content and the partial meat quality characteristics[J]. Feed Industry， 2013（9）： 49-51. （in Chinese）
Key words Intramuscular fat （IMF）; Meat sheep; Molecular markers; Breed; Inosine acid
Intramuscular fat （IMF） is one of the main indicators of flavour of lamb muscle[1]. In the normal range， the content of intramuscular fat has a direct positive correlation with the tenderness， taste and juicyness of lamb. When a certain amount of fat is deposited between muscle fibers， the marble pattern increases， and the tenderness and juicyness of the meat are improved[2]. Previous 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 percentage and a high internal fat content （that is， good meat texture）. Inosinic acid （IMP）， also known as hypoxanthine nucleotide， has a variety of sources. It can be synthesized from scratch in animals or formed by ATP after slaughter. It is an important flavor substance that mainly provides umami taste in chickens， ducks， fish， cattle and sheep （the first International Symposium on umami taste in 1985 listed the taste provided by inosinic acid as umami）[3-4]， but few studies have been conducted on the screening of molecular markers for intramuscular fat and inosinic acid content in cattle and sheep， especially sheep of specific breeds.
Discovery of ANGPTL4 （Angiopoietin-like Protein 4） Polymorphism Site in German Merino Sheep
ANGPTL4 has a Chinese name： angiopoietin-like protein 4. Existing research shows that it is widely present in mammals and has been linked to lipid metabolism， glucose metabolism and even tumors. The ANGPTL4 polymorphism in German Merino sheep and its association with intramuscular fat and inosinic acid have been studied. Specifically， 355 healthy， adult purebred German Merino sheep were selected in the test ranch. Jugular blood was collected from the jugular vein before slaughter. 5 ml of blood was added to a centrifuge tube containing anticoagulant and stored at -20 ℃. The longest back muscles were stored at -80 ℃ or directly used for the determination of inosinic acid in fresh meat.
Determination of the intramuscular fat content： Soxhlet extraction method was used to detect the intramuscular fat content of the longest muscle in sheeps back. 3-5 g of the sample was ground to fine powder in liquid nitrogen， and put into a weighed dry filter paper bag， which was then weighed again. The bag containing the wet sample was dried for 2 h， and then， the filter paper bag containing the dry sample was weighed. A Soxhlet extractor was used to extract the sample with ether for 6 h at room temperature. 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 dry filter paper bag after extraction）/（Weight of the filter paper bag containing the wet sample-Weight of the filter paper bag）.
Determination of the inosinic acid content in the longest muscle of sheeps back： The inosinic acid content in the longest muscle of sheeps back was determined by referring to the prior art using HPLC. 2 g of meat sample was cut and added into 10 ml of 5% perchloric acid， followed by homogenization. The homogenate was centrifuged at 4 000 r/min for 5 min， obtaining the supernatant， which was filtered. 5 ml of 5% perchloric acid was added to the precipitation， followed by centrifugation at 4 000 r/min for 5 min， giving the supernatant， which was merged with the previous supernatant. The pH of the mixed supernatant was adjusted to 6.5， and diluted to 25 ml with purified water， followed by mixing and HPCL detection. An Agilent 1200HPLC instrument equipped with C18 column was used. The detection was performed using a mobile phase of 5% methanol and 95% phosphoric acid-triethylamine solution at a rate of 0.7 ml/min with a temperature of 25 ℃ and a detection wavelength of 254 nm. A standard curve was established using an inosinic acid standard， for the calculation of inosinic acid content （mg/g fresh meat）.
The molecular biology experiment was entrusted to Beijing Weiyue Gene Technology Co.， Ltd. Specifically， DNA was extracted from blood samples using phenol-chloroform method， and the DNA concentration was detected by agarose gel electrophoresis and stored at -20 ℃. According to the sequence information of sheep ANGPTL4 on Genbank （KF873613， CDS： 237-1469， the present application： SEQ ID NO.1-2）， primers were designed using Primer Premier 5.1 for amplification and PCR-SSCP analysis. Two polymorphic sites， G303A （based on KF873613 full-length mRNA sequence， Ala-Thr， changed from non-polar amino acid to polar amino acid） and G396T （based on KF873613 full-length mRNA sequence， Gly-Trp， changed from non-polar amino acid to critical polar amino acid） related to intramuscular fat and inosinic acid content， were found. Genotype Frequency and Allele Frequency of Polymorphic Loci
The genotype frequencies and allele frequencies of G303A and G396T on ANGPTL4 in the experimental German Merino flock （355） are shown in Table 1：
It can be seen from the data in Table 3 that the relationships between the least squares mean of the intramuscular fat content of the longest muscles at the two sites were GG<GA<AA and GG<GT<TT， respectively. The differences between GG and GA and between GA and AA at 303 were both significant （P<0.05）， and the differences between GG and GT and between GT and TT at position 396 were also significant （P<0.05）， while the difference between AG and AA was not significant （P=0.166）. Both polymorphic loci have good potential as molecular markers for breeding.
As can be seen from the data in the table above， the relationships between the least squares mean of the longest muscle inosine content at the two sites were GG<GA<AA and GG<GT<TT， respectively. The differences between GG and GA and between GA and AA at 303 were both significant （P<0.05）， and the difference between GT and TT was significant at position 396 （P<0.05）， while the difference between GG and GT was not significant （P=0.135）. 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， a blood sample from the jugular vein of the sheep was sent to Beijing Weiyue Gene Technology Co.， Ltd. The primers of SEQ ID NO.5-8 were amplified and sequenced to detect genotypes at G303A and G396T on ANGPTL4. Based on the genotype， one ram and five ewes with genotype AA at position 303 and genotype TT at position 396， one ram and 14 ewes with genotype AA at position 303 and genotype GT at position 396， and 2 rams and 6 ewes with genotype GA at 303 and TT at 396， were selected. The above rams and ewes were used for natural mating and breeding. Eight samples at the age of 6 months were investigated to have the average intramuscular fat contents in the longest back muscles and gluteus maximus muscles of 9.31% ± 0.28% and 5.82% ± 0.18%， respectively and the inosinic acid content of 1.45 mg/g fresh meat. From experience， the values exceeded the German Merino sheep raised in this laboratory by about 1.3， 0.8 percentage points， and 0.12 mg/g of fresh meat over the same period， respectively; the taste was improved to a certain extent; and there were no significant changes in body weight， wool and other characteristics.
References
[1] 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）
[2] GERBENS F， et al. Assoeiations of heart and adipoeyte fatty acid-binding protein gene expression with intramuscular fat content in pigs[J]. Anim Sei， 2001（79）： 347-354
[3] YUKIKO M， et al. Multiple taste functions of the umami substances in muscle extracts of yellowtail and bastard halibut[J]. Fish Science， 2010（76）： 521-528.
[4] QU GJ， MENG KY， YU D. The correlation analysis about different hybridized combinative beef cattle intramuscular fat content and the partial meat quality characteristics[J]. Feed Industry， 2013（9）： 49-51. （in Chinese）