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Chinese to English: 博若莱新酒节，2016佳美新酿本周四直运福楼！ General field: Marketing Detailed field: Food & Drink
Source text - Chinese 博若莱是单一品种酿造的葡萄酒，不同于混酿，充分体现了佳美葡萄果味轻盈、单宁弱的个性。酿制单一品种葡萄酒对土壤、天气、栽种技术、酿制技术等都有更高的要求。所有博若莱新酒的葡萄均以人工采收。由于不经过木桶陈年，博若莱出品后达到酒体轻薄、果香浓郁的舒适口感，特别适合葡萄酒入门者。
Translation - English Unlike blends, Beaujolais is a varietal wine, light bodied, fruity and low in tannin. Varietal wines are usually more demanding in the soil, the weather, the planting technology, and the fermenting process. All the grapes for the Beaujolais wines must be hand-harvested, and then they will go through a winemaking process called carbonic maceration, also called whole berry fermentation. This technique preserves the fresh, fruity quality of the wine, without extracting bitter tannins from the grape skins, which is especially popular with beginners.
Chinese to English: Menu translation General field: Marketing Detailed field: Food & Drink
Source text - Chinese Langoustine d’Islande n°0 (1pièce) 135
Iceland scampi No.0 cooked to order (1 piece)现烹No.0冰岛海螯虾（1只）
Carabineros de Mediterranée (1 pièce) 68
Red Mediterannean Carabineros prawn (1 piece)地中海深海红虾（1只）
Huître de Tasmanie (1 pièce) RMB 48
Tasmanian No.3, Australian (1 piece)澳大利亚塔斯马尼亚No.3生蚝（1只）
Huître marine
Boudeuse oyster with tuna, salmon roe, RMB 65
apple caviar, ponzu sauce布德斯生蚝，搭配金枪鱼，鲑鱼籽，苹果鱼子酱，日式酱汁
Huître Vichyssoise
Fine de Claire No.3 oyster with leek and potato RMB 65
panna cotta, Baeri caviar芬迪克雷No.3生蚝搭配韭葱，土豆，意式奶冻，鲟鱼子酱
Plateau Ecailler 绚味海鲜拼盘
1 Tasmanian oyster, 1 Fine de Claire No.3 oyster
1 New Zealand langoustine, 1 marinated Pacific scallop,
100g of shrimp & 125g of whelk
278
塔斯马尼亚生蚝1只，芬迪克雷No.3生蚝1只，新西兰螯虾1只，渍太平洋扇贝1只，100克海虾和125g海螺
Carabineros de Mediterranée (1 pièce) 68
Red Mediterannean Carabineros prawn (1 piece)
地中海深海红虾（1只）
Plateau Des Mers 大洋海鲜拼盘
3 Gillardeau oysters, 3 Fine de Claire No.3 oysters,
1 Australian crab, 200g of shrimp
& 250g of whelk
888
吉拉多生蚝3只，芬迪克雷No.3生蚝3只，澳大利亚蟹1只，200g海虾和250g海螺
Plateau De La “Grande Bleue”“邃蓝”海鲜拼盘
1 Boston lobster, 1 Australian crab, 4 New Zealand langoustines,
4 red Mediterranean Carabineros prawn
200g of shrimp & 250g of whelks
1566
波士顿龙虾1只，澳大利亚蟹1只，新西兰螯虾4只，地中海深海红虾4只，200g海虾和250g海螺
Plateau Suprême 极味海鲜拼盘
6 Gillardeau oysters, 6 Ancelin oysters,
6 Tasmanian oysters, 6 Fine de Claire No.3 oysters,
1 Boston lobster, 1 Australian crab
6 New Zealand langoustines
200g of shrimp & 250g of whelk
2799
吉拉多生蚝6只, 安瑟兰生蚝6只，塔斯马尼亚生蚝6只，芬迪克雷No.3生蚝6只，波士顿龙虾1只，澳大利亚蟹1只，新西兰螯虾6只，200g海虾和250g海螺
Plateau Voyage 旅味海鲜拼盘
2 Gillardeau oysters, 2 marinated Pacific scallops,
2 red Mediterranean Carabineros prawns, 2 abalones,
2 Ark shells, 200g of shrimp & 250g of whelk
666
吉拉多生蚝2只，渍太平洋扇贝2只，地中海深海红虾2只，鲍鱼两只，赤贝2只，200g海虾和250g海螺
Translation - English Langoustine d’Islande n°0 (1pièce) 135
Iceland scampi No.0 cooked to order (1 piece)现烹No.0冰岛海螯虾（1只）
Carabineros de Mediterranée (1 pièce) 68
Red Mediterannean Carabineros prawn (1 piece)地中海深海红虾（1只）
Huître de Tasmanie (1 pièce) RMB 48
Tasmanian No.3, Australian (1 piece)澳大利亚塔斯马尼亚No.3生蚝（1只）
Huître marine
Boudeuse oyster with tuna, salmon roe, RMB 65
apple caviar, ponzu sauce布德斯生蚝，搭配金枪鱼，鲑鱼籽，苹果鱼子酱，日式酱汁
Huître Vichyssoise
Fine de Claire No.3 oyster with leek and potato RMB 65
panna cotta, Baeri caviar芬迪克雷No.3生蚝搭配韭葱，土豆，意式奶冻，鲟鱼子酱
Plateau Ecailler 绚味海鲜拼盘
1 Tasmanian oyster, 1 Fine de Claire No.3 oyster
1 New Zealand langoustine, 1 marinated Pacific scallop,
100g of shrimp & 125g of whelk
278
塔斯马尼亚生蚝1只，芬迪克雷No.3生蚝1只，新西兰螯虾1只，渍太平洋扇贝1只，100克海虾和125g海螺
Carabineros de Mediterranée (1 pièce) 68
Red Mediterannean Carabineros prawn (1 piece)
地中海深海红虾（1只）
Plateau Des Mers 大洋海鲜拼盘
3 Gillardeau oysters, 3 Fine de Claire No.3 oysters,
1 Australian crab, 200g of shrimp
& 250g of whelk
888
吉拉多生蚝3只，芬迪克雷No.3生蚝3只，澳大利亚蟹1只，200g海虾和250g海螺
Plateau De La “Grande Bleue”“邃蓝”海鲜拼盘
1 Boston lobster, 1 Australian crab, 4 New Zealand langoustines,
4 red Mediterranean Carabineros prawn
200g of shrimp & 250g of whelks
1566
波士顿龙虾1只，澳大利亚蟹1只，新西兰螯虾4只，地中海深海红虾4只，200g海虾和250g海螺
Plateau Suprême 极味海鲜拼盘
6 Gillardeau oysters, 6 Ancelin oysters,
6 Tasmanian oysters, 6 Fine de Claire No.3 oysters,
1 Boston lobster, 1 Australian crab
6 New Zealand langoustines
200g of shrimp & 250g of whelk
2799
吉拉多生蚝6只, 安瑟兰生蚝6只，塔斯马尼亚生蚝6只，芬迪克雷No.3生蚝6只，波士顿龙虾1只，澳大利亚蟹1只，新西兰螯虾6只，200g海虾和250g海螺
Plateau Voyage 旅味海鲜拼盘
2 Gillardeau oysters, 2 marinated Pacific scallops,
2 red Mediterranean Carabineros prawns, 2 abalones,
2 Ark shells, 200g of shrimp & 250g of whelk
666
吉拉多生蚝2只，渍太平洋扇贝2只，地中海深海红虾2只，鲍鱼两只，赤贝2只，200g海虾和250g海螺
Chinese to English: 新疆野生羊肚菌系统发育分析 Phylogenetic Analysis on Xinjiang Wild Morchella General field: Science Detailed field: Biology (-tech,-chem,micro-)
Source text - Chinese 基金项目：国家自然科学基金资助项目（31160011）资助
Fund project: Sponsored by NSFC(National Science Fundation of China) 31160011
新疆野生羊肚菌系统发育分析
Phylogenetic Analysis on Xinjiang Wild Morchella
摘 要：为确定新疆野生羊肚菌的分类地位，根据子实体形态特征并结合ITS+ef1-a+rpb1+rpb2多基因联合分析技术，对根据形态特征挑选的、采自新疆伊犁、塔城、石河子等地的代表新疆野生羊肚菌物种多样性的23株野生羊肚菌进行多基因系统进化分析。多基因联合分析结果表明，新疆野生羊肚菌共有7种物种组成，包括黑色羊肚菌5个物种（Mel-13、Mel-19、Mel-33、Mel-31和Mel-2），黄色羊肚菌2个物种（Mes-6和Mes-17）。根据国内最新研究资料，其中2种为中国新纪录种，分别为Mes-17和Mel-2。
Abstract: 23 strains of wild true morels(Morchella) representing Morchella generic diversity in Xinjiang were selected from areas such as Xinjiang Ili, Tacheng, Shihezi and etc for their distinct morphological characteristics. Based on the morphology study of fruit bodies, a joint phylogenetic analysis multiple-genes of ITS+ef1-a+rpb1+rpb2 was carried out to determine the taxonomic status of Xinjiang wild morels. The results showed that there were seven species among the 23 strains of Xinjiang wild morel, including five species of Elata Clade (black morels): Mel-13, Mel-19, Mel-33, Mel-31 and Mel-2, and two species of Esculenta Clade (yellow morels): Mes-6 and Mes-17. According to the latest domestic research, Mes-17 and Mel-2 are new record species in China.
关键词：新疆，野生羊肚菌，系统发育分析，多基因联合
Keywords: Xinjiang, Wild Morchella, Phylogenetic Analysis, joint analysis of Multiple-Genes
羊肚菌属（Morchella Dill.ex Pers.: Fr.），属子囊菌门（Ascomycota），盘菌纲（Pezizomycetes），盘菌目(Pezizales)，羊肚菌科(Morchellaceae)（Hibbett et al., 2007）。因其表面呈蜂窝状，外形常为圆顶或尖顶，与羊肚颇为相似，故得名羊肚菌，其模式种是羊肚菌Morchella esculenta（杜习慧等，2014）。羊肚菌子实体肉质脆嫩，味道鲜美，是世界公认的珍贵、稀有食药用真菌（Royse & May, 1990; 戴玉成和杨祝良，2008）。
Morchella (Morchella Dill.ex Pers.: Fr.) is classified to Ascomycota phylum; Pezizomycetes class; Pezizales order; Morchellaceae family ( Hibbett et al. , 2007). Its Chinese name: lamp tripe fungus comes from its dome or apex shape and honeycomb appearance which resembles lamp tripe(Du Xi Hui et al., 2014). The fruit bodies of Morchella are highly valued and well known in the world for its succulent meat, delicious taste and medical function. (Royse & May, 1990; Dai Yucheng and Yang Zhu Liang, 2008).
据报道，中国分布有30种羊肚菌（杜习慧等，2014）。新疆特殊的地理环境孕育了丰富的野生羊肚菌资源，有关新疆野生羊肚菌属的物种构成，有的文献记载（卯晓岚，2000）有5种；有的文献（赵震宇，2001；兰进等，1999；谢占玲，2007）记载有6种。无论5种还是6种，都是依据子实体的外观形状、菌丝、子囊、子囊孢子及侧丝的形态特征（沈洪等，2007）界定的形态学物种。但因羊肚菌子实体在发育过程中形态特征常随环境条件和个体发育年龄发生一定的变化，导致出现同菌异名及同名异菌现象（Du et al.,2012）。
It is reported that there are 30 different species of Morchella in China(Du Xihui et al., 2014). The distinct geographical environment of Xinjiang bred a rich resource of wild morels.. Five or six species have been reported in some literatures, and the species number of Morchella were categorized according to the shape and morphological characteristics of fruiting body, hyphae, ascus, ascospore and paraphyses (Shen Hong et al., 2007). However, the morphological characteristics of the fruit bodies often change with the environmental conditions and ontogenetic development, resulting in the strains with different names or different strains of the same name (Du et al., 2012).
近年来，分子生物学技术的飞速发展带领了真菌分类和系统发育研究的革命性变革（Yang ZL，et al., 2011; 杨祝良，2013），引领食用菌相关研究方法的推陈出新，适用于不同分类阶元的基因、内含子和基因间隔区序列不断被开发和选用，通过基于多个基因片段的联合矩阵构建系统发育树，进而进行物种界定的多基因联合分析技术应运而生（Taylor et al.,2000）。这种多基因联合分析的有效性为羊肚菌属的系统分类研究提供了新的思路（Taskin et al., 2010,2012; O′ Donnel et al., 2011）。
In recent years, the rapid development of molecular biology techniques brought revolutionary changes to fungus classification and phylogenetic studies( Yang ZL, et al, 2011;. Yang Zhu Liang, 2013) and inspired new thoughts to the research methods of edible fungus.The selection of genes, introns and intergenic region sequence used for different taxa; phylogenetic tree construction by joint matrix construction system based on multi-gene fragments; the introduce of multi-gene joint analysis technology for defining species (Taylor et al ., 2000) were developed. The joint analysis of multile-genes provides a new insight into the classification study of Morchella( Taskin et al, 2010,2012;.. O 'Donnel et al, 2011).
杜习慧等采用ITS+ef1-a+rpb1+rpb2+LSU多基因联合分析法对我国及北半球其他地区的羊肚菌样品进行了分子系统学研究，发现羊肚菌属至少有61个系统发育种，共分为3个支系，识别出11个新物种，加深了对中国羊肚菌物种多样性的认识。相对于ITS单一基因对羊肚菌属的分类鉴别方法，多基因联合分析法能够更明确地确定羊肚菌属种的分类地位，具有明显的优势。
Du Xi-hui et al conducted a molecular systematic studies on the morel specimens from China and other Northern Hemispheric regions using the joint analysis of multiple-genes of ITS + ef1-a + rpb1 + rpb2 + LSU. The study found that there were at least 61 Morchella phylogenetic species divided into three branches, and 11 of them are newly identified. The understanding of Chinese morel generic diversity was deepened. Compared to ITS single gene identification method, the joint analysis of multiple-genes shows significant advantages in defining taxonomic status
有关新疆野生羊肚菌属的分类研究也逐渐由传统的形态学分类研究发展到DNA序列分析研究阶段（刘伟，2009；冯丽，2009；苏俊，2011），为新疆野生羊肚菌的正确分类揭开了崭新的一页。本研究以2011-2013年间自新疆野生羊肚菌主产区伊犁、乌鲁木齐、石河子、阿勒泰和塔城地区采集到能够代表新疆野生羊肚菌物种多样性的野生羊肚菌为材料，提取子实体基因组DNA，采用ITS+ef1-a+rpb1+rpb2进行多基因联合分析并结合形态学特征，进一步探讨羊肚菌的物种多样性，旨在较为准确地确定其科学分类地位，进而为新疆野生羊肚菌人工栽培及进一步的开发利用提供科学依据。
The classification study of Xinjiang wild Morchella has gradually developed from the traditional morphological research method to DNA sequence analysis (Liu Wei, 2009; Feng Li, 2009; Su Jun, 2011), a significant step towards the correct taxonomy of Xinjiang wild morels. The specimens of this study were selected from the main appellations in Xinjiang like Yili, Urumqi, Shihezi, Altay and Tacheng during 2011-2013, and those wild morels demonstrate the species diversity of the fungus. In order to identify the taxonomic status more accurately and provide scientific basis for artificial cultivation, fruiting body genomic DNA was extracted for further investigation of the genetic diversity of Xinjiang wild Morchella using iTS + ef1-a + rpb1 + rpb2 multiple-genes joint analysis combined with morphology studies.
1材料与方法Materials and Methods
1.1 材料与仪器 Materials and equipment
野生羊肚菌子实体：23份形态特征各异且能够代表新疆野生羊肚菌物种多样性的样品分别于2011-2013年采自新疆伊犁、乌鲁木齐、石河子、阿勒泰和塔城地区，样品编号、来源、形态学分类鉴定结果见表1。
Wild morel fruit bodies: 23 morphologically distinct specimens representing the genetic diversity of Xinjiang wild morel were collected from Yili, Urumqi, Shihezi, Altay and Tacheng during 2011-2013. Sample number, origin and morphology classification result are shown in Table 1.
酶和试剂：2×Easy Taq Mix、液氮、2×CTAB DNA提取液、裂解液购自北京康为世纪生物公司；SanPrep柱式DNA胶回收试剂盒、蛋白酶K溶液（20 mg/ml）、β-巯基乙醇（100 mg/mL）、RNA酶（10 mg/ml）购自上海生工生物工程有限公司；70%乙醇及其他试剂均为国产或进口分析纯试剂。
Enzymes and reagents: 2 × Easy Taq Mix, liquid nitrogen, 2 × CTAB DNA extract, lysate from Beijing ComWin Biotech Co.,Ltd, SanPrep column DNA gel extraction kit, proteinase K solution (20 mg / ml), β- mercaptoethanol (100 mg / mL), RNA enzyme (10 mg / ml) from Sangon Biotech (Shanghai) Co.,Ltd, 70% ethanol and other analytical reagents.
主要仪器设备：组织破碎仪（德国莱弛 MM400 用于样品的破碎）；水浴锅（上海精宏 DK-S24）；涡旋振荡器（德国IKA MS1 minishaker 用于样品的混匀）；冷冻离心机（德国Sigma Z-16KC 用于DNA提取时冷冻离心）；PCR仪（BIO-RAD Thermal cycle 用于DNA扩增）；电泳仪凝胶成像系统 美国Bio-Rad T2A 用于电泳凝胶图像的分析）。
Main equipment: Bullet Blender (German RETSCH MM400); Water Bath (Shanghai JING HONG DK-S24); Vortex (German IKA MS1 minishaker for sample mixing); Refrigerated Centrifuge (German Sigma Z-16KC for DNA extraction centrifuge); PCR (BIO-RAD Thermal cycle for DNA amplification); electrophoresis gel imaging system(United States Bio-Rad T2A for gel electrophoresis image analysis) .
1.2实验方法 Method
1.2.1形态学分类Morphology classification
根据Bunyard（1996）的分类方法对23份供试样品进行分类。
23 specimens were classified according to the Bunyard (1996) classification method.
1.2.1野生羊肚菌子实体基因组DNA提取及检测 Genome DNA extraction and detection of the wild morel fruiting body
基因组DNA的提取参照Du et al.(2012)的方法并加以改进：分别称取0.12~0.15 g野生羊肚菌子实体样品于2.0 mL离心管中，于液氮中冷冻2 min，置于组织破碎仪中研磨45 s 至粉末状，加入1000 µL 2×CTAB DNA提取缓冲液、β-巯基乙醇20 µL，蛋白酶K 10 µL，涡旋混匀，冰浴保存10 min，然后4℃，12000r/min离心10 min；弃上清，沉淀中加2×CTAB裂解液（65℃预热）500 µL，β-巯基乙醇20 µL，蛋白酶K10 µL，混匀后置65℃水浴抽提30 min，每10 min翻转一次，以保证充分裂解；冷却，加入500 µL 氯仿：异戊醇（24:1）混合液，12000r/min离心10 min；取上清，冷却，加入500 µL 氯仿：异戊醇（24:1）混合液，12000r/min离心10 min；取上清，加入0.6倍体积预冷的异丙醇，混匀，静置10 min，12000r/min离心10 min；弃上清，沉淀用75%的乙醇洗涤两次，10000r/min离心10 min；弃上清，通风干燥，40 µL ddH2O溶解DNA，加入3 µL RNA酶，37℃水浴消化30 min后用1.0%的琼脂糖凝胶电泳检测。以ddH2O将获得的DNA稀释10倍备用。
Genome DNA extraction referred to and improved from Du et al (2012): Weigh 0.12 ~ 0.15 g of wild morel fruiting body specimens in 2.0 mL centrifuge tubes, and froze them in liquid nitrogen for 2 min. Blend in the bullet blender for 45 s until it become powder, and then add 1000 μL 2 × CTAB DNA extraction buffer, 20 μL β-mercaptoethanol, and 10 μL proteinase K to vortex and ice bath for 10 min. After that, centrifuge at 12000r / min for 10 min under 4 ℃, discard the supernatant and add 500 μL 2×CTAB lysis buffer (65 ℃ preheating), 20 μL β-mercaptoethanol, and 10 μL protease K to the precipitate. Mix thoroughly in the 65 ℃ water bath to extract for 30 min. Flip every 10 min in order to ensure thorough dissociation, and wait until it is cooled down. Add 500 μL of chloroform(isoamyl alcohol mixture 24: 1), and centrifuge at 12000r / min for 10 min. Extract the supernatant, cool down, and add 500 μL of chloroform(isoamyl alcohol mixture 24 : 1). Centrifuge at 12000r / min for 10 min, extract the supernatant and add 0.6×total volume of cold isopropanol. Mix and leave standing for 10 min. Centrifuge at 12000r / min for 10 min. Precipitate the supernatant with 75 % ethanol, wash twice, and centrifuge at 10000r / min for 10 min. Discard the supernatant, ventilation dry, and dissolve in 40 μL ddH2O DNA. Add 3 μL RNA enzyme, and water bath at 37 ℃ for 30 min after digestion with 1.0% agarose gel electrophoresis. Dilute DNA10-fold in ddH2O for later use.
取DNA样品5 µL，上样缓冲液（含0.25%溴酚蓝，40%蔗糖）2 µL，混匀，点入含0.75 µL核酸染料的0.8%琼脂糖凝胶中，同时点入λDNA/HindIII作为标记，用1×TAE缓冲液，在80 V电压下电泳40 min，在凝胶成像仪下观察并照相。
Prepare 5 μL DNA, mix with 2 μL loading buffer (0.25% bromophenol blue, 40% sucrose), and add to 0.8% agarose gel with 0.75 μL nucleic acid dye. Mark with λDNA / HindIII, use 1 × TAE buffer, electrophoresis at 80 V for 40 min, and observe and photograph in a gel imager.
1.2.2目的基因扩增及测序 Objective gene amplification and sequencing
用于多基因联合分析的目的基因包括ITS,ef1-a,rpb1,rpb2，引物（表2）由上海生工生物工程技术服务有限公司合成。
PCR扩增反应所使用的引物名称及碱基组成见表2。
Target genes for joint analysis of multiple-genes include ITS, ef1-a, rpb1, rpb2. All primers (Table 2) were synthesized by Shanghai Sangon Biotech Co.,Ltd.
Primers for PCR amplification are shown in Table 2.
以羊肚菌子实体基因组DNA为模板，进行ITS,ef1-a,rpb1,rpb2序列的PCR扩增，反应条件如下：
PCR扩增反应体系（20 µL）为：2×Easy Taq Mix 10 µL，ITS1（10 μM）0.8 µL，ITS4（10 μM）0.8 µL，模板DNA 0.6 µL，ddH2O 7.8 µL。
ITS序列PCR扩增反应程序为：95℃预变性4 min，95℃变性30 s，57℃复性30 s，72℃延伸60 s，31个循环后72℃延伸7 min结束反应。
ef1-a,rpb1,rpb2序列PCR扩增反应程序为：94℃预变性3 min，94℃变性60 s，50℃复性30 s，72℃延伸60 s，35个循环后72℃延伸10 min结束反应。
Morchella fruiting body genome DNA are used as templates for ITS, ef1-a, rpb1 and rpb2 gene sequence PCR. The reaction conditions were as follows:
PCR amplification system (20 μL): 2 × Easy Taq Mix 10 μL, ITS1 (10 μM) 0.8 μL, ITS4 (10 μM) 0.8 μL, template DNA 0.6 μL, ddH2O 7.8 μL.
ITS sequences PCR protocol: 95 ℃ pre-denaturation for 4 min, and denaturation at 95 ℃ for 30 s. 57 ℃ annealing for 30 s, and 31 cycles of 72 ℃ extension for 60 s followed by a 72 ℃ extension for 7 min to complete.
ef1-a, rpb1, rpb2 sequence PCR protocol: Pre-denaturation at 94 ℃ for 3 min and then 94 ℃ denaturation for 60 s. 50 ℃ annealing 30 s, and 35 cycles of 72 ℃ extension for 60 s followed by a 72 ℃ extension for another 10 min to complete.
PCR产物经1.0%琼脂糖凝胶电泳分离，用DNA凝胶回收试剂盒分别回收纯化目的片段，将扩增产物连接到pUCm-T载体上，转化大肠杆菌，经PCR鉴定后委托上海生工生物工程技术服务有限公司完成正反向双向测序，测序引物与PCR扩增引物相同。
Amplicons were electrophoresed in 1.0% agarose, and target DNA fragments were recovered and purified by gel purification kit. The products were connected to the pUCm-T vector and then transformed into E. coli for bi-directional sequencing done by Shanghai Sangon Biotech Co.,Ltd after PCR identification. Primers used for sequencing are same as the primers used for PCR amplification.
1.2.3序列分析与系统发育树的构建
Sequence analysis and phylogenetic tree construction
供试材料测序后分别利用DNAStar Lasergene.v7.1软件包中的Seqmen进行拼接处理，并对照测序色谱图仔细核查每一个碱基以确保所得序列的准确性，分别得到目的基因ITS,ef1-a,rpb1,rpb2片段全序列，保存为FASTA格式。将从GenBank中下载的序列、自测序列通过Bioedit Sequence Alignment Editor（Version 5.09）软件包中的ClustalW进行序列比对，并进行手工调整次序以保证对应碱基的同源性，数据中的空位“gap”作为缺失数据“missing data”处理。所有性状均视为无序和非加权。最后将比对好的序列通过Clustal X（Version 1.81）软件以Nexus文件格式输出。
After sequencing, assembly work was done by Seqmen in the DNAStar Lasergene.v7.1 software package, and every base was checked with chromatograms to ensure a correct sequence. The full sequence of target gene ITS,ef1-a,rpbl, and rpb2 were obtained and saved as FASTA format. Alignment between sequences downloaded from GenBank and the PCR sequences was completed by ClustalW in the Bioedit Sequence Alignment Editor (Version 5.09) software package, and manual adjustments were made to guarantee homology. Data "gap" were processed as "missing data". All characters were considered as unordered and non-weighted. Finally, sequences alignment using Clustal X (Version 1.81) software were exported as Nexus format.
皱盖钟菌作为外类群，利用PAUP4.0软件上进行最大简约性分析（Maximum parsimony analysis，MP）。分析方法按以下参数设置：依据最大简约性原则，将所有参与分析的性状作等权无序处理，空位“gap”作为缺失数据“missing data”处理；选用“collapse branches if minimum length is zero”（如果最小枝长为0则折叠分支）选项，采用启发式搜索（hueristic search）,1000次随机加入序列，（tree-bisectionreconnection,TBR）枝长交换（branch-swapping）构建系统进化树，靴带分析（boostrap）进行分支的可靠性评价，启发式搜索1000次重复取样。
The Maximum Parsimony Analysis (MP) were conducted using PAUP4.0 software towards the outgroup Verpa Bohemicax. Parameters are set as follows: according to the maximum parsimony principle, all traits were considered unordered and nonweighted, and data "gap" were processed as "missing data"; choose option "collapse branches if minimum length is zero" (branch fold if the minimum length is 0), use heuristic search, and add random sequence for 1000 times. Utilize branch length exchange (branch-swapping) for phylogenetic tree construction, and bootstrap analysis (boostrap) for branch reliability evaluation. Repeat heuristic sampling for1000-times.
2 结果与分析Results and Analysis
2.1形态学分类morphology classification
根据Bunyard（1996）的分类方法，按照子实体形态差异、色泽等特征将样品分为2个大类群，黑色羊肚菌类群和黄色羊肚菌类群。
According to Bunyard (1996) classification method, the specimens are divided into two big categories which are Elata Clade (black morels) and Esculenta Clade (yellow morels) based on their morphological differences in fruit bodies, colors and other characteristics.
2.2子实体基因组DNA提取及目的基因PCR扩增Fruiting body genome DNA extraction and target gene PCR
由于野生羊肚菌中多糖、胶质等次生代谢物含量较高，容易与DNA吸附，给基因组DNA的提取和纯化带来一定困难。研究中采用改进的CTAB法，电泳结果显示该法可以完成羊肚菌子实体基因组DNA的提取（图1 A B）。
The extraction and purification of fruiting body genome DNA were met with certain challenges because wild morels contain a higher content of secondary metabolites, such as polysaccharides and gelatin, which are easily absorbed to DNA. Therefore, this research adopted an improved CTAB method. The electrophoresis result showed that the genome DNA were successfully extracted (Fig. 1 A B).
分别以23株供试样品基因组DNA为模板，采用相应引物进行PCR扩增后均得到清晰的、与预期大小一致的特异性条带。其中，黑色羊肚菌的ITS序列范围为750-1000bp，黄色羊肚菌的ITS序列约1200bp(图2)，RPB1,RPB2片段条带大小约为800bp，EF1a片段约为1200bp(图3-5)。
The genomic DNA of the 23 samples was used as templates and primers were chosen respectively for PCR amplification. Clear bands were observed and they were consistent with the expected sizes. The range of black morel ITS sequences is 750-1000bp, Yellow morel ITS sequences is about 1200bp (Figure 2), RPB1, RPB2 about 800bp, EF1a about 1200bp (Figure 3-5 ).
2.3 序列分析与系统发育树的构建
Construction sequence analysis and phylogenetic tree construction
23份样品序列分别利用DNAStar Lasergene.v7.1软件包中的Seqmen进行拼接处理，并对照测序色谱图仔细核查每一个碱基以确保所得序列的准确性，分别得到EF1a,RPB1,RPB2片段。RPB1,RPB2片段条带大小约为800bp，EF1a片段约为1200bp，测序结果与PCR结果一致。将样品序列提交NCBI数据库，分别利用BLAST程序进行序列同源性比较。
Assembly work of the 23 specimens was conducted by Seqmen in the DNAStar Lasergene.v7.1 software package, and every base was checked with chromatograms to ensure a correct sequence. EF1a,RPB1,RPB2 were obtained respectively, among which RPB2 is about 800bp and EF1a about 1200bp. Sequencing result matches well with the PCR results. The sequence data were submitted to the NCBI database for homology analysis using the BLAST program.
将从GenBank中下载的序列、自测序列通过Bioedit Sequence Alignment Editor（Version 5.09）软件包中的ClustalW进行序列比对，手工调整并手动删除排列结果中5′ 和3′ 的非对位排列，数据中的空位“gap”作为缺失数据“missing data”处理。最后将比对好的序列通过Clustal X（Version 1.81）软件以Nexus文件格式输出。
Alignment between sequences downloaded from GenBank and the PCR sequences was completed by ClustalW in the Bioedit Sequence Alignment Editor (Version 5.09) software package. Manual adjustments were made to delete the 5′ and 3′ non-aligned position, and data vacancy ‘gap’ were dealt with as ‘missing data’. Final step is exporting sequences aligned by Clustal X (Version 1.81) software as Nexus format.
由于黄色羊肚菌和黑色羊肚菌的ITS序列具有高度差异性，如果将二者的序列矩阵合并为一个矩阵进行分析，容易导致很多信息位点被排除，因此将二者的联合序列数据分别进行分析。矩阵序列长度分别为3380bp和3087bp。联合矩阵序列通过PAUP4.0软件进行最大简约性分析（Maximum parsimony analysis）构建羊肚菌属系统发育树，如图5.4所示。
Aligned ITS of the yellow and black morels were analyzed separately due to high sequence difference. A lot of information positions could have been lost if the two sequence matrices were combined as one for analysis. The lengths of the matrix sequences were 3380bp and 3087bp. Maximum parsimony analysis was used for aligned matrix sequence to construct Morchella phylogenetic tree by PAUP4.0 software. The result was shown in Figure 5.4.
由系统发育树可以看出，以羊肚菌属基部类群M.rufobrunnea为外类群，20份黑色羊肚菌样品与来自NCBI数据库中的羊肚菌属物种聚类，M106、M111、M117、M15、M151、M188、M193、M58序列差异较小，亲缘关系较近，与Mel-13、Mel-26共同聚为第I类群（黑色羊肚菌）；M72、M176、M30、M38、M59、M74与Mel-19聚为一类（黑色羊肚菌）；M144、M197、M25、M68与Mel-33共同聚为一类（黑色羊肚菌）；M192与Mel-31(M.pulchella)聚为一类；样品M194以96%的Bootstrap值与Mel-2聚为一类（介于黑色羊肚菌和黄色羊肚菌之间）。由此可知，黑色羊肚菌类群主要由Mel-13、Mel-19、Mel-33、Mel-31和Mel-2共计5个物种构成。根据最新研究结果（杜习慧，2012），Mel-2为中国新纪录种。
Based on the phylogenetic tree, M.rufobrunnea were classified as an outgroup. 20 black morels and morels from the NCBI database have been clustered into different groups: M106, M111, M117, M15 , M151, M188, M193, M58 which are similar and genetically close were clustered together with Mel-13, Mel-26 into the first group (black morel); M72, M176, M30, M38, M59, M74 and Mel-19 in one group (black morel); M144, M197, M25, M68 and Mel-33 in one group (black morel); M192 and Mel-31 (M.pulchella) in another one group; M194, with 96% Bootstrap value, were classified with Mel-2 into the last group (between black and yellow morels). So far there are five groups of black morel as in Mel-13, Mel-19, Mel-33, Mel-31 and Mel-2. Besides, latest findings showed that Mel-2 is a new record species in China (Du Xi Hui, 2012),.
3份黄色羊肚菌与来自NCBI的黄色羊肚菌属其他物种聚类分析结果表明，样品M24、M62与Mes-6、Mes-7(M.prava)共同聚为一类，但与Mes-6亲缘关系较近；样品M1以95%的Bootstrap值与Mes-17(M.vulgaris)聚类在同一类群（图5.5）。根据最新研究结果（杜习慧，2012），Mes-17为中国新记录种。
The cluster analysis of 3 yellow morel specimens and morels from NCBI database showed that the sample M24, M62 and Mes-6, Mes-7 (M.prava) were classified into one group, but closer to Mes-6; M1, with 95% Bootstrap value, went into the same group as Mes-17 (M.vulgaris) (Figure 5.5). Shown by the lastest research, Mes-17 was also recorded as a new species in China(Du Xi Hui, 2012).
综合黑色羊肚菌、黄色羊肚菌聚类分析结果可知，新疆上述五地区羊肚菌属共有7种物种组成，包括黑色羊肚菌5个物种（Mel-13、Mel-19、Mel-33、Mel-31和Mel-2），黄色羊肚菌2个物种（Mes-6和Mes-17）。其中，黑色羊肚菌类群中Mel-2，黄色羊肚菌类群中Mes-17分别为中国新记录种。Two cluster analysis above indicate that, in the five regions of Xinjiang, there are altogether 7 morel species including five black morels (Mel-13, Mel-19, Mel-33, Mel-3 and Mel-2), and two yellow morels (Mes-6 and Mes-17). The black morel Mel-2 and the yellow morel Mes-17 are two new record species in China. .
3.讨论 Discussion
野生羊肚菌发育过程中形态特征随环境条件和个体发育年龄发生一定程度的变化，根据传统的分类方法很难对其进行准确的分类鉴定。鉴于此，本研究采用ITS+ RPB1+RPB2+EF1a四个基因片段进行多基因联合分析，更准确、全面地对新疆野生羊肚菌归属进行了分析，证明，新疆上述五地区羊肚菌属共有7个物种组成，包括黑色羊肚菌5个物种，黄色羊肚菌2个物种。黑色羊肚菌物种构成为Mel-19,Mel-31,Mel-13, Mel-33和Mel-2；黄色羊肚菌物种构成为Mes-17和Mes-6。构成野生羊肚菌类群的7个物种中，分布于新疆塔城地区的Mel-2及分布于新疆霍城县的Mes-17在中国尚未有报道，为中国新纪录种。该研究结果不仅丰富了新疆野生羊肚菌多样性的菌种资源库信息，还为野生羊肚菌的人工栽培及进一步开发利用提供科学依据。然而，新疆地大物博，物种多样性丰富，羊肚菌属的分类研究任重而道远。
Morphological characteristics of wild morels would experience certain changes with environmental conditions and development stages during the development process. Therefore, the present joint analysis of four genes(ITS + RPB1 + RPB2 + EF1)provides a more accurate and comprehensive analysis for Xinjiang wild morel classification. the results show that there are seven morel species in the five Xinjiang morel appellations, including five species of black morel and two species of yellow morel. The five black morel species are Mel-13, Mel-19, Mel-33, Mel-3 and Mel-2; two yellow morel species are Mes-6 and Mes-17. Among all seven wild morel species in Xinjiang, Mel-2 found in Tacheng area and Mes-17 grown in Xinjiang Huocheng area have never been reported before in China. These results not only enriched the genetic resources of Xinjiang wild morel, but also provide scientific basis for artificial cultivation and further utilization of the wild morels. However, due to the vast land and rich biodiversity in Xinjiang, the classification of wild Morchella is still a long-term undertaking.
新疆野生羊肚菌，尽管资源量在逐年减少，面临灭绝的局面，但从物种水平仍然维持了较高的物种多样性。仅5个采样区分布有7个羊肚菌物种，并且有两种是中国新纪录种。首先是新疆特殊的地理特征和生境特征为其物种多样性供提供了孕育的场所，其次，古老的历史起源、涵盖沿天山一带的广布性和大尺度的地理跨度是另一重要影响因素。
Although the amount of wild morels in Xinjiang on the decrease every year and the species face the problem of extinction, wild Morchella still maintain a high level of generic diversity. Five appellations can generate as much as seven distinct morel species and even contain two new ones. The first reason comes to the geographic features and the unique habitats which provide the breeding place for species diversity. Second, the long history of origin and the widespread geographical span covering Tianshan Mountains is another important factor.
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Translation - English 基金项目：国家自然科学基金资助项目（31160011）资助
Fund project: Sponsored by NSFC(National Science Fundation of China) 31160011
新疆野生羊肚菌系统发育分析
Phylogenetic Analysis on Xinjiang Wild Morchella
摘 要：为确定新疆野生羊肚菌的分类地位，根据子实体形态特征并结合ITS+ef1-a+rpb1+rpb2多基因联合分析技术，对根据形态特征挑选的、采自新疆伊犁、塔城、石河子等地的代表新疆野生羊肚菌物种多样性的23株野生羊肚菌进行多基因系统进化分析。多基因联合分析结果表明，新疆野生羊肚菌共有7种物种组成，包括黑色羊肚菌5个物种（Mel-13、Mel-19、Mel-33、Mel-31和Mel-2），黄色羊肚菌2个物种（Mes-6和Mes-17）。根据国内最新研究资料，其中2种为中国新纪录种，分别为Mes-17和Mel-2。
Abstract: 23 strains of wild true morels(Morchella) representing Morchella generic diversity in Xinjiang were selected from areas such as Xinjiang Ili, Tacheng, Shihezi and etc for their distinct morphological characteristics. Based on the morphology study of fruit bodies, a joint phylogenetic analysis multiple-genes of ITS+ef1-a+rpb1+rpb2 was carried out to determine the taxonomic status of Xinjiang wild morels. The results showed that there were seven species among the 23 strains of Xinjiang wild morel, including five species of Elata Clade (black morels): Mel-13, Mel-19, Mel-33, Mel-31 and Mel-2, and two species of Esculenta Clade (yellow morels): Mes-6 and Mes-17. According to the latest domestic research, Mes-17 and Mel-2 are new record species in China.
关键词：新疆，野生羊肚菌，系统发育分析，多基因联合
Keywords: Xinjiang, Wild Morchella, Phylogenetic Analysis, joint analysis of Multiple-Genes
羊肚菌属（Morchella Dill.ex Pers.: Fr.），属子囊菌门（Ascomycota），盘菌纲（Pezizomycetes），盘菌目(Pezizales)，羊肚菌科(Morchellaceae)（Hibbett et al., 2007）。因其表面呈蜂窝状，外形常为圆顶或尖顶，与羊肚颇为相似，故得名羊肚菌，其模式种是羊肚菌Morchella esculenta（杜习慧等，2014）。羊肚菌子实体肉质脆嫩，味道鲜美，是世界公认的珍贵、稀有食药用真菌（Royse & May, 1990; 戴玉成和杨祝良，2008）。
Morchella (Morchella Dill.ex Pers.: Fr.) is classified to Ascomycota phylum; Pezizomycetes class; Pezizales order; Morchellaceae family ( Hibbett et al. , 2007). Its Chinese name: lamp tripe fungus comes from its dome or apex shape and honeycomb appearance which resembles lamp tripe(Du Xi Hui et al., 2014). The fruit bodies of Morchella are highly valued and well known in the world for its succulent meat, delicious taste and medical function. (Royse & May, 1990; Dai Yucheng and Yang Zhu Liang, 2008).
据报道，中国分布有30种羊肚菌（杜习慧等，2014）。新疆特殊的地理环境孕育了丰富的野生羊肚菌资源，有关新疆野生羊肚菌属的物种构成，有的文献记载（卯晓岚，2000）有5种；有的文献（赵震宇，2001；兰进等，1999；谢占玲，2007）记载有6种。无论5种还是6种，都是依据子实体的外观形状、菌丝、子囊、子囊孢子及侧丝的形态特征（沈洪等，2007）界定的形态学物种。但因羊肚菌子实体在发育过程中形态特征常随环境条件和个体发育年龄发生一定的变化，导致出现同菌异名及同名异菌现象（Du et al.,2012）。
It is reported that there are 30 different species of Morchella in China(Du Xihui et al., 2014). The distinct geographical environment of Xinjiang bred a rich resource of wild morels.. Five or six species have been reported in some literatures, and the species number of Morchella were categorized according to the shape and morphological characteristics of fruiting body, hyphae, ascus, ascospore and paraphyses (Shen Hong et al., 2007). However, the morphological characteristics of the fruit bodies often change with the environmental conditions and ontogenetic development, resulting in the strains with different names or different strains of the same name (Du et al., 2012).
近年来，分子生物学技术的飞速发展带领了真菌分类和系统发育研究的革命性变革（Yang ZL，et al., 2011; 杨祝良，2013），引领食用菌相关研究方法的推陈出新，适用于不同分类阶元的基因、内含子和基因间隔区序列不断被开发和选用，通过基于多个基因片段的联合矩阵构建系统发育树，进而进行物种界定的多基因联合分析技术应运而生（Taylor et al.,2000）。这种多基因联合分析的有效性为羊肚菌属的系统分类研究提供了新的思路（Taskin et al., 2010,2012; O′ Donnel et al., 2011）。
In recent years, the rapid development of molecular biology techniques brought revolutionary changes to fungus classification and phylogenetic studies( Yang ZL, et al, 2011;. Yang Zhu Liang, 2013) and inspired new thoughts to the research methods of edible fungus.The selection of genes, introns and intergenic region sequence used for different taxa; phylogenetic tree construction by joint matrix construction system based on multi-gene fragments; the introduce of multi-gene joint analysis technology for defining species (Taylor et al ., 2000) were developed. The joint analysis of multile-genes provides a new insight into the classification study of Morchella( Taskin et al, 2010,2012;.. O 'Donnel et al, 2011).
杜习慧等采用ITS+ef1-a+rpb1+rpb2+LSU多基因联合分析法对我国及北半球其他地区的羊肚菌样品进行了分子系统学研究，发现羊肚菌属至少有61个系统发育种，共分为3个支系，识别出11个新物种，加深了对中国羊肚菌物种多样性的认识。相对于ITS单一基因对羊肚菌属的分类鉴别方法，多基因联合分析法能够更明确地确定羊肚菌属种的分类地位，具有明显的优势。
Du Xi-hui et al conducted a molecular systematic studies on the morel specimens from China and other Northern Hemispheric regions using the joint analysis of multiple-genes of ITS + ef1-a + rpb1 + rpb2 + LSU. The study found that there were at least 61 Morchella phylogenetic species divided into three branches, and 11 of them are newly identified. The understanding of Chinese morel generic diversity was deepened. Compared to ITS single gene identification method, the joint analysis of multiple-genes shows significant advantages in defining taxonomic status
有关新疆野生羊肚菌属的分类研究也逐渐由传统的形态学分类研究发展到DNA序列分析研究阶段（刘伟，2009；冯丽，2009；苏俊，2011），为新疆野生羊肚菌的正确分类揭开了崭新的一页。本研究以2011-2013年间自新疆野生羊肚菌主产区伊犁、乌鲁木齐、石河子、阿勒泰和塔城地区采集到能够代表新疆野生羊肚菌物种多样性的野生羊肚菌为材料，提取子实体基因组DNA，采用ITS+ef1-a+rpb1+rpb2进行多基因联合分析并结合形态学特征，进一步探讨羊肚菌的物种多样性，旨在较为准确地确定其科学分类地位，进而为新疆野生羊肚菌人工栽培及进一步的开发利用提供科学依据。
The classification study of Xinjiang wild Morchella has gradually developed from the traditional morphological research method to DNA sequence analysis (Liu Wei, 2009; Feng Li, 2009; Su Jun, 2011), a significant step towards the correct taxonomy of Xinjiang wild morels. The specimens of this study were selected from the main appellations in Xinjiang like Yili, Urumqi, Shihezi, Altay and Tacheng during 2011-2013, and those wild morels demonstrate the species diversity of the fungus. In order to identify the taxonomic status more accurately and provide scientific basis for artificial cultivation, fruiting body genomic DNA was extracted for further investigation of the genetic diversity of Xinjiang wild Morchella using iTS + ef1-a + rpb1 + rpb2 multiple-genes joint analysis combined with morphology studies.
1材料与方法Materials and Methods
1.1 材料与仪器 Materials and equipment
野生羊肚菌子实体：23份形态特征各异且能够代表新疆野生羊肚菌物种多样性的样品分别于2011-2013年采自新疆伊犁、乌鲁木齐、石河子、阿勒泰和塔城地区，样品编号、来源、形态学分类鉴定结果见表1。
Wild morel fruit bodies: 23 morphologically distinct specimens representing the genetic diversity of Xinjiang wild morel were collected from Yili, Urumqi, Shihezi, Altay and Tacheng during 2011-2013. Sample number, origin and morphology classification result are shown in Table 1.
酶和试剂：2×Easy Taq Mix、液氮、2×CTAB DNA提取液、裂解液购自北京康为世纪生物公司；SanPrep柱式DNA胶回收试剂盒、蛋白酶K溶液（20 mg/ml）、β-巯基乙醇（100 mg/mL）、RNA酶（10 mg/ml）购自上海生工生物工程有限公司；70%乙醇及其他试剂均为国产或进口分析纯试剂。
Enzymes and reagents: 2 × Easy Taq Mix, liquid nitrogen, 2 × CTAB DNA extract, lysate from Beijing ComWin Biotech Co.,Ltd, SanPrep column DNA gel extraction kit, proteinase K solution (20 mg / ml), β- mercaptoethanol (100 mg / mL), RNA enzyme (10 mg / ml) from Sangon Biotech (Shanghai) Co.,Ltd, 70% ethanol and other analytical reagents.
主要仪器设备：组织破碎仪（德国莱弛 MM400 用于样品的破碎）；水浴锅（上海精宏 DK-S24）；涡旋振荡器（德国IKA MS1 minishaker 用于样品的混匀）；冷冻离心机（德国Sigma Z-16KC 用于DNA提取时冷冻离心）；PCR仪（BIO-RAD Thermal cycle 用于DNA扩增）；电泳仪凝胶成像系统 美国Bio-Rad T2A 用于电泳凝胶图像的分析）。
Main equipment: Bullet Blender (German RETSCH MM400); Water Bath (Shanghai JING HONG DK-S24); Vortex (German IKA MS1 minishaker for sample mixing); Refrigerated Centrifuge (German Sigma Z-16KC for DNA extraction centrifuge); PCR (BIO-RAD Thermal cycle for DNA amplification); electrophoresis gel imaging system(United States Bio-Rad T2A for gel electrophoresis image analysis) .
1.2实验方法 Method
1.2.1形态学分类Morphology classification
根据Bunyard（1996）的分类方法对23份供试样品进行分类。
23 specimens were classified according to the Bunyard (1996) classification method.
1.2.1野生羊肚菌子实体基因组DNA提取及检测 Genome DNA extraction and detection of the wild morel fruiting body
基因组DNA的提取参照Du et al.(2012)的方法并加以改进：分别称取0.12~0.15 g野生羊肚菌子实体样品于2.0 mL离心管中，于液氮中冷冻2 min，置于组织破碎仪中研磨45 s 至粉末状，加入1000 µL 2×CTAB DNA提取缓冲液、β-巯基乙醇20 µL，蛋白酶K 10 µL，涡旋混匀，冰浴保存10 min，然后4℃，12000r/min离心10 min；弃上清，沉淀中加2×CTAB裂解液（65℃预热）500 µL，β-巯基乙醇20 µL，蛋白酶K10 µL，混匀后置65℃水浴抽提30 min，每10 min翻转一次，以保证充分裂解；冷却，加入500 µL 氯仿：异戊醇（24:1）混合液，12000r/min离心10 min；取上清，冷却，加入500 µL 氯仿：异戊醇（24:1）混合液，12000r/min离心10 min；取上清，加入0.6倍体积预冷的异丙醇，混匀，静置10 min，12000r/min离心10 min；弃上清，沉淀用75%的乙醇洗涤两次，10000r/min离心10 min；弃上清，通风干燥，40 µL ddH2O溶解DNA，加入3 µL RNA酶，37℃水浴消化30 min后用1.0%的琼脂糖凝胶电泳检测。以ddH2O将获得的DNA稀释10倍备用。
Genome DNA extraction referred to and improved from Du et al (2012): Weigh 0.12 ~ 0.15 g of wild morel fruiting body specimens in 2.0 mL centrifuge tubes, and froze them in liquid nitrogen for 2 min. Blend in the bullet blender for 45 s until it become powder, and then add 1000 μL 2 × CTAB DNA extraction buffer, 20 μL β-mercaptoethanol, and 10 μL proteinase K to vortex and ice bath for 10 min. After that, centrifuge at 12000r / min for 10 min under 4 ℃, discard the supernatant and add 500 μL 2×CTAB lysis buffer (65 ℃ preheating), 20 μL β-mercaptoethanol, and 10 μL protease K to the precipitate. Mix thoroughly in the 65 ℃ water bath to extract for 30 min. Flip every 10 min in order to ensure thorough dissociation, and wait until it is cooled down. Add 500 μL of chloroform(isoamyl alcohol mixture 24: 1), and centrifuge at 12000r / min for 10 min. Extract the supernatant, cool down, and add 500 μL of chloroform(isoamyl alcohol mixture 24 : 1). Centrifuge at 12000r / min for 10 min, extract the supernatant and add 0.6×total volume of cold isopropanol. Mix and leave standing for 10 min. Centrifuge at 12000r / min for 10 min. Precipitate the supernatant with 75 % ethanol, wash twice, and centrifuge at 10000r / min for 10 min. Discard the supernatant, ventilation dry, and dissolve in 40 μL ddH2O DNA. Add 3 μL RNA enzyme, and water bath at 37 ℃ for 30 min after digestion with 1.0% agarose gel electrophoresis. Dilute DNA10-fold in ddH2O for later use.
取DNA样品5 µL，上样缓冲液（含0.25%溴酚蓝，40%蔗糖）2 µL，混匀，点入含0.75 µL核酸染料的0.8%琼脂糖凝胶中，同时点入λDNA/HindIII作为标记，用1×TAE缓冲液，在80 V电压下电泳40 min，在凝胶成像仪下观察并照相。
Prepare 5 μL DNA, mix with 2 μL loading buffer (0.25% bromophenol blue, 40% sucrose), and add to 0.8% agarose gel with 0.75 μL nucleic acid dye. Mark with λDNA / HindIII, use 1 × TAE buffer, electrophoresis at 80 V for 40 min, and observe and photograph in a gel imager.
1.2.2目的基因扩增及测序 Objective gene amplification and sequencing
用于多基因联合分析的目的基因包括ITS,ef1-a,rpb1,rpb2，引物（表2）由上海生工生物工程技术服务有限公司合成。
PCR扩增反应所使用的引物名称及碱基组成见表2。
Target genes for joint analysis of multiple-genes include ITS, ef1-a, rpb1, rpb2. All primers (Table 2) were synthesized by Shanghai Sangon Biotech Co.,Ltd.
Primers for PCR amplification are shown in Table 2.
以羊肚菌子实体基因组DNA为模板，进行ITS,ef1-a,rpb1,rpb2序列的PCR扩增，反应条件如下：
PCR扩增反应体系（20 µL）为：2×Easy Taq Mix 10 µL，ITS1（10 μM）0.8 µL，ITS4（10 μM）0.8 µL，模板DNA 0.6 µL，ddH2O 7.8 µL。
ITS序列PCR扩增反应程序为：95℃预变性4 min，95℃变性30 s，57℃复性30 s，72℃延伸60 s，31个循环后72℃延伸7 min结束反应。
ef1-a,rpb1,rpb2序列PCR扩增反应程序为：94℃预变性3 min，94℃变性60 s，50℃复性30 s，72℃延伸60 s，35个循环后72℃延伸10 min结束反应。
Morchella fruiting body genome DNA are used as templates for ITS, ef1-a, rpb1 and rpb2 gene sequence PCR. The reaction conditions were as follows:
PCR amplification system (20 μL): 2 × Easy Taq Mix 10 μL, ITS1 (10 μM) 0.8 μL, ITS4 (10 μM) 0.8 μL, template DNA 0.6 μL, ddH2O 7.8 μL.
ITS sequences PCR protocol: 95 ℃ pre-denaturation for 4 min, and denaturation at 95 ℃ for 30 s. 57 ℃ annealing for 30 s, and 31 cycles of 72 ℃ extension for 60 s followed by a 72 ℃ extension for 7 min to complete.
ef1-a, rpb1, rpb2 sequence PCR protocol: Pre-denaturation at 94 ℃ for 3 min and then 94 ℃ denaturation for 60 s. 50 ℃ annealing 30 s, and 35 cycles of 72 ℃ extension for 60 s followed by a 72 ℃ extension for another 10 min to complete.
PCR产物经1.0%琼脂糖凝胶电泳分离，用DNA凝胶回收试剂盒分别回收纯化目的片段，将扩增产物连接到pUCm-T载体上，转化大肠杆菌，经PCR鉴定后委托上海生工生物工程技术服务有限公司完成正反向双向测序，测序引物与PCR扩增引物相同。
Amplicons were electrophoresed in 1.0% agarose, and target DNA fragments were recovered and purified by gel purification kit. The products were connected to the pUCm-T vector and then transformed into E. coli for bi-directional sequencing done by Shanghai Sangon Biotech Co.,Ltd after PCR identification. Primers used for sequencing are same as the primers used for PCR amplification.
1.2.3序列分析与系统发育树的构建
Sequence analysis and phylogenetic tree construction
供试材料测序后分别利用DNAStar Lasergene.v7.1软件包中的Seqmen进行拼接处理，并对照测序色谱图仔细核查每一个碱基以确保所得序列的准确性，分别得到目的基因ITS,ef1-a,rpb1,rpb2片段全序列，保存为FASTA格式。将从GenBank中下载的序列、自测序列通过Bioedit Sequence Alignment Editor（Version 5.09）软件包中的ClustalW进行序列比对，并进行手工调整次序以保证对应碱基的同源性，数据中的空位“gap”作为缺失数据“missing data”处理。所有性状均视为无序和非加权。最后将比对好的序列通过Clustal X（Version 1.81）软件以Nexus文件格式输出。
After sequencing, assembly work was done by Seqmen in the DNAStar Lasergene.v7.1 software package, and every base was checked with chromatograms to ensure a correct sequence. The full sequence of target gene ITS,ef1-a,rpbl, and rpb2 were obtained and saved as FASTA format. Alignment between sequences downloaded from GenBank and the PCR sequences was completed by ClustalW in the Bioedit Sequence Alignment Editor (Version 5.09) software package, and manual adjustments were made to guarantee homology. Data "gap" were processed as "missing data". All characters were considered as unordered and non-weighted. Finally, sequences alignment using Clustal X (Version 1.81) software were exported as Nexus format.
皱盖钟菌作为外类群，利用PAUP4.0软件上进行最大简约性分析（Maximum parsimony analysis，MP）。分析方法按以下参数设置：依据最大简约性原则，将所有参与分析的性状作等权无序处理，空位“gap”作为缺失数据“missing data”处理；选用“collapse branches if minimum length is zero”（如果最小枝长为0则折叠分支）选项，采用启发式搜索（hueristic search）,1000次随机加入序列，（tree-bisectionreconnection,TBR）枝长交换（branch-swapping）构建系统进化树，靴带分析（boostrap）进行分支的可靠性评价，启发式搜索1000次重复取样。
The Maximum Parsimony Analysis (MP) were conducted using PAUP4.0 software towards the outgroup Verpa Bohemicax. Parameters are set as follows: according to the maximum parsimony principle, all traits were considered unordered and nonweighted, and data "gap" were processed as "missing data"; choose option "collapse branches if minimum length is zero" (branch fold if the minimum length is 0), use heuristic search, and add random sequence for 1000 times. Utilize branch length exchange (branch-swapping) for phylogenetic tree construction, and bootstrap analysis (boostrap) for branch reliability evaluation. Repeat heuristic sampling for1000-times.
2 结果与分析Results and Analysis
2.1形态学分类morphology classification
根据Bunyard（1996）的分类方法，按照子实体形态差异、色泽等特征将样品分为2个大类群，黑色羊肚菌类群和黄色羊肚菌类群。
According to Bunyard (1996) classification method, the specimens are divided into two big categories which are Elata Clade (black morels) and Esculenta Clade (yellow morels) based on their morphological differences in fruit bodies, colors and other characteristics.
2.2子实体基因组DNA提取及目的基因PCR扩增Fruiting body genome DNA extraction and target gene PCR
由于野生羊肚菌中多糖、胶质等次生代谢物含量较高，容易与DNA吸附，给基因组DNA的提取和纯化带来一定困难。研究中采用改进的CTAB法，电泳结果显示该法可以完成羊肚菌子实体基因组DNA的提取（图1 A B）。
The extraction and purification of fruiting body genome DNA were met with certain challenges because wild morels contain a higher content of secondary metabolites, such as polysaccharides and gelatin, which are easily absorbed to DNA. Therefore, this research adopted an improved CTAB method. The electrophoresis result showed that the genome DNA were successfully extracted (Fig. 1 A B).
分别以23株供试样品基因组DNA为模板，采用相应引物进行PCR扩增后均得到清晰的、与预期大小一致的特异性条带。其中，黑色羊肚菌的ITS序列范围为750-1000bp，黄色羊肚菌的ITS序列约1200bp(图2)，RPB1,RPB2片段条带大小约为800bp，EF1a片段约为1200bp(图3-5)。
The genomic DNA of the 23 samples was used as templates and primers were chosen respectively for PCR amplification. Clear bands were observed and they were consistent with the expected sizes. The range of black morel ITS sequences is 750-1000bp, Yellow morel ITS sequences is about 1200bp (Figure 2), RPB1, RPB2 about 800bp, EF1a about 1200bp (Figure 3-5 ).
2.3 序列分析与系统发育树的构建
Construction sequence analysis and phylogenetic tree construction
23份样品序列分别利用DNAStar Lasergene.v7.1软件包中的Seqmen进行拼接处理，并对照测序色谱图仔细核查每一个碱基以确保所得序列的准确性，分别得到EF1a,RPB1,RPB2片段。RPB1,RPB2片段条带大小约为800bp，EF1a片段约为1200bp，测序结果与PCR结果一致。将样品序列提交NCBI数据库，分别利用BLAST程序进行序列同源性比较。
Assembly work of the 23 specimens was conducted by Seqmen in the DNAStar Lasergene.v7.1 software package, and every base was checked with chromatograms to ensure a correct sequence. EF1a,RPB1,RPB2 were obtained respectively, among which RPB2 is about 800bp and EF1a about 1200bp. Sequencing result matches well with the PCR results. The sequence data were submitted to the NCBI database for homology analysis using the BLAST program.
将从GenBank中下载的序列、自测序列通过Bioedit Sequence Alignment Editor（Version 5.09）软件包中的ClustalW进行序列比对，手工调整并手动删除排列结果中5′ 和3′ 的非对位排列，数据中的空位“gap”作为缺失数据“missing data”处理。最后将比对好的序列通过Clustal X（Version 1.81）软件以Nexus文件格式输出。
Alignment between sequences downloaded from GenBank and the PCR sequences was completed by ClustalW in the Bioedit Sequence Alignment Editor (Version 5.09) software package. Manual adjustments were made to delete the 5′ and 3′ non-aligned position, and data vacancy ‘gap’ were dealt with as ‘missing data’. Final step is exporting sequences aligned by Clustal X (Version 1.81) software as Nexus format.
由于黄色羊肚菌和黑色羊肚菌的ITS序列具有高度差异性，如果将二者的序列矩阵合并为一个矩阵进行分析，容易导致很多信息位点被排除，因此将二者的联合序列数据分别进行分析。矩阵序列长度分别为3380bp和3087bp。联合矩阵序列通过PAUP4.0软件进行最大简约性分析（Maximum parsimony analysis）构建羊肚菌属系统发育树，如图5.4所示。
Aligned ITS of the yellow and black morels were analyzed separately due to high sequence difference. A lot of information positions could have been lost if the two sequence matrices were combined as one for analysis. The lengths of the matrix sequences were 3380bp and 3087bp. Maximum parsimony analysis was used for aligned matrix sequence to construct Morchella phylogenetic tree by PAUP4.0 software. The result was shown in Figure 5.4.
由系统发育树可以看出，以羊肚菌属基部类群M.rufobrunnea为外类群，20份黑色羊肚菌样品与来自NCBI数据库中的羊肚菌属物种聚类，M106、M111、M117、M15、M151、M188、M193、M58序列差异较小，亲缘关系较近，与Mel-13、Mel-26共同聚为第I类群（黑色羊肚菌）；M72、M176、M30、M38、M59、M74与Mel-19聚为一类（黑色羊肚菌）；M144、M197、M25、M68与Mel-33共同聚为一类（黑色羊肚菌）；M192与Mel-31(M.pulchella)聚为一类；样品M194以96%的Bootstrap值与Mel-2聚为一类（介于黑色羊肚菌和黄色羊肚菌之间）。由此可知，黑色羊肚菌类群主要由Mel-13、Mel-19、Mel-33、Mel-31和Mel-2共计5个物种构成。根据最新研究结果（杜习慧，2012），Mel-2为中国新纪录种。
Based on the phylogenetic tree, M.rufobrunnea were classified as an outgroup. 20 black morels and morels from the NCBI database have been clustered into different groups: M106, M111, M117, M15 , M151, M188, M193, M58 which are similar and genetically close were clustered together with Mel-13, Mel-26 into the first group (black morel); M72, M176, M30, M38, M59, M74 and Mel-19 in one group (black morel); M144, M197, M25, M68 and Mel-33 in one group (black morel); M192 and Mel-31 (M.pulchella) in another one group; M194, with 96% Bootstrap value, were classified with Mel-2 into the last group (between black and yellow morels). So far there are five groups of black morel as in Mel-13, Mel-19, Mel-33, Mel-31 and Mel-2. Besides, latest findings showed that Mel-2 is a new record species in China (Du Xi Hui, 2012),.
3份黄色羊肚菌与来自NCBI的黄色羊肚菌属其他物种聚类分析结果表明，样品M24、M62与Mes-6、Mes-7(M.prava)共同聚为一类，但与Mes-6亲缘关系较近；样品M1以95%的Bootstrap值与Mes-17(M.vulgaris)聚类在同一类群（图5.5）。根据最新研究结果（杜习慧，2012），Mes-17为中国新记录种。
The cluster analysis of 3 yellow morel specimens and morels from NCBI database showed that the sample M24, M62 and Mes-6, Mes-7 (M.prava) were classified into one group, but closer to Mes-6; M1, with 95% Bootstrap value, went into the same group as Mes-17 (M.vulgaris) (Figure 5.5). Shown by the lastest research, Mes-17 was also recorded as a new species in China(Du Xi Hui, 2012).
综合黑色羊肚菌、黄色羊肚菌聚类分析结果可知，新疆上述五地区羊肚菌属共有7种物种组成，包括黑色羊肚菌5个物种（Mel-13、Mel-19、Mel-33、Mel-31和Mel-2），黄色羊肚菌2个物种（Mes-6和Mes-17）。其中，黑色羊肚菌类群中Mel-2，黄色羊肚菌类群中Mes-17分别为中国新记录种。Two cluster analysis above indicate that, in the five regions of Xinjiang, there are altogether 7 morel species including five black morels (Mel-13, Mel-19, Mel-33, Mel-3 and Mel-2), and two yellow morels (Mes-6 and Mes-17). The black morel Mel-2 and the yellow morel Mes-17 are two new record species in China. .
3.讨论 Discussion
野生羊肚菌发育过程中形态特征随环境条件和个体发育年龄发生一定程度的变化，根据传统的分类方法很难对其进行准确的分类鉴定。鉴于此，本研究采用ITS+ RPB1+RPB2+EF1a四个基因片段进行多基因联合分析，更准确、全面地对新疆野生羊肚菌归属进行了分析，证明，新疆上述五地区羊肚菌属共有7个物种组成，包括黑色羊肚菌5个物种，黄色羊肚菌2个物种。黑色羊肚菌物种构成为Mel-19,Mel-31,Mel-13, Mel-33和Mel-2；黄色羊肚菌物种构成为Mes-17和Mes-6。构成野生羊肚菌类群的7个物种中，分布于新疆塔城地区的Mel-2及分布于新疆霍城县的Mes-17在中国尚未有报道，为中国新纪录种。该研究结果不仅丰富了新疆野生羊肚菌多样性的菌种资源库信息，还为野生羊肚菌的人工栽培及进一步开发利用提供科学依据。然而，新疆地大物博，物种多样性丰富，羊肚菌属的分类研究任重而道远。
Morphological characteristics of wild morels would experience certain changes with environmental conditions and development stages during the development process. Therefore, the present joint analysis of four genes(ITS + RPB1 + RPB2 + EF1)provides a more accurate and comprehensive analysis for Xinjiang wild morel classification. the results show that there are seven morel species in the five Xinjiang morel appellations, including five species of black morel and two species of yellow morel. The five black morel species are Mel-13, Mel-19, Mel-33, Mel-3 and Mel-2; two yellow morel species are Mes-6 and Mes-17. Among all seven wild morel species in Xinjiang, Mel-2 found in Tacheng area and Mes-17 grown in Xinjiang Huocheng area have never been reported before in China. These results not only enriched the genetic resources of Xinjiang wild morel, but also provide scientific basis for artificial cultivation and further utilization of the wild morels. However, due to the vast land and rich biodiversity in Xinjiang, the classification of wild Morchella is still a long-term undertaking.
新疆野生羊肚菌，尽管资源量在逐年减少，面临灭绝的局面，但从物种水平仍然维持了较高的物种多样性。仅5个采样区分布有7个羊肚菌物种，并且有两种是中国新纪录种。首先是新疆特殊的地理特征和生境特征为其物种多样性供提供了孕育的场所，其次，古老的历史起源、涵盖沿天山一带的广布性和大尺度的地理跨度是另一重要影响因素。
Although the amount of wild morels in Xinjiang on the decrease every year and the species face the problem of extinction, wild Morchella still maintain a high level of generic diversity. Five appellations can generate as much as seven distinct morel species and even contain two new ones. The first reason comes to the geographic features and the unique habitats which provide the breeding place for species diversity. Second, the long history of origin and the widespread geographical span covering Tianshan Mountains is another important factor.
参考文献References
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Years of experience: 10. Registered at ProZ.com: Nov 2016.
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Peking University, School of Life Sciences, Bachelor Degree GPA: 3.5/4.0
ο Excellent Students Scholarship in 2013(15% of class);
Middlebury Institute of International Studies at Monterey, Master Degree GPA: 3.8/4.0
China Association Tests for Translators and Interpreters （CATTI） level II interpreter
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TA 2015.9-now Translation Theories and Practice／Medical Interpretation Teaching assistant
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Career Fresher Media Company Editor & BD
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ο Translation and liaison interpretation
Interpretation for the 10th China-US Conference on Medical Professionalism
Interpretation for the Swiss Ambassador
Simultaneous interpretation for 2016 Forbidden City Pharmacist Forum
Translated a medical thesis on professional-patient relationships.
Part time translation for China Family Newspaper
Part time translation & interpretation for Quality Resource International
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Language Native Chinese; Near native English; Intermediate Low French.
Professional Training Translation & interpretation II at the New Oriental (Xindongfang) Institution 140 hours
Consecutive & Simultaneous Interpretation at the For You Global Institution 100 hours
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