Habitat utilization and recommendations for conservation of Indo-Pacific humpback dolphin along northern Hong Kong-Zhuhai-Macao Bridge
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摘要:
为了解在港珠澳大桥建成后,中华白海豚 (Sousa chinensis) 对大桥水域北侧栖息地的利用情况,及其可能穿越大桥的潜在通道。在大桥北侧沿线水域布置了A、B、C、D 4个固定被动声学监测位点,总共进行了2 947 h的声学录音。在4个监测位点发现中华白海豚声学事件的概率分别为15.31% (A)、17.30% (B)、8.97% (C)、5.85% (D),表明大桥北侧沿线水域依然是中华白海豚的重要栖息地,且其活动存在时空差异。在不同监测位点发现的中华白海豚声学事件并无明显的昼夜差异。在桥梁结构的A、B和C 3个监测位点,低潮位阶段发现中华白海豚的概率分别为1.56%、1.82%和0.78%,均高于高潮位阶段 (0.94%、1.56%和0.26%);A和B点在涨潮和落潮阶段发现中华白海豚的概率相近,但C和D点在涨潮阶段的发现概率分别为4.55%和3.64%,均高于落潮阶段 (3.12%和1.30%),表明潮汐变化对中华白海豚在桥梁结构水域的栖息地选择上有一定影响且存在空间差异。根据声学监测结果推测,A和B点之间的水域可能是中华白海豚穿越大桥的重要区域,建议加强对该区域的巡查和管理。
Abstract:To understand the utilization of the habitat in the waters along the Hong Kong-Zhuhai-Macao Bridge by Indo-Pacific humpback dolphin (Sousa chinensis) and its potential migration pathways to cross the bridge. We set four fixed passive acoustic monitoring points (A, B, C, D) along the northern waters of the Hong Kong-Zhuhai-Macao Bridge, and recorded a total of 2 947 hours of acoustic data in November 2020 and March 2021, respectively. The probabilities of detecting acoustic events in S. chinensis at four monitoring points are as follows: 15.31% (A), 17.30% (B), 8.97% (C), 5.85% (D), which indicates that the waters along the northern of the bridge is an important habitat for S. chinensis with spatiotemporal differences. There is no significant diurnal and nocturnal difference in the acoustic events of S. chinensis observed at different monitoring sites. The probabilities of detecting S. chinensis at low tide levels of monitoring sites A, B and C on bridge structures were 1.56%, 1.82% and 0.78%, respectively, all higher than those at high tide levels (0.94%, 1.56% and 0.26%). The probabilities of discovering S. chinensis during high tide and low tide stages at sites A and B were similar, but those during high tide stage at sites C and D were 4.55% and 3.64%, both higher than those during low tide stage (3.12% and 1.30%), indicating that tidal changes have a certain impact on the habitat selection of S. chinensis in bridge structure waters with spatial differences. The result also indicates that S. chinensis may prefer the area between sites A and B to cross the bridge than the other area. Inspection and management of its passageway should be strengthened.
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微卫星(microsatellite)DNA是由1~6个核苷酸为单位多次串联重复的简单序列,又称简短串联重复序列(short tandem repeat,STR)、简单序列重复(simple sequence repeat,SSR) 或简单序列长度多态性(simple sequence length polymorphism,SSLP),是20世纪80年代末发展起来的一种分子标记。微卫星具有高度的多态性[1]和共显性,在真核生物基因组中随机分布,每代变异超过2%[2]。其分析可以分辨一个碱基对的差异[3],且等位基因的条带易于识别和解释,比其它分子标记带来更多的信息量[4]。因此在种群遗传结构分析、种群遗传多样性检测、遗传图谱的构建及生产性状位点的连锁分析与QTL定位分析中得到了广泛的应用[5-7]。
由于不同种类之间的微卫星引物序列通用性较差。因此必须首先从实验生物基因组中获得微卫星DNA序列,设计引物,筛选多态性微卫星标记。然而筛选微卫星座位的工作繁重,其使用受到一定的限制。随着分子生物学技术的发展,相继产生了一些新的微卫星DNA分离方法。本文对几种微卫星位点分离技术进行介绍并对其进行分析比较,为选择适合的方法提供参考。
1. 微卫星位点的分离方法
1.1 生物信息学方法(Data mining)与近缘种交叉扩增(cross amplification)
获得微卫星最简捷的途径就是通过互连网从公共数据库(如EMBL、GenBank、EST数据库等)查找微卫星DNA,如河豚(Fugu rubripes)[8]、中国明对虾(Fenneropenaeus orientalis)[9]和栉孔扇贝(Chlamys farreri)[10]等。徐鹏等[9]利用生物信息学方法从10 446个中国明对虾ESTs序列中筛选微卫星DNA,共发现微卫星序列229个,占整个ESTs数据的2.19%,其中含双碱基重复序列146个和3碱基重复序列58个,分别占在ESTs微卫星序列总数的63.76%和25.33%,大部分为完美型(perfect)的重复序列。根据筛选的微卫星序列设计19对引物并进行多态性检测,在有扩增产物的16对引物中,首次筛选得到8个中国明对虾微卫星标记。其次,利用近缘种的已知微卫星引物进行交叉扩增(cross amplification)筛选也可获得一定的有效微卫星引物。鲁双庆等[11]研究了鲤(Cyprinus carpio)微卫星引物对远缘种黄鳝(Monopteras albus)的适用性。结果显示,31对鲤微卫星引物中有11对引物能对黄鳝DNA模板扩增出特异性带谱。每对引物扩增的等位基因数3~13个,平均每个位点5.6个,显示了较高的多态性。林凯东等[12]首次将鲤的微卫星引物用于草鱼(Ctenopharyngodon idellus)基因组分析。
1.2 小片段DNA克隆法(small DNA fragments cloning)
此方法的基本原理是构建目标生物基因组小片段DNA文库,通过杂交筛选出含有微卫星序列的阳性克隆。首先用限制性内切酶充分消化DNA,琼脂糖凝胶电泳,选取400~900 bp片段克隆,构建基因组文库。然后将重组克隆转移到杂交膜上,采用32P标记的重复序列探针如(AT)n进行杂交,放射自显影,获得阳性克隆、测序,设计PCR引物并进行扩增筛选(图 1)。
孙效文等[13]利用上述方法从鲤鱼文库中筛选2 000个菌落,获得阳性克隆45个,有22个含有微卫星,其中完美型的占63.6%,非完美型的占22.7%,混合型的占13.7%。陈微等[14]用此方法从牙鲆(Paralichthys olivaceus)文库中筛选阳性克隆45个,共得到20个微卫星序列,其中完美型13个,非完美型5个,混合型2个。PANIEGO等[15]也用此方法分离向日葵(Helianthus annnus)微卫星标记。测序503个微卫星克隆,设计了271对微卫星引物。
利用微卫星重复序列作探针筛选基因组文库是一种耗时费力的方法[16]。获得阳性克隆的比例通常较少(0.04%~12%之间)。对于那些基因组中微卫星DNA含量不是很丰富的种类(比如鸟类和植物),或需要大量的微卫星标记用于研究种群遗传结构[17-18]或构建遗传图谱[19]时,用此方法分离微卫星难以满足需要。
1.3 以RAPD为基础的PCR分离微卫星法(RAPD-based PIMA approach)
为了避免基因组文库的构建和筛选的繁琐性,一些国外学者先后报道用随机扩增多态DNA(RAPD)方法去扩增未知的微卫星序列[20-21]。其中用PCR分离微卫星(PCR isolation of microsatellite approach,PIMA)[22]就是用RAPD引物从目标生物基因组中获得随机扩增片段,这些扩增产物克隆到T-载体上,然后用含有重复序列引物和载体引物筛选阳性克隆、测序(图 1,PIMA支路)。RAPD片段比随机基因组克隆含有较多的微卫星重复序列[23-25]。与传统的方法相比,PIMA省略了DNA酶切、片段大小选择以及接头连接这些步骤,虽然实验操作简单但研究报道较少。
1.4 引物伸长法(Primer extension approach)
基因组DNA酶切后,选一定大小的片段插入到质粒(pBluescript)或噬菌体(M13mp18)载体上,然后转化、洗脱获得含有单链环状DNA(ssDNA)并构建文库。以ssDNA为模板,与含有重复序列的寡聚核苷酸或带有生物素标记的重复序列进行伸长反应,获得富含微卫星位点双链DNA的次级文库。最后Southern杂交或PCR筛选阳性克隆并测序(图 2)。
OSTRANDER等[26]和PAETKAU[27]运用引物伸长法分别对犬齿类和鲍鱼基因组文库进行实验,并分别获得40%~50%和最高达100%阳性克隆。OSTRANDER等构建了含60 000克隆的单链DNA文库,对于基因组频率低于1%的特定重复序列有600座位(含所需的重复序列)在富集文库中表现出来。并且该方法对分离富含2个寡核苷酸重复序列如(AC)n比较有效,对分离3个和4个寡核苷酸重复序列是否有效还不肯定。此方法实验步骤繁琐使其适用性受到一定限制。
1.5 选择性杂交法(selective hybridization method)
选择性杂交法第一步与传统方法一样,也是基因组DNA经过酶切后与接头或载体连接,用连有接头的DNA片段与标记的重复序列探针选择性杂交,捕获含有微卫星序列的DNA片段。最后PCR扩增、克隆。对重组克隆可直接测序、PCR筛选或Southern blot筛选(图 3)。
此方法实验步骤简单,报道较多[28-32]。其中酶切片段与接头或载体连接是非常重要的一步,因为连接插入片段的量不足和多联体的形成都会限制下一步的实验。酶切片段大小的选择可以在酶切后[30]或在连接后[31]进行,在连接后选择片段大小有利于去除非特异性连接。重复序列探针与DNA的杂交既可在尼龙膜上[28, 32]也可在包被有链霉亲和素的磁珠上[30-31]进行。LI等[33-34]采用选择杂交法已成功分离出8个皱纹盘鲍(Haliotis discus hannai)微卫星标记。
1.6 磁珠富集法(magnetic beads enrichment method)
磁珠富集法分离微卫星分子标记是一种简单高效的方法[31, 35], 已经应用于一些植物和动物微卫星分子标记的分离[26, 36-37]。实验生物基因组DNA经酶切,连上接头,PCR扩增,与生物素标记的重复序列探针杂交,磁珠富集,构建PCR富集文库(图 4)。然后再进行筛选即可得到微卫星分子标记。可用不同的酶和接头进行酶切、连接。如果按AFLP的方法进行酶切、连接、扩增,再富集,即为FIASCO法(fast isolation by AFLP of sequences containing repeats)[38]。包括单酶切扩增产物富集[38]和双酶切预扩增产物富集[36]。
孙效文等[13]用此方法筛选鲤鱼微卫星标记。获得微卫星314个,完美型占79.0 %,非完美型占14.3 %,混合型占6.7 %,重复次数超过10的有293个,占93.3 %。孙效文等[38]用磁珠富集法分离草鱼微卫星DNA标记。筛选获得阳性克隆132个,86.36 %含有微卫星序列。高国庆等[36]用FIASCO法从AFLP片段中分离花生(Arachis hypogaeal)微卫星DNA标记,结果回收纯化14个片段,测序后发现都含有简单重复序列。从预扩增的AFLP片段中富集SSR可获得较多的含简单重复序列的片段,而选择性扩增的AFLP片段不经富集直接测序的方法效率较低[39]。此方法在其他种类(比如一些鸟类、鱼类、甲壳类和红珊瑚)的富集率在50%~90%[38]。这些研究表明,用生物素-磁珠富集法克隆微卫星效率高,成本低,所获微卫星质量高。
2. 结语
直接克隆法耗时费力且筛选的效率也比较低。引物伸长法虽有所报道,其实验操作繁琐使其适用性受到限制。磁珠富集法是一种高效而简单快速的分离方法,已经应用于一些植物和动物微卫星分子标记的分离。整个过程可在一星期完成。如果利用篮白斑筛选克隆,从理论上讲,每一个白色菌斑都应当含有微卫星序列。但是操作过程中一些因素会影响筛选微卫星效率。最主要的影响因素是磁珠的平衡及洗液和洗涤温度的严格控制。从国内发表的文献上看,目前从基因组分离微卫星的主要方法是小片段克隆法和磁珠富集法以及稍作改进的一些方法。还有一些从基因组中克隆微卫星的方法,但最有效的还是基于PCR扩增的磁珠富集法。
从整体上看,微卫星分子标记在陆生动植物的分离和应用比较早,水产动物起步较晚,大多还处于分离筛选的初级阶段。在国内,只有鲤[41],对虾[9, 42],栉孔扇贝[10]等少数种类已分离出微卫星分子标记。网上基因库可利用的资源也还非常有限,远不能满足研究和应用的需要。而我国是一个渔业大国,水产动物种类多,遗传差异大,在遗传多样性分析、连锁图谱构建、数量性状基因(quantitative trait loci, QTL)定位分析、分子标记辅助育种等方面需要大量的微卫星标记。因此,选择有效的分离方法可以加速水产动物尤其主要养殖种类的微卫星分子标记的筛选及其应用进程。随着水产养殖生物的基因组测序和大规模cDNA测序,生物信息学方法分离微卫星DNA及其在近缘种的扩增筛选将越来越受到重视。
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表 1 港珠澳大桥北侧沿线水域4个声学监测位点的被动声学数据采集情况
Table 1 Acoustic data collection of four acoustic monitoring sites along north side of Hong Kong-Zhuhai-Macao Bridge
监测位点
Acoustic
monitoring
site第一阶段
First period第二阶段
Second period声音文件
数量
Number of
acoustic
files开始时间
Start time结束时间
End time开始时间
Start time结束时间
End timeA 2020-11-06 2020-11-24 2021-03-01 2021-03-09 640 B 2020-11-06 2020-11-29 2021-03-01 2021-03-09 769 C 2020-11-06 2020-11-29 2021-03-01 2021-03-09 769 D 2020-11-06 2020-11-29 2021-03-01 2021-03-09 769 -
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