CN104697997B - The method of quick discriminating Meretrix species - Google Patents
The method of quick discriminating Meretrix species Download PDFInfo
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- CN104697997B CN104697997B CN201510147304.5A CN201510147304A CN104697997B CN 104697997 B CN104697997 B CN 104697997B CN 201510147304 A CN201510147304 A CN 201510147304A CN 104697997 B CN104697997 B CN 104697997B
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Abstract
A kind of method of quick discriminating Meretrix species, it is characterised in that comprise the following steps：1. clam vivisection；2. oral siphon tentaculum peristomiale film-making；3. microexamination；4. species determines, according to oral siphon tentaculum peristomiale morphological feature, its species is determined.Compared with prior art, the advantage of the invention is that：Not only detected accurately using the microexamination of oral siphon tentaculum peristomiale, and with the advantages of technical difficulty is small, operating procedure is simple, visual result is accurate, especially have considerably less than traditional mitochondrial COI gene PCR sequencing PCR and AFLP labelling methods in experiment total time-consuming.
Description
Technical field
The present invention relates to a kind of discrimination method of clam species.
Background technology
Meretrix (Meretrix) species is one of the maximum species that made a variation in bivalve shellfish, because of the influence of h and E
And causing it very big difference on Morphology of shell, shell are color patterned to be present, domestic and foreign scholars are in the discriminating of its species, system for a long time
Very big dispute be present on the problems such as classification, fauna analysis.Chinese scholar defines that there is also larger point to Meretrix species
Discrimination.
Zhuan Qi is modest written《Fauna Sinica》(Beijing：Science Press, 2001：229-236.) in be related to mollusk
Bivalvia Veneridae the Meretrix species that China is distributed is incorporated into as clam (M.meretrix), beautiful clam (M.lusoria)
With 3 kinds of axe clam (M.1amarckii).
Qi Zhongyan (Qi Zhongyan) is written《Seashells of China》(Beijing:Maritime Press, 2004:
306)；Zhang Suping is written《Chinese Sea shellfish illustrated handbook》(Beijing:Maritime Press, 2008:353-354.)；The institute such as Xu Fengshan
Write《Chinese marine products Bivalve figure will》(Beijing:Science Press, 2008:249-251).
Above-mentioned three documents have very big difference to the word description and picture information of axe clam (M.1amarckii) form.
《Oceanologia et Limnologia Sinica》The 4th phase of volume 37 in July, 2006, written " Meretrix (Meretrix) 16S of Pan's treasured equality
The Phylogenetic Analysis of rRNA genes and ITS1 sequences " and《Zoological research》In June, 2009, the 3rd phase, 30 (3)：233-239 " bases
In the Meretrix (Mollusca of mitochondrial COI gene sequence：Veneridae) Phylogenetic Relationships ", above-mentioned two documents are based on
Chondriogen fragment and ribosomes ITS sequence compare, it is found that the affiliation of clam and beautiful clam is close, it is proposed that draw them
It is classified as same species.
《Oceanologia et Limnologia Sinica》In January, 2009, the 1st phase of volume 40, woods will China wait written by " Guangxi clam (Meretrix)
FAFLP and ITS analyses ", the document is found by fAFLP and ITS analyses, Guangxi White shell clam and other existing record clams
There is larger difference in phenotype and genetic structure, thus it is speculated that be the novel species of Meretrix one.
《Aquatic product journal》In October, 2011, " point of Zhejiang and two kinds of Guangxi clam written by the 10th phase, Dong Yinghui etc. of volume 35
Cangnan County, Zhejiang Province clam is defined as axe text by son identification and Morphometric analysis ", the document using mitochondrial COI gene sequence analysis
Clam, so that the distributed areas northward extension of axe clam has been arrived, Southern Zhejiang is coastal, have modified only existing described in existing literature
The saying of China South Sea Zhu Sheng distributions.
In summary, the division of Meretrix species and identification are still more chaotic, and this is studied to plasm resource protection and related science
Bring serious puzzlement.
At present, mainly using mitochondria 16S rDNA, COI gene orders and AFLP molecular labelings etc. in different Meretrix kinds
Difference between class carries out species identification.Pertinent literature may be referred to《Oceanologia et Limnologia Sinica》In May, 2011, the 3rd phase of volume 42, Zhu
In beautiful etc. written " the hereditary difference analyses of 4 color patterned strains of shell of clam (Meretrix meretrix) "；It can also refer to
《Aquatic product journal》In October, 2011, written by the 10th phase, Dong Yinghui etc. of volume 35 " Molecular Identification of Zhejiang and two kinds of Guangxi clam and
Morphometric analysis ".
Although the above method is accurate, application is commonplace, there is that operation difficulty is big, time-consuming, and testing result is not directly perceived
The deficiencies of.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of simple to operate, detection knot for the above-mentioned state of the art
The method of fruit intuitively quick discriminating Meretrix species.
Technical scheme is used by the present invention solves above-mentioned technical problem：A kind of side of quick discriminating Meretrix species
Method, it is characterised in that comprise the following steps：
1. clam vivisection, clam live body to be checked cuts off anterior adductor muscle after cleaning, and breaks two shells into two with one's hands, then closed shell after cutting off
Flesh, also, ensure not damage the oral siphon of rear end；
2. oral siphon tentaculum peristomiale film-making, drawing transparent clam body fluid, drip in forming drop on slide, clip is a piece of
Oral siphon distally tentacle part, inside pipe wall are placed in the drop of slide upwards；
3. microexamination, stir oral siphon tentaculum peristomiale and remove mucus and dirt, microscope or amplification Microscopic observation its
Morphological feature, microscope here can use common stereomicroscope or light microscope (4 × 10 times)；
4. species determines, according to oral siphon tentaculum peristomiale morphological feature, its species is determined.
Further, 4. species is defined below step：
If oral siphon tentaculum peristomiale is in ox horn shape, there is obvious black pigment band at the tapering no branch in end, middle part, then is to draw
Fourth scientific name is Meretrix meretrix clam；
Top such as cauliflower branch, then it is axe clam as oral siphon tentaculum peristomiale is dispersed in cluster；
Then it is white shell clam as there is short branch sample branch on oral siphon tentaculum peristomiale top；
If oral siphon tentaculum peristomiale is in long taper, the tapering no branch in end, there is the Huang being dispersed at middle part without black pigment band
Color pigment granule, then it is curtain clam.
Further, the body fluid of the step 2. clam, which uses, cuts after closed shell flesh 2~3min in clam vivisection from body
The transparency liquid of interior outflow, as isotonic NMF, prevent moisture rapid evaporation and oral siphon tentaculum peristomiale is dehydrated shrinkage.
Preferably, 2. described oral siphon distally tentacle area is long 0.2cm × wide 0.3~0.4cm to step.
Compared with prior art, the advantage of the invention is that：
1st, method is simple, simple operation, and it is numerous to need not move through DNA extractions, PCR amplifications, sequencing or the electrophoresis of molecular identificalion etc.
Trivial program, directly made and microexamination by simple water load；
2nd, testing requirements are low, detection difficulty is small, without a large amount of expensive instrument and equipments such as centrifuge, electrophoresis apparatus, only need light
Just stereomicroscope, light microscopic even magnifying glass；
3rd, take less, it is time saving and energy saving in 5min or so with regard to Identification of Species can be completed；
4th, visual result is accurate, is not as a result that DNA sequence dna hard to understand compares and running gel figure, but directly perceived and easily distinguish
The microscopic morphology figure of discrimination, but qualification result is equally accurately and reliably.
Brief description of the drawings
Fig. 1 is the microphoto of clam (Meretrix meretrix) oral siphon tentaculum peristomiale form in embodiment 1
Fig. 2 is axe clam (M.lamarckii) oral siphon tentaculum peristomiale form microphoto in embodiment 1.
Fig. 3 is the microphoto of white shell clam (M.spp.) oral siphon tentaculum peristomiale form in embodiment 1.
Fig. 4 is the microphoto of curtain clam (M.lyrata) oral siphon tentaculum peristomiale form in embodiment 1.
Fig. 5 is the microphoto of Changle of Fujian Province triangle clam oral siphon tentaculum peristomiale form in embodiment 2.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Embodiment 1, the experiment material of the present embodiment are 4 kinds of clams of Meretrix of China's southeastern coast collection, are text respectively
Clam (picking up from Nantong Rudong sea area, longitude and latitude is 32 ° of 30 ' N, 121 ° of 11 ' E), axe clam (pick up from Wenzhou District of Zhejiang Province Cang Nanhai
Domain, longitude and latitude are 27 ° of 33 ' N, 120 ° of 36 ' E), curtain clam (pick up from Zhanjiang East Sea Island marine site, longitude and latitude is 21 ° of 04 ' N,
110 ° of 20 ' E), white shell clam (picking up from Beihai Fisheries Base Guangxi Province military camp marine site, longitude and latitude is 21 ° of 27 ' N, 109 ° of 26 ' E), shell grow 3~7cm.
Implementation steps are as follows：
1st, clam vivisection：4 kinds of clam live bodies to be checked are cleaned up, are placed in dissecting pan, before being cut off with scalpel
Closed shell flesh, slowly break two shells into two with one's hands, then cut off posterior adductor muscle；
2nd, oral siphon tentaculum peristomiale film-making：2min after dissection, the transparent blood/mucus mixture of clam is drawn with dropper
1mL, drop 1 are dripped in clean slide center；With the small small pieces of scissors clip oral siphon distally tentacle part one, size is length
0.2cm × wide 0.3cm, inside pipe wall are placed in the drop in slide center upwards, and transparent blood/mucus mixture uses clam
From the transparency liquid flowed out in vivo after 2~3min of incision closed shell flesh in vivisection；
3rd, microexamination：Water pipe tentaculum peristomiale is gently dialled in dissecting needle, mucus and dirt are removed, in common stereomicroscope
The morphological feature of 4 kinds of clam oral siphon tentaculum peristomiales of lower observation；
4th, species determines：Compare oral siphon tentaculum peristomiale form difference, distinguish 4 kinds of clams.
5th, oral siphon tentaculum peristomiale microscopic inspection and the method and step and result ratio of traditional mitochondrial COI gene sequencing method
Compared with：
4 kinds of clams are distinguished and identified with oral siphon tentaculum peristomiale microscopic inspection, it is as a result as follows：
As shown in figure 1, clam oral siphon tentaculum peristomiale is in ox horn shape, the tapering no branch in end, there is obvious black at middle part
Pigment zone (PB：Pigment Belt, pigment zone)；
As shown in Fig. 2 axe clam oral siphon tentaculum peristomiale is dispersed in cluster, top such as cauliflower branch；
As shown in figure 3, there is the branch sample branch at end on white shell clam oral siphon tentaculum peristomiale top；
As shown in figure 4, curtain clam oral siphon tentaculum peristomiale is in long taper, the tapering no branch in end, but middle part is without black color
Plain band, and have the xanthein particle being dispersed in.
This 4 kinds of clam samples also have been subjected to mitochondrial COI gene PCR sequencing PCR and carried out species identification, and species information is bright
Really.As can be seen here, oral siphon tentaculum peristomiale microscopic inspection is applied to distinguish Meretrix species, as a result accurately and reliably.
Table 1 compares the oral siphon tentaculum peristomiale microscopic inspection of the present invention and traditional mitochondrial COI gene PCR sequencing PCR
Method and step and result accuracy, as a result show oral siphon tentaculum peristomiale microscopic inspection have technical difficulty it is small, operation step
The advantages of rapid simple, visual result is accurate, especially there is obvious advantage in experiment total time-consuming.
Table 1, oral siphon tentaculum peristomiale microscopic inspection and the method and step and result of traditional mitochondrial COI gene sequencing method
Accuracy compares
Embodiment in June, 2,2012, from Changle of Fujian Province city Zhang Gang towns, (25 ° 54 ' 32.78 " N, 119 ° 39 ' 04.53 " E) is adopted
Collect a kind of clam (local to claim " triangle clam "), form and shell color have certain difference with other Meretrix species.In order to permanently happy
" triangle clam " is accurately differentiated, oral siphon tentaculum peristomiale microscopic inspection and two kinds of hands of AFLP molecular marking techniques is respectively adopted
Duan Jinhang research.
Oral siphon tentaculum peristomiale microscopic inspection observes the oral siphon tentaculum peristomiale of 4 " triangle clam ", takes 15min,
With reference to shown in Fig. 5, as a result show, the oral siphon tentaculum peristomiale of " triangle clam " is dispersed in cluster, top such as cauliflower-shaped branch, with figure
Axe clam oral siphon tentaculum peristomiale is identical in 2, and " triangle clam " is defined as into axe clam accordingly.
Using AFLP labelling techniques, to " triangle clam ", clam, axe clam, curtain clam, each 15 of 5 colonies of white shell clam
Sample has carried out finger-print comparison and genetic similarty analysis, takes 5d, as a result shows, the AFLP finger-prints of " triangle clam "
Closely similar with axe clam, genetic similarty reaches 0.9554, is significantly higher than 0.5499~0.5844 with other colonies, therefore
" triangle clam " is defined as axe clam.
Result above shows that the operation of oral siphon tentaculum peristomiale microscopic inspection is simpler, result is more directly perceived accurate.
Claims (3)
- A kind of 1. method of quick discriminating Meretrix species, it is characterised in that comprise the following steps：1. clam vivisection, clam live body to be checked cuts off anterior adductor muscle after cleaning, and breaks two shells into two with one's hands, then cuts off posterior adductor muscle, and The oral siphon of rear end is not damaged；2. oral siphon tentaculum peristomiale film-making, drawing transparent clam body fluid, drip in forming drop on slide, clip it is a piece of enter water Pipe distally tentacle part, inside pipe wall are placed in the drop of slide upwards；3. microexamination, stir oral siphon tentaculum peristomiale and remove mucus and dirt, in microscope or amplification Microscopic observation its form Feature；4. species determines, according to oral siphon tentaculum peristomiale morphological feature, its species is determined；4. species is defined below step：If oral siphon tentaculum peristomiale is in ox horn shape, the tapering no branch in end, there is obvious black pigment band at middle part, then is Latin Name is Meretrix meretrix clam；Top such as cauliflower branch, then it is axe clam as oral siphon tentaculum peristomiale is dispersed in cluster；Then it is white shell clam as there is short branch sample branch on oral siphon tentaculum peristomiale top；If oral siphon tentaculum peristomiale is in long taper, the tapering no branch in end, there is the yellow color being dispersed at middle part without black pigment band Crude granule, then it is curtain clam.
- 2. the method for quick discriminating Meretrix species according to claim 1, it is characterised in that step 2. described clam Body fluid, which uses, to be cut after closed shell flesh 2~3min in clam vivisection from the transparency liquid flowed out in vivo.
- 3. the method for quick discriminating Meretrix species according to claim 1, it is characterised in that step is 2. described to enter water Pipe distally tentacle area is long 0.2cm × wide 0.3~0.4cm.
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