WO2023087790A1 - 一种用于筛选总多糖含量高的黄绿卷毛菇的dna条形码 - Google Patents

一种用于筛选总多糖含量高的黄绿卷毛菇的dna条形码 Download PDF

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WO2023087790A1
WO2023087790A1 PCT/CN2022/109999 CN2022109999W WO2023087790A1 WO 2023087790 A1 WO2023087790 A1 WO 2023087790A1 CN 2022109999 W CN2022109999 W CN 2022109999W WO 2023087790 A1 WO2023087790 A1 WO 2023087790A1
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fragment containing
repeat elements
primer
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杨满军
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杨满军
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Definitions

  • the invention relates to the technical field of edible fungus germplasm resource screening, in particular to a DNA barcode used for screening yellow-green mushrooms with high total polysaccharide content.
  • Yellow-green mushrooms are mainly distributed in the Qinghai-Tibet Plateau.
  • the main production areas are Damxung County in the Cambodia Autonomous Region, Qilian County in Qinghai City, and Shiqu County in Sichuan province. The quality of these three main production areas is also the best.
  • Yellow-green mushroom is a high-quality edible fungus with unique flavor, which cannot be artificially cultivated at present.
  • the main indicators for evaluating the nutritional value, flavor and biological activity of the yellow-green mushroom include: high content of total soluble protein, total soluble amino acid, total polyphenol, total polysaccharide and total fat, and strong antioxidant activity.
  • DNA barcode molecular identification technology is a molecular biology technique based on DNA barcode (conserved and stable genetic DNA sequence in the genome) to identify species and good quality. It is an effective supplement and expansion of traditional breeding methods, and it can accurately and effectively identify samples when their morphology is incomplete or lacks morphological structure (processed products such as powder, etc.).
  • ITS ribosomal RNA internal transcriptional spacer
  • RFLP restriction fragment length polymorphism
  • RAPD random amplified polymorphic DNA
  • the present invention provides a DNA barcode used for screening Pleurotus pilosula with high total polysaccharide content.
  • a DNA barcode for screening indicators of the total polysaccharide content of Pleurotus volvulus includes:
  • the present invention is based on fluorescent PCR amplification of all simple sequence repeats (simple sequence repeats, SSR) in the whole genome of Pleurotus volvulus, establishes a DNA barcode effectively corresponding to the total polysaccharide content, and the amplified fragments are consistent with the DNA barcode of the present invention
  • SSR simple sequence repeats
  • Another object of the present invention is to provide a primer set for amplifying the DNA barcode of the above-mentioned screening indicator for the total polysaccharide content of Pleurotus volvulus, the nucleotide sequence of the primer set includes:
  • the nucleotide sequence of the primer set includes: such as SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 15.
  • Different primer sets of the present invention can be used alone or in combination to screen the total polysaccharide content of Pleurotus chinensis, and when all the primer sets are used together, the screening accuracy is the highest.
  • Another object of the present invention is to provide a method for screening yellow-green mushrooms with the index of total polysaccharide content, comprising the steps of:
  • S2 Using S1 genomic DNA as a template, the above-mentioned one or more sets of primers are respectively subjected to fluorescent PCR amplification reactions to obtain amplification products;
  • the amplified products described in S3 and S2 are detected by capillary fluorescence electrophoresis, and determined by the number of fragments of the amplified product, the number of SSR sites, the SSR repeat elements and the number of repeats thereof.
  • step S3 are:
  • the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 amplified to obtain a 263bp fragment containing 5 ACC repeat elements, a 266bp fragment containing 6 ACC repeat elements and a 269bp fragment containing 7 ACC repeat elements;
  • primer set of SEQ ID NO: 6 and SEQ ID NO: 7 amplified to obtain a 255bp fragment containing 7 TG repeat elements and a 257bp fragment containing 8 TG repeat elements;
  • the primer set of SEQ ID NO: 10 and SEQ ID NO: 11 amplified to obtain a 222bp fragment containing 6 AT repeat elements and a 232bp fragment containing 12 AT repeat elements;
  • the reaction system of step S2 fluorescent PCR amplification reaction is:
  • the concentrations of the upstream primer, the downstream primer and the fluorescent M13 primer are all 10uM.
  • the fluorescent PCR amplification reaction procedure of step S2 is:
  • Another object of the present invention is to provide the application of the above-mentioned DNA barcode and/or the above-mentioned primer set in the preparation of products for screening high-quality yellow-green mushrooms based on the total polysaccharide content index.
  • Another object of the present invention is to provide a product for screening high-quality yellow-green mushrooms with the total polysaccharide content index, which contains the above-mentioned one or more sets of primers, and meets the standards:
  • the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 amplified to obtain a 263bp fragment containing 5 ACC repeat elements, a 266bp fragment containing 6 ACC repeat elements and a 269bp fragment containing 7 ACC repeat elements;
  • primer set of SEQ ID NO: 6 and SEQ ID NO: 7 amplified to obtain a 255bp fragment containing 7 TG repeat elements and a 257bp fragment containing 8 TG repeat elements;
  • the primer set of SEQ ID NO: 10 and SEQ ID NO: 11 amplified to obtain a 222bp fragment containing 6 AT repeat elements and a 232bp fragment containing 12 AT repeat elements;
  • the product is a kit.
  • the present invention discloses a DNA barcode for screening yellow-green mushrooms with high total polysaccharide content. Compared with the prior art, the present invention can accurately and quickly
  • the DNA barcoding technology to identify the strains of Pleurotus pubescens and achieve high-quality breeding has the characteristics of low cost, high efficiency, easy operation, stable results, good reliability and repeatability.
  • the present invention Compared with the traditional breeding method and other existing DNA barcode technologies, the present invention has the advantages of saving time, effort, money, accuracy and high efficiency, and plays an active role in the identification of the origin of high-quality yellow-green mushrooms and genetic breeding. It also provides an effective method for the identification and protection of germplasm resources.
  • Figure 1 is a diagram showing the comparison results of the total polysaccharide content of the embodiment of the present invention, comparative examples 1 and 2, wherein from left to right are comparative example 1, comparative example 2 and the embodiment.
  • Fig. 2 accompanying drawing is that the present invention utilizes primer pair 1 PCR amplification comparison example 1, 2 and embodiment comparative result figure.
  • Fig. 3 Accompanying drawing of Fig. 3 is that the present invention utilizes primer to 2 PCR amplification comparative examples 1, 2 and embodiment comparative result figure.
  • Fig. 4 Accompanying drawing of Fig. 4 is that the present invention utilizes primer pair 3 PCR amplification comparative examples 1, 2 and embodiment comparative result figure.
  • Fig. 5 accompanying drawing is that the present invention utilizes primer pair 4 PCR amplification comparative example 1, 2 and embodiment comparative result figure.
  • the embodiment of the present invention discloses a DNA barcode for screening yellow-green mushrooms with high total polysaccharide content.
  • Genome sequencing was carried out on the samples of Dangxiong County in Cambodia Autonomous Region, Qilian County in Qinghai City, and Shiqu County in Sichuan province.
  • the SSR loci in the genome sequences were analyzed using the MISA program.
  • the samples from the above three origins were respectively amplified using effective primers and detected by capillary electrophoresis.
  • the simple sequence repeat (SSR) site corresponding to the total polysaccharide content was established through analysis.
  • 4 pairs of primers were obtained (see Table 1), and the fragment polymorphism obtained by using these 4 pairs of primers to amplify the sample genome can assist in the screening of Pleurotus chinensis with high total polysaccharide content.
  • Comparative example 1 sample from Damxung County, Cambodia Autonomous Region (treatment method is the same as above).
  • Comparative Example 2 Samples from Qilian County, Qinghai City (the processing method is the same as above).
  • the content of total polysaccharides in the extract was determined by the sulfuric acid phenol method, referring specifically to Zhao Qiduo et al. (Journal of Yichun University, 2011, 33(08):74-76), and converted to mg/g.
  • the total polysaccharide content in the yellow-green mushroom in Shiqu County, Sichuan province is 242.88 ( ⁇ 2.43) mg per gram, which is determined as a test example, and the total polysaccharide content in the yellow-green mushroom in Dangxiong County, Cambodia Autonomous Region is 148.49 ( ⁇ 3.85) mg
  • Each gram is determined as comparative example 1
  • the total polysaccharide content in the yellow-green mushroom in Qilian County, Qinghai province is 195.95 ( ⁇ 3.28) mg per gram is determined as comparative example 2 (see accompanying drawing 1).
  • Fluorescent PCR amplification reaction system (10 ⁇ L): 2 ⁇ Taq PCR Master Mix 5 ⁇ L, template (genomic DNA) 1 ⁇ L, upstream primer 0.1 ⁇ L, downstream primer 0.4 ⁇ L (concentration of both upstream and downstream primers is 10 uM), fluorescent M13 primer ( Concentration 10uM) 0.4 ⁇ L, dilute to 10 ⁇ L with sterile deionized water;
  • Reaction conditions pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 s, drop PCR annealing at 62 to 55°C for 30 s, extension at 72°C for 30 s, a total of 10 cycles; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, a total of 25 cycles 72°C final extension for 20min; 4°C incubation for 6h for fluorescence capillary electrophoresis detection.
  • the internal standard is LIZ-500 Molecular weight internal standard (also known as molecular weight internal control, internal lane standards) is composed of 16 double-stranded DNA fragments labeled with LIZ fluorescein (orange), and the molecular weights are: 35, 50, 75, 100 , 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500bp.
  • the size of the fragment in the electrophoresis graph of the amplification result is equal to the actual bp number of the amplified fragment plus the M13 fluorescent primer (about 18bp), the error range is 1-2bp, the amplified capillary electrophoresis peak is combined with the sequencing result, and the number of peaks indicates that the gene heterozygote is amplified number of fragments.
  • the amplification result of primer 1 is shown in Figure 2.
  • 3 fragments (3 peaks) were amplified, containing 3 SSR sites, and the SSR repeating element was ACC.
  • the characteristics of the amplified fragments obtained in the test example are 263bp and 269bp fragments containing 5 and 7 repetitions.
  • Primer 1 amplified fragment (The length of the statistical fragment in the electropherogram includes the M13 fluorescent primer. The specific sequence display removes the M13 fluorescent primer sequence (18bp), and the underlined part is the SSR repeat element.)
  • the amplification result of primer 2 is shown in Figure 3.
  • primer 2 was used for fluorescent PCR amplification
  • 2 fragments (2 peaks) were amplified, containing 2 SSR sites, and the SSR repeating element was TG.
  • the characteristic information of the amplified fragments obtained in the test example are 255bp fragments and 257bp fragments of 7 and 8 repeats of TG, respectively.
  • Primer 2 amplified fragment (The statistical fragment length of the electropherogram includes the M13 fluorescent primer, the specific sequence shows that the M13 fluorescent primer sequence (18bp) is removed, and the underlined part is the SSR repeat element.)
  • the amplification result of primer 3 is shown in Figure 4.
  • primer 3 was used for fluorescent PCR amplification, 2 fragments (2 peaks) were amplified, containing 2 SSR sites, and the SSR repeating element was TA.
  • the characteristic information of the amplified fragment obtained in the test example is 222bp and 232bp fragments of 6 and 12 repeats of AT respectively.
  • Primer 3 amplified fragment (The statistical fragment length of the electropherogram includes the M13 fluorescent primer, the specific sequence shows that the M13 fluorescent primer sequence (18bp) is removed, and the underlined part is the SSR repeat element.)
  • the amplification result of primer 4 is shown in Figure 5.
  • primer 4 was used for fluorescent PCR amplification, 2 fragments (2 peaks) were amplified, containing 2 SSR sites, and the SSR repeating element was GAT.
  • the characteristic information of the amplified fragments obtained in the test example are 217bp and 223bp fragments containing 5 and 7 repeats respectively.
  • Primer 4 amplified fragment (where the statistical fragment length of the electropherogram includes the M13 fluorescent primer, and the M13 fluorescent primer sequence (18bp) is removed from the specific sequence display, and the statistical fragment length of the 223bp fragment electropherogram includes the M13 fluorescent primer, and the specific sequence display removes it
  • Primer 1 amplifies a 263bp fragment containing 5 ACC repeat elements (as shown in SEQ ID NO: 3), a 266bp fragment containing 6 ACC repeat elements (as shown in SEQ ID NO: 4) and a 7-time ACC repeat
  • the 269bp fragment (as shown in SEQ ID NO: 5) of the element, primer 2 amplifies the 255bp fragment (as shown in SEQ ID NO: 8) containing 7 TG repeat elements and the 257bp fragment containing 8 TG repeat elements ( Shown in SEQ ID NO: 9);
  • Primer 3 amplifies the 222bp fragment (as shown in SEQ ID NO: 12) containing 6 AT repeat elements and the 232bp fragment containing 12 AT repeat elements (as SEQ ID NO: 13);
  • Primer 4 amplifies a 217bp fragment containing five GAT repeat elements (as shown in SEQ ID NO: 16) and a 223bp fragment containing seven GAT repeat elements (as shown
  • the DNA barcode of the total polysaccharide content of the yellow-green mushroom was verified by blind test.
  • samples from Shiqu County, Sichuan province with a total polysaccharide content higher than or equal to 242.88 mg/g were used as the test group;
  • the samples from Qilian County in the province were the comparison group 1 and the comparison group 2, and 16 samples were taken from each group for blind test;
  • the second step test using primers (SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 15) was amplified and subjected to capillary electrophoresis.
  • the primer set can be amplified using one or more pairs of combinations to distinguish the blind test sample from the DNA barcode characteristics of the test example;
  • the third step is unblinding, and the results are shown in Table 3.

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Abstract

本发明公开了一种用于筛选总多糖含量高的黄绿卷毛菇的DNA条形码，属于食用菌种质资源筛选技术领域，可用于优质黄绿卷毛菇的遗传育种和种质资源的鉴定及保护。

Description

一种用于筛选总多糖含量高的黄绿卷毛菇的DNA条形码 技术领域
本发明涉及食用菌种质资源筛选技术领域，更具体的说是涉及一种用于筛选总多糖含量高的黄绿卷毛菇的DNA条形码。
背景技术
黄绿卷毛菇，色泽呈金黄色，又称为黄蘑菇、金蘑菇。黄绿卷毛菇主要分布于青藏高原，主产区为西藏自治区当雄县、青海省祁连县以及四川省石渠县等，也以这三个主产区的品质最佳。黄绿卷毛菇是一种风味独特的优质食用菌，当前不能人工栽培。评价黄绿卷毛菇营养价值风味及生物学活性的主要指标包括：总可溶性蛋白、总可溶性氨基酸、总多酚、总多糖和总脂肪含量高，抗氧化活性强。按传统方法，要筛选出优质菌株十分困难，此外，由于其所分布的主产区海拔较高，样本采集也十分困难。为了实现黄绿卷毛菇的开发利用，利用DNA条形码分子鉴定技术辅助筛选优质黄绿卷毛菇菌株显得尤为重要和迫切。黄绿卷毛菇不同产地具有不同营养价值，不同风味，不同生物学活性，不同市场价格。以往对黄绿卷毛菇的选育主要利用形态学方法结合上述有益指标测定进行。
但是受到特殊的青藏高气候原环境的影响不同地区所产的黄绿卷毛菇常常出现同名异物和同物异名的现象，因此形态学鉴别法难以有效区分。更为困难的是，不能通过形态学方法来筛选出总可溶性蛋白、总可溶性氨基酸、总多酚、总多糖和总脂肪含量高，抗氧化活性强的优质菌株。DNA条形码分子鉴定技术是基于DNA条形码(基因组中保守且稳定遗传DNA序列)来进行物种和优良品质识别鉴定的分子生物学技术。它是传统育种方法的有效补充和拓展，能够在样品形态不完整或缺乏形态结构(加工制品如粉末等)时对样品进行精准和有效地鉴定。
现有的DNA条形码技术中，ITS(核糖体RNA内转录间隔区)和线粒体体中的非编码区或保守基因序列主要用于物种物鉴定；限制性片段长度多态性(restriction fragment length polymorphism，RFLP)操作十分繁复，结果的可靠性和可重复性较差，随机扩增多态性DNA(random amplified polymorphic DNA，RAPD)易受干扰，对操作者技术水平要求较高，在辅助育种工作中难以推广；单核苷酸多态性(single nucleotide polymorphism，SNP)对设备要求高，成本也高。
因此针对传统育种方法选育黄绿卷毛菇菌株不够准确费时费力的缺点，如何提供一种可以准确、快捷鉴别黄绿卷毛菇的所属菌株，同时实现优质品质选育的DNA条形码，具有成本低，效率高，操作简便，结果稳定可靠性重复性好的特点是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种用于筛选总多糖含量高的黄绿卷毛菇的DNA条形码。
为了实现上述目的，本发明采用如下技术方案：
一种筛选黄绿卷毛菇总多糖含量指标的DNA条形码，DNA条形码的核苷酸序列包括：
如SEQ ID NO：3和SEQ ID NO：5组合；
和/或SEQ ID NO：4和SEQ ID NO：5组合；
和/或SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5组合；
和/或SEQ ID NO：8；
和/或SEQ ID NO：9；
和/或SEQ ID NO：8和SEQ ID NO：9组合；
和/或SEQ ID NO：12；
和/或SEQ ID NO：13；
和/或SEQ ID NO：12和SEQ ID NO：13组合；
和/或SEQ ID NO：16和SEQ ID NO：17组合中的一种或多种。
本发明基于黄绿卷毛菇全基因组中所有简单重复序列(simple sequence repeat，SSR)进行荧光PCR扩增，确立了与总多糖含量有效对应的DNA条形码，扩增所得片段与本发明的DNA条形码进行比对，可以快速、准确地筛选出黄绿卷毛菇总多糖含量高的菌株，为黄绿卷毛菇的育种提供有利辅助。
本发明的又一目的是，提供扩增上述筛选黄绿卷毛菇总多糖含量指标的DNA条形码的引物组，引物组的核苷酸序列包括：
如SEQ ID NO：1和SEQ ID NO：2，
和/或SEQ ID NO：6和SEQ ID NO：7，
和/或SEQ ID NO：10和SEQ ID NO：11，
和/或SEQ ID NO：14和SEQ ID NO：15中的一组或多组。
作为本发明优选的技术方案，引物组的核苷酸序列包括：如SEQ ID NO：1和SEQ ID NO：2、SEQ ID NO：6和SEQ ID NO：7，SEQ ID NO：10和SEQ ID NO：11、SEQ ID NO：14和SEQ ID NO：15。
本发明不同的引物组可以单独或组合使用筛选黄绿卷毛菇的总多糖含量，当所有引物组共同使用时，筛选的准确率最高。
本发明的再一目的是，提供一种以总多糖含量指标筛选黄绿卷毛菇的方法，包括如下步骤：
S1、提取待测样品基因组DNA；
S2、以S1基因组DNA为模板，上述的一组或多组引物分别进行荧光PCR扩增反应，得扩增产物；
S3、S2所述扩增产物经毛细管荧光电泳检测，通过扩增产物的片段数、SSR位点数、SSR重复元件及其重复次数进行判定。
作为本发明优选的技术方案，步骤S3的判定标准为：
SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含5次ACC重复元件的263bp片段、含6次ACC重复元件的266bp片段和含7次ACC重复元件的269bp片段；
和/或SEQ ID NO：6和SEQ ID NO：7引物组扩增得到含7次TG重复元件的255bp片段和含8次TG重复元件的257bp片段；
和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含6次AT重复元件的222bp片段和含12次AT重复元件的232bp片段；
和/或SEQ ID NO：14和SEQ ID NO：15引物组扩增得到含5次GAT重复元件的217bp片段和含7次GAT重复元件的223bp片段时，判定该黄绿卷毛菇为总多糖含量高的黄绿卷毛菇。
作为本发明优选的技术方案，步骤S2荧光PCR扩增反应的反应体系为：
2×Taq PCR Master Mix 5μL，基因组DNA 1μL，上游引物0.1μL，下游引物0.4μL，带荧光的M13引物0.4μL，用无菌去离子水定容至10μL。
更优选的，上游引物、下游引物和带荧光的M13引物浓度均为10uM。
作为本发明优选的技术方案，步骤S2荧光PCR扩增反应程序为：
95℃预变性3min；95℃变性30s，62至55℃降落PCR退火30s，72℃延伸30s，共10个循环；95℃变性30s，52℃退火30s，72℃延伸30s，共25个循环；72℃终延伸20min；4℃保温6h后用于荧光毛细管电泳检测。
本发明的再一目的是，提供上述DNA条形码和/或上述引物组在制备以总多糖含量指标筛选优质黄绿卷毛菇的产品中的应用。
本发明的再一目的是，提供一种以总多糖含量指标筛选优质黄绿卷毛菇的产品，含有上述的一组或多组引物组，且符合标准：
SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含5次ACC重复元件的263bp片段、含6次ACC重复元件的266bp片段和含7次ACC重复元件的269bp片段；
和/或SEQ ID NO：6和SEQ ID NO：7引物组扩增得到含7次TG重复元件的255bp片段和含8次TG重复元件的257bp片段；
和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含6次AT重复元件的222bp片段和含12次AT重复元件的232bp片段；
和/或SEQ ID NO：14和SEQ ID NO：15引物组扩增得到含5次GAT重复元件的217bp片段和含7次GAT重复元件的223bp片段中的一个或多个
作为本发明优选的技术方案，产品为试剂盒。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种用于筛选总多糖含量高的黄绿卷毛菇的DNA条形码，相较于现有技术本发明可以准确、快捷鉴别黄绿卷毛菇的所属菌株，同时实现优质品质选育的DNA条 形码技术，具有成本低，效率高，操作简便，结果稳定可靠性重复性好的特点。
本发明与传统育种方法及其他现有DNA条形码技术相比较，它具有省时、省力、省钱、准确、高效的优点，在优质黄绿卷毛菇原产地鉴别和遗传育种上发挥积极作用，同时也为种质资源的鉴定及保护提供了一种有效方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例、对比例1和2的总多糖含量对比结果图，其中由左至右依次为对比例1、对比例2和实施例。
图2附图为本发明利用引物对1 PCR扩增对比例1、2和实施例对比结果图。
图3附图为本发明利用引物对2 PCR扩增对比例1、2和实施例对比结果图。
图4附图为本发明利用引物对3 PCR扩增对比例1、2和实施例对比结果图。
图5附图为本发明利用引物对4 PCR扩增对比例1、2和实施例对比结果图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例公开了一种用于筛选总多糖含量高的黄绿卷毛菇的DNA条形码。
实施例1
黄绿卷毛菇DNA条形码的构建
采集西藏自治区当雄县、青海省祁连县、四川省石渠县的黄绿卷毛菇样品进行基因组测序，使用MISA程序对基因组序列中的SSR位点进行分析。
设计引物对这些SSR位点进行PCR扩增，保留能扩增出对应片段的引物，舍弃无效引物。
选取西藏自治区当雄县、青海省祁连县、四川省石渠县的黄绿卷毛菇样品测定总多糖含量。
利用有效引物对上述三个产地样品分别扩增并通过毛细管电泳检测。经分析建立总多糖含量对应的简单重复序列(simple sequence repeat，SSR)位点。最终获得4对引物(见表1)，利用这4对引物对样品基因组进行扩增所得片段多态性可辅助筛选总多糖含量高的黄绿卷毛菇。
表1黄绿卷毛菇多糖含量高的菌株筛选特异引物
Figure PCTCN2022109999-appb-000001
实施例2
黄绿卷毛菇高总多糖含量高的菌株SSR特异引物扩增
(1)总多糖提取与鉴定
以四川省石渠县样品为本发明试验例，将子实体样品冻干粉碎过50目筛，以1克干粉加20mL双蒸水，用300W超声波辅助提取30min，然后5000转每分钟离心30min后取上清液制备成总多糖提取液。
对比例1：西藏自治区当雄县样品(处理方法同上)。
对比例2：青海省祁连县样品(处理方法同上)。
提取物中总多糖含量测定采用硫酸苯酚法，具体参照赵启铎等(宜春学院学报,2011,33(08):74-76)，并换算为毫克每克。其中四川省石渠县黄绿卷毛菇中总多糖含量为242.88(±2.43)毫克每克，确定为试验例，西藏自治区当雄县黄绿卷毛菇中总多糖含量为148.49(±3.85)毫克每克确定为对比例1、青海省祁连县黄绿卷毛菇中总多糖含量为195.95(±3.28)毫克每克确定为对比例2(参见附图1)。
(2)利用生工生物工程(上海)有限公司Ezup柱式真菌基因组DNA抽提试剂盒(货号B518259)提取黄绿卷毛菇样品基因组，稀释至20ng/μL用于荧光PCR扩增。
(3)利用表1中引物进行荧光PCR扩增SSR DNA条形码。
荧光PCR扩增反应体系(10μL)：2×Taq PCR Master Mix 5μL，模板(基因组DNA)1μL，上游引物0.1μL，下游引物0.4μL(上下游引物浓度均为10uM)，带荧光的M13引物(浓度10uM)0.4μL，用无菌的去离子水定容至10μL；
反应条件：95℃预变性3min；95℃变性30s，62至55℃降落PCR退火30s，72℃延伸30s，共10个循环；95℃变性30s，52℃退火30s，72℃延伸30s，共25个循环；72℃终延伸20min；4℃保温6h后用于荧光毛细管电泳检测。
(4)将PCR产物进行定量稀释后，取1μL PCR稀释产物加9μL甲酰胺(含1％内标)变性后上DNA测序仪ABI 3730xl进行毛细管荧光电泳检测。内标为LIZ-500分子量内标(又称分子量内对照，internal lane standards)由16条带有LIZ荧光素(橙色)标记的双链DNA片段组成，分子量分别是：35、50、75、100、139、150、160、200、250、300、340、350、400、450、490和500bp。扩增结果电泳图中片段大小等于所扩增片段实际bp数加上M13荧光引物(约18bp)，误差范围1～2bp，扩增毛细管电泳峰结合测序结果，峰数量表示该基因杂合子扩增片段数量。
(5)采用以上方法对试验例1、对比例1和对比例2的黄绿卷毛菇进行鉴定。
引物1扩增结果如附图2所示，当使用引物1进行荧光PCR扩增时，扩增得到3个片段(3个峰)，含有3个SSR位点，SSR重复元件为ACC。其中试验例所得扩增片段的特征为含5和7次重复的263bp和269bp片段。
引物1扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp)，下划线部分为SSR重复元件。)
263bp扩增片段序列：
CACCGCACCATCTGCTGATAATGAATACTTGT ACCACCACCACCACCAACACCACGCCCACTCGTTTTCCTGCGTTTCCCTGCGGCACATTTTCCTCAATCACAATCACAGTCACCGCTTCCACAGTCCCTCCTAGTCTTGATGTTGCTCATTTCCCTGTGTGGTCTCGTTTCTTGGTACACACAGGACCGCCACCGACCTCCCGCCACCTCTCCGTCATTTAACTTCCCGTTATATGCTCATACCCACACTGACGAATCACC，如SEQ ID No.3；
266bp扩增片段序列：
CACCGCACCATCTGCTGATAATGAATACTTGT ACCACCACCACCACC ACCAACACCACGCCCACTCGTTTTCCTGCGTTTCCCTGCGGCACATTTTCCTCAATCACAATCACAGTCACCGCTTCCACAGTCCCTCCTAGTCTTGATGTTGCTCATTTCCCTGTGTGGTCTCGTTTCTTGGTACACACAGGACCGCCACCGACCTCCCGCCACCTCTCCGTCATTTAACTTCCCGTTATATGCTCATACCCACACTGACGAATCACC，如SEQ ID No.4；
269bp扩增片段序列：
CACCGCACCATCTGCTGATAATGAATACTTGT ACCACCACCACCACC ACCACCAACACCACGCCCACTCGTTTTCCTGCGTTTCCCTGCGGCACATTTTCCTCAATCACAATCACAGTCACCGCTTCCACAGTCCCTCCTAGTCTTGATGTTGCTCATTTCCCTGTGTGGTCTCGTTTCTTGGTACACACAGGACCGCCACCGACCTCCCGCCACCTCTCCGTCATTTAACTTCCCGTTATATGCTCATACCCACACTGACGAATCACC，如SEQ ID No.5。
引物2扩增结果如附图3所示，当使用引物2进行荧光PCR扩增时，扩增得到2个片段(2个峰)，含有2个SSR位点，SSR重复元件为TG。其中试验例所得扩增片段的特征信息为分别7和8次重复TG的255bp片段和257bp片段。
引物2扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp)，下划线部分为SSR重复元件。)
255bp扩增片段序列：
TAACTATTTCCCGGCTCGGCTCTGCAGACAAATTTCAGGGTGTTCCTTTTCCTGATCCATACATGCCGAACCACACCTGTGACCTGCCGTGCACTATGCCGATAAACTTCATCGTTTATCCAAGTCTCTCCCACAGGAGTTATATTCGAGACTCCGTGTAAACCTTATGTACGTCACAAGTATGCCAAGTAGTCAGCTTCTTCGCA TGTGTGTGTGTGTGCGGTTTGTGCTAAATCAAGCGCGAACCTTCCTTTC，如SEQ ID No.8；
257bp扩增片段序列：
TAACTATTTCCCGGCTCGGCTCTGCAGACAAATTTCAGGGTGTTCCTTTTCCTGATCCATACATGCCGAACCACACCTGTGACCTGCCGTGCACTATGCCGATAAACTTCATCGTTTATCCAAGTCTCTCCCACAGGAGTTATATTCGAGACTCCGTGTAAACCTTATGTACGTCACAAGTATGCCAAGTAGTCAGCTTCTTCGCA TGTGTGTGTGTGTGTGCGGTTTGTGCTAAATCAAGCGCGAACCTTCCTTTC，如SEQ ID No.9。
引物3扩增结果如附图4所示，当使用引物3进行荧光PCR扩增时，扩增得到2个片段(2个峰)，含有2个SSR位点，SSR重复元件为TA。其中试验例所得扩增片段的特征信息为分别6和12次重复AT的222bp和232bp片段。
引物3扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp)，下划线部分为SSR重复元件。)
222bp扩增片段序列：
AGCATTCCAGAGACGTCAGCGTCTGTCATATTGATAAAGTTAAAGTAATTTGTGCTTGAAGTCATATCATATAACTATTCGGCAGCTCAAAGACTATGAATGTGCTTACCGGTACCCTAGAGTATATTGAATAAGATTGAAT TA TATATATATACATATATAATAATGGAACGCACACATATCCGTATTCAGAGCGATGATACGCAGTCCATCTTGACGTCT，如SEQ ID No.12；
232bp扩增片段序列：
AGCATTCCAGAGACGTCAGCGTCTGTCATATTGATAAAGTTAAAGTAATTTGTGCTTGAAGTCATATCATATAACTATTCGCAGCTCAAAGACTATGAATGTGCTTACCGGTACCCTAGAGTATATTGAATAAGATTGAAT TAT ATATATATATATATATATATACATATATAATAATGAACGCACACATATCCGTATTCAGAGCGATGATACGCAGTCCATCTTGACGTCT，如SEQ ID No.13；
引物4扩增结果如附图5所示，当使用引物4进行荧光PCR扩增时，扩增得到2个片段(2个峰)，含有2个SSR位点，SSR重复元件为GAT。其中试验例所得扩增片段的特征信息为分别含5和7次重复的217bp和223bp片段。
引物4扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp),其中223bp片段电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(19bp)，误差为1bp，下划线部分为SSR重复元件。)
217bp扩增片段序列：
GAAGTGGTAGTGGGACTGGCGAAGAAGTCAGCGAAACCCGGAGGAAGGAGGAAGAAGAGGAAGGAGGAGGATGTAGGAGATGATGATGAAGAGGAA GATGATGATGATGATGACGATGATGATGAACTTTGGAGAGGAAGAACATCCATCTTTCCGGACGCGCTTCTGGCTCGATGTCCAGTCTCGGCCACCCAATTTCACCTCGCTATCCCTTCCA，如SEQ ID No.16；
223bp扩增片段序列：
GAAGTGGTAGTGGGACTGGCGAAGAAGTCAGCGAAACCCGGAGGAAGGAGGAAGAAGAGGAAGGAGGAGGATGTAGGAGATGATGATGAAGAGGAA GATGATGATGATGATGATGATGACGATGATGATGAACTTTGGAGAGGAAGAACATCCATCTTTCCGGACGCGCTTCTGGCTCGATGTCCAGTCTCGGCCACCCAATTTCACCTCGCTATCCCTTCCA，如SEQ ID No.17；
通过对试验例、对比例1和对比例2图谱和测序结果综合分析，获得总多糖含量高的黄绿卷毛菇的DNA条形码特征信息如表2。
表2总多糖含量高的黄绿卷毛菇的DNA条形码特征
Figure PCTCN2022109999-appb-000002
Figure PCTCN2022109999-appb-000003
引物1扩增出含5次ACC重复元件的263bp片段(如SEQ ID NO：3所示)、含6次ACC重复元件的266bp片段(如SEQ ID NO：4所示)和含7次ACC重复元件的269bp片段(如SEQ ID NO：5所示)，引物2扩增出含7次TG重复元件的255bp片段(如SEQ ID NO：8所示)和含8次TG重复元件的257bp片段(如SEQ ID NO：9所示)；引物3扩增出含6次AT重复元件的222bp片段(如SEQ ID NO：12所示)和含12次AT重复元件的232bp片段(如SEQ ID NO：13所示)；引物4扩增出含5次GAT重复元件的217bp片段(如SEQ ID NO：16所示)和含7次GAT重复元件的223bp片段(如SEQ ID NO：17所示)。
此外，同时使用上述引物1、2、3、4进行综合检测判断时，黄绿卷毛菇总多糖含量指标筛选的准确性最好。
实施例3
黄绿卷毛菇总多糖含量指标筛选验证
通过盲试试验验证黄绿卷毛菇总多糖含量的DNA条形码。
第一步盲试，以总多糖含量高于或等于242.88毫克每克的四川省石渠县样品为试验组，以低于242.88毫克每克(显著性p<0.05)的西藏自治区当雄县和青海省祁连县样品为对比1组和对比2组，每组各取16份样品进行盲试；
第二步测试，利用引物(SEQ ID NO：1和SEQ ID NO：2，SEQ ID NO：6和SEQ ID NO：7，SEQ ID NO：10和SEQ ID NO：11，SEQ ID NO：14和SEQ ID NO：15)扩增并进行毛细管电泳。引物组可使用一对或多对组合扩增以试验例DNA条形码特征区分盲试样品；
第三步揭盲，结果如表3所示，以总多糖含量条形码特征区分总多糖含量的48份样品揭盲结果全部正确。由此说明总多糖含量的DNA条形码适用于总多糖含量性状的筛选。
表3总多糖含量DNA条形码特征揭盲鉴定结果
Figure PCTCN2022109999-appb-000004
Figure PCTCN2022109999-appb-000005
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种筛选黄绿卷毛菇总多糖含量指标的DNA条形码，其特征在于，所述DNA条形码的核苷酸序列包括：
    如SEQ ID NO：3和SEQ ID NO：5组合；
    和/或SEQ ID NO：4和SEQ ID NO：5组合；
    和/或SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5组合；
    和/或SEQ ID NO：8；
    和/或SEQ ID NO：9；
    和/或SEQ ID NO：8和SEQ ID NO：9组合；
    和/或SEQ ID NO：12；
    和/或SEQ ID NO：13；
    和/或SEQ ID NO：12和SEQ ID NO：13组合；
    和/或SEQ ID NO：16和SEQ ID NO：17组合中的一种或多种。
  2. 一种扩增如权利要求1所述筛选黄绿卷毛菇总多糖含量指标的DNA条形码的引物组，其特征在于，所述引物组的核苷酸序列包括：
    如SEQ ID NO：1和SEQ ID NO：2；
    和/或SEQ ID NO：6和SEQ ID NO：7；
    和/或SEQ ID NO：10和SEQ ID NO：11，
    和/或SEQ ID NO：14和SEQ ID NO：15中的一组或多组。
  3. 根据权利要求2所述的引物组，其特征在于，所述引物组的核苷酸序列包括：如SEQ ID NO：1和SEQ ID NO：2、SEQ ID NO：6和SEQ ID NO：7，SEQ ID NO：10和SEQ ID NO：11、SEQ ID NO：14和SEQ ID NO：15。
  4. 一种以总多糖含量指标筛选黄绿卷毛菇的方法，其特征在于，包括如下步骤：
    S1、提取待测样品基因组DNA；
    S2、以S1基因组DNA为模板，选择权利要求2所述的一组或多组引物分别进行荧光PCR扩增反应，得扩增产物；
    S3、S2所述扩增产物经毛细管荧光电泳检测，通过扩增产物的片段数、SSR位点数、SSR重复元件及其重复次数进行判定。
  5. 根据权利要求4所述的以总多糖含量指标筛选黄绿卷毛菇的方法，其特征在于，所述步骤S3的判定标准为：
    SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含5次ACC重复元件的263 bp片段、含6次ACC重复元件的266 bp片段和含7次ACC重复元件的269 bp片段；
    和/或SEQ ID NO：6和SEQ ID NO：7引物组扩增得到含7次TG重复元件的255 bp片段和含8次TG重复元件的257 bp片段；
    和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含6次AT重复元件的222 bp片段和含12次AT重复元件的232 bp片段；
    和/或SEQ ID NO：14和SEQ ID NO：15引物组扩增得到含5次GAT重复元件的217 bp片段和含7次GAT重复元件的223 bp片段时，判定该黄绿卷毛菇为总多糖含量高的黄绿卷毛菇。
  6. 根据权利要求4所述的以总多糖含量指标筛选黄绿卷毛菇的方法，其特征在于，步骤S2所述荧光PCR扩增反应的反应体系为：
    2×Taq PCRMasterMix 5μL，基因组DNA 1μL，上游引物0.1μL，下游引物0.4μL，带荧光的M13引物0.4μL，用无菌去离子水定容至10μL。
  7. 根据权利要求6所述的以总多糖含量指标筛选黄绿卷毛菇的方法，其特征在于，所述上游引物、下游引物和带荧光的M13引物浓度均为10uM。
  8. 根据权利要求4所述的以总多糖含量指标筛选黄绿卷毛菇的方法，其特征在于，步骤S2所述荧光PCR扩增反应程序为：
    95℃预变性3min；95℃变性30s，62至55℃降落PCR退火30s，72℃延伸30s，共10个循环；95℃变性30s，52℃退火30s，72℃延伸30s，共25个循环；72℃终延伸20min；4℃保温6h后用于荧光毛细管电泳检测。
  9. 权利要求1所述DNA条形码和/或权利要求2所述引物组在制备以总多糖含量指标筛选黄绿卷毛菇的产品中的应用。
  10. 一种以总多糖含量指标筛选优质黄绿卷毛菇的产品，其特征在于，含有权利要求2所述的一组或多组引物组，且符合标准：
    SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含5次ACC重复元件的263 bp片段、含6次ACC重复元件的266 bp片段和含7次ACC重复元件的269 bp片段；
    和/或SEQ ID NO：6和SEQ ID NO：7引物组扩增得到含7次TG重复元件的255 bp片段和含8次TG重复元件的257 bp片段；
    和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含6次AT重复元件的222 bp片段和含12次AT重复元件的232 bp片段；
    和/或SEQ ID NO：14和SEQ ID NO：15引物组扩增得到含5次GAT重复元件的217 bp片段和含7次GAT重复元件的223 bp片段中的一个或多个。
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