Identification of Cashmere and Wool by DNA Barcode

ABSTRACT In the textile market, it is common to use wool instead of precious cashmere, and it is difficult to identify those accurately by traditional methods such as appearance identification. To differentiate the cashmere and wool accurately and efficiently, a polymerase chain reaction (PCR) method based on the universal primers of DNA barcode was established according to the variations in the base sequence of mitochondrial COX I gene between goats and sheep. The mitochondrial DNA of cashmere and wool was extracted and then amplified by a pair of universal primers designed based on the COX I gene of goats and sheep. The amplified products were thus obtained with the length of 380 bp as predicted and subsequently sequenced. The sequencing results were verified with BOLD databases for homology comparison and the species of cashmere and wool were identified successfully. In conclusion, the PCR technology based on DNA barcode established in this study is quick and accurate in differentiating the species of cashmere and wool.


Introduction
As a kind of special animal fiber, cashmere has many excellent characteristics, so it is often used in the production of textiles. The products of cashmere are quite precious and expensive. For this reason, there are cases of adulteration of cashmere products in the market, usually by using cheap wool instead of cashmere. Therefore, the distinction between cashmere and wool is an important part of the identification of animal sources of textiles. In addition, animal fiber is easy to obtain and not perishable in many cases, so the species identification of animal fiber can provide clues and a basis for wild animal identification and protection (Jun et al. 2011), species evolution analysis (Clack, MacPhee, and Poinar 2012), archeology (Li 2019), and forensic identification (Melton et al. 2005).
The structure and properties of cashmere and wool are similar, so it is difficult to identify them accurately by burning and dissolving methods usually applied in textile identifications (Meiju et al. 2012). There are some traditional methods such as appearance identification usually uses an optical microscope and electron microscope to observe the color, curvature, cross-section, scale, and medullary cavity of the hair fibers. However, it is difficult to identify the hair fibers which were processed into textiles or came from closely related species by using these methods. Moreover, appearance identification has high requirements for technicians, which is easily affected by subjective factors, and the efficiency of identification and the reliability of the results are poor (Hongbo and Yaqin 2008). Infrared spectroscopy is another traditional methods to identify hair fiber by analyzing the characteristic atlas of animal hair measured by OMNI sampler of FT-IR Fourier transform infrared spectrometer. This method obtains the structural information of organic components on the surface of the samples by collecting the reflection signal of the sample surface. However, this method is easily affected by the state of the fiber itself. Usually, there is no significant differences in the characteristic atlas of animals of the same family. Therefore, this method also has great limitations (Haitao, Xiaoming, and Senlin 2011).
In contrast, the identification technique based on DNA has high specificity and sensitivity and is much more reliable and efficient. The DNA in hair is mainly concentrated in hair follicle cells, and the hair shaft contains only a small amount of mitochondrial DNA (Yalin 2008). The hair fibers used for textiles are generally without hair follicles after a series of processing, so comparing the differences in the base sequences of mitochondrial DNA is more suitable for the identification of animal hair fibers. The difficulty of this method is extracting high-quality DNA from the animal hair shafts. Although the content of DNA in the hair shaft is very low, a small amount of DNA can be obtained if the extraction method is appropriate. The extracted DNA can meet the requirements of subsequent polymerase chain reaction (PCR) amplification.
Some scholars have identified cashmere and wool according to the differences in the DNA base sequences between goats and sheep. Selvi Subramanian identified cashmere fiber and wool fiber by PCR-RFLP according to the mitochondrial Cyt b gene (Subramanian, Karthik, and Vijayaraaghavan 2005). Rajiv Kumar established a PCR technique for the detection of cashmere fiber in blended fabrics using the specific primers of the mitochondrial 12s rRNA gene (Kumar et al. 2015). Wan Ji distinguished cashmere and wool by TaqMan PCR technology according to the mitochondrial 12s rRNA gene (Ji et al. 2011). Qing-Rong Geng identified cashmere and wool by duplex PCR and PCR-RFLP according to the D-loop gene and 12s rRNA gene (Geng 2016;Geng, Yuan, and Chen 2012).
Till now, no study has reported the use of the mitochondrial cytochrome c oxidase I (COX I) gene to distinguish between cashmere and wool. The mitochondrial COX I gene of 650 bp has been recognized as a barcode region for species identification in animals (Marshall 2005). It provides more than 97% of species specificities to birds, mammals, fish, and various arthropods (Meusnier et al. 2008). In this study, we tried to extract the mitochondrial DNA from cashmere and wool respectively, and a pair of universal primers of the mitochondrial COX I gene were designed for both goats and sheep. Cashmere and wool were thus identified successfully, and a PCR technique for the identification of cashmere and wool by mitochondrial COX I gene was well established.

Animal fiber source
The cashmere from goats (Capra hircus) and wool from sheep (Ovis aries) used in this study are provided by the China National Silk Museum. Both cashmere and wool were collected from Inner Mongolia, China.

DNA extraction
The DNA extraction of cashmere and wool based on the protocol of QIAamp Fast DNA Stool Mini Kit (cat. no. 51604) of QIAGEN Company, the method was improved and DNA was extracted. The specific extraction steps are as follows: The samples of cashmere and wool were washed with deionized water until there were no obvious impurities, then the samples were washed and soaked with anhydrous ethanol. After natural drying, the samples were cut up with scissors and packed into 1.5 ml EP tubes for use. Each 1.5 ml EP tube contained 0.5-3.0 g samples. The samples were incubated and digested for more than 24 hours with 600 μl inhibitEX Buffer, 20 μl DTT solution (2 M), 20 μl protease K solution (20 mg/ml), and 600 μl Buffer AL. Subsequently, the supernatants were separated by centrifugation (14,000rpm, 15-25°C, 1 min), and 600 μl anhydrous ethanol was added to the supernatants. The mixed solutions were transferred into the collection columns and centrifuged (14,000rpm, 15-25°C, 1 min), and then washed with 500 μl Buffer AW1 and 500 μl Buffer AW2. Finally, DNA was eluted from the collection columns with 100 μl Buffer ATE heated to 56°C, and the DNA samples were obtained.

Primer designing
The sequences of the mitochondrial COX I gene of goat (Capra hircus) and sheep (Ovis aries) were searched from GeneBank (https://www.ncbi.nlm.nih.gov/). The sequences were compared by NCBI Nucleotide BLAST and the universal primers for the mitochondrial COX I gene of goat and sheep were designed using NCBI Primer-BLAST. Forward primer: 5′-ATCGGCACCCTCTACCTTCT-3′, Reverse primer: 5′-GCTCCTGCATGGGCTAGATT-3′.

PCR amplification
PCR amplification was performed in a final volume of 20 μl containing 5 μl of DNA template (Table 1), 0.5 μl of forward primer (10 μM), 0.5 μl of reverse primer (10 μM), 2 μl of TaKaRa 10× Ex Taq Buffer, 2 μl of TaKaRa dNTP Mixture, 0.5 μl of TaKaRa Ex Taq Mix, and 9.5 μl of ddH2O. PCR cycling parameters were as follows: 35 cycles each consisted of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with an initial hot start at 94°C for 1 min and a final extension at 72°C for 5 min.

PCR products detection
The PCR products were electrophoresed in 2% agarose gel. Electrophoretic bands were observed by gel imager, and PCR products were recovered from the target bands using V-ELUTE Gel Mini Purification Kit (ZPV202). To ensure the accuracy of sequencing, PCR products related to the T vector and then transformed into competent cells. After culture and identification, positive colonies were selected for further identification, culture, and plasmid extraction. Sangon Biotech (Shanghai) Co., Ltd. has been commissioned to sequence the plasmid containing the target gene fragment.

DNA extraction results
DNA was extracted from cashmere and wool, and the concentration of DNA was determined by a Qubit fluorescence quantitative analyzer. The results are shown in Table 1. Because the DNA concentration will be affected by the sample quality and the volume of DNA eluent, DNA-specific gravity is used to calculate and compare the contents of extracted DNA. The DNA-specific gravity is the amount of DNA that can be extracted per gram of hair. The formula is as follows: DNA À specific gravity ¼ Qubit DNA concentration ðng=μlÞ � eluent volume ðμlÞ hair weight ðgÞ The DNA-specific gravity of the extracted DNA of cashmere and wool in this study is 35.42 ng/g and 22.49 ng/g respectively. The DNA purity of the samples was analyzed by a NanoDrop nucleic acid protein analyzer. The genomic DNA of the samples was electrophoresed by Agilent 2100 Bioanalyzer and the results are shown in Figure 1.

PCR amplification results
Cashmere DNA and wool DNA were amplified by PCR using universal primers of mitochondrial COX I gene of goat and sheep. The agarose gel electrophoresis of the PCR products is shown in Figure 2. Cashmere and wool both have a clear band between 250-500bp. It can be preliminarily concluded that the PCR products of both cashmere and wool DNA have the same length and the length range is 250-500bp. The sizes of the two PCR products were determined to be 380bp by sequencing.

Sequencing results of amplification products
The PCR products of cashmere and wool were sequenced, and the base sequences of the two PCR products are shown in Figure 3 (OP740835 and OP740836). It can be observed that the universal primers were used to amplify cashmere DNA and wool DNA respectively, and the amplification products with a length of 380bp were obtained. The two sequences were compared by NCBI Nucleotide BLAST, and 35 different bases sites between the two sequences have been found. The specific differences between the two sequences are shown in Table 2. These different sites are the basis for species identifications.

Homology comparison
The sequences of the amplified products of two samples were input into BOLD database (http://www. boldsystems.org/index.php) for homology comparison. Judge whether the animals with the highest      Sample\Site  135  141  147  171  180  183  186  189  196  207  213  228  cashmere  T  C  C  A  G  T  A  G  G  C  A  C  wool  C  T  T  G  A  C  T  A  A  T  G  T  Sample\Site  255  270  279  282  286  288  294  309  315  345  360  cashmere  T  C  C  T  C  A  T  A  A  T  T  wool  C  T  A  C  T  G  C  G  G  C  C similarity are consistent with the animal sources of the two samples used in this study, and then judge the genetic relationship between the samples and the most matching animals according to the highest matching degree. The results are shown in Tables 3, Figure 4, Tables S1, and Tables S2. The species with the highest matching degree of cashmere DNA was Capra hircus, and the highest matching degree was 99.73%. The species with the highest matching degree of wool DNA was Ovis aries, and the highest matching degree was 99.04%. According to "the homology of animals within the same species is more than 96%, and the homology of species belonging to different genera of the same family is 70%-90%, and the homology of species belonging to different families is 65%-70%" (Fu-Zai et al. 2009), it can be determined that the identification results of the two fiber samples are consistent with their actual animal sources. Therefore, the fiber sources of animal species can be successfully determined by this experimental method.

Discussion
In recent years, the application of DNA technology to species identification of biological samples has developed rapidly. Mitochondrial deoxyribonucleic acid (mtDNA) is widely used in species  identification derived from its particular molecular properties, including its high evolutionary rate, uniparental inheritance, and small sizes. COX I gene is a specific DNA segment of mtDNA. The sequence of the COX I gene with a length of about 650 bp from the 5' is highly conserved and has the abundant mutation sites, so it is called DNA barcode and is widely used for species classification and identifications (Hebert, Ratnasingham, and deWaard 2003;Rabie 2019). This provides a theoretical and material bases for species identification of special samples such as animal fiber.
In this study, the species of cashmere and wool have been successfully identified. Firstly, the DNA of two kinds of animal fibers was extracted, and then analyzed, and the results showed that the amount of DNA in the two samples was very scarce. The results of DNA-specific gravity show that the DNA that can be extracted from per gram of hair is even far less than 1 μg. That is to say, the extraction of DNA from hair is challenging because of the limited quantity. The A260/A280 values of the cashmere are higher than 2.0. This ratio indicates the RNA contamination of the sample. In this study, RNA contamination has little effect on the specific amplification of DNA. The A260/A230 values of the two samples are lower than 2.0. This shows that the samples are easily affected by external pollution due to the few extracted DNA, and the contaminants in the samples may come from guanidine salts in nucleic acid extraction reagents.
In addition, according to the results of 2100 electrophoresis, there is no obvious DNA bands on the electrophoresis bands of the two samples, and the background colors of the electrophoresis bands are dark. Also, there is no obvious peak in the peak diagram, but there are weak peaks with a span of 1,000-10,380 bp (the composition of DNA shorter than 10,380bp can be detected by Agilent 2100 Bioanalyzer). Theoretically, the DNA in the hair shaft should be mitochondrial DNA with a length of about 16.5 kbp. But, due to the high degree of keratosis of the hair shaft, the high degradation of DNA in hair is very serious. This shows that the high degradation of DNA in the hair shaft is very serious.
The results show that the DNA in cashmere and wool is very few and has degraded seriously, which greatly limits the application of DNA in hair fiber identifications. As the sensitivity and specificity of PCR technology are very high, and only a very small amount of sample DNA is needed to complete the amplifications.
The DNA of the two samples was amplified by PCR using the designed universal primers of goat and sheep, and the target bands could be observed by 2% agarose gel electrophoresis. The target bands were recovered and sequenced, and the base sequences of PCR products were obtained. The sequences were compared by the BOLD database, and the results showed that the two samples could be accurately identified the animal origin.
Compared with traditional methods, using a DNA barcode to identify cashmere and wool avoids the influence of subjective factors, and more importantly the detection accuracy and sensitivity of this method are very high, so the results are more objective and reliable. Moreover, this method is simple and easy to operate, so accurate results could be obtained in less time. Universal primers can effectively identify cashmere and wool and improve the detection efficiency and sensitivity. However, it is not entirely reliable to identify species only by the target bands from agarose gel electrophoresis. Therefore, we obtained accurate sequence information of the target bands by the Sanger sequencing after PCR amplification and using the BOLD database for homology comparisons, so that the species of cashmere and wool were identified successfully. Our study laid a foundation for the promotion and application of gene technology in the field of identification of cashmere and wool, and provided strong technical support for the qualitative detection of cashmere and wool based on gene technology. It is also of great significances can be used to identify ancient textiles and other animal samples.

Highlights
• Universal primers for the identification of cashmere and wool were designed using COX I gene for the first time.
• DNA was successfully extracted from hair shaft samples with degraded seriously.
• Trace amounts of samples can be successfully identified.
• A PCR technique for the identification of cashmere and wool by DNA barcode was established.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The work was supported by the scientific project of Preservation of Cultural Relics of Zhejiang Province [2021010]; and the Natural Science Foundation of Zhejiang Province [Z20C170008].