CN111128310B - Method for identifying eucalyptus tree species and genetic relationship between species - Google Patents
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Abstract
The invention provides a method for identifying the species and the genetic relationship between the species of a eucalyptus tree, which comprises the following steps: establishing a database, comparing parameters and judging the genetic relationship; the method combines a scanning electron microscope and an optical microscope for use, accurately observes and measures the morphological characteristics of the pollen from different angles, and comprises the important characteristic of pollen germination, so that the pollen identification result is accurate, rapid and effective.
Description
Technical Field
The invention relates to the technical field of plants, in particular to a method for identifying eucalyptus tree species and genetic relationship between the eucalyptus tree species.
Background
Eucalyptus (Eucalyptus) is an important afforestation tree species in the world, large-area planting is carried out in China, according to statistics of the Eucalyptus division 2015 of the Chinese forest institute, the planting area of the Eucalyptus reaches 6750 ten thousand acres, and the Eucalyptus contributes a large amount of wood to China. The improvement of eucalyptus varieties is particularly important due to the planting area in such a scale, the improvement of the eucalyptus varieties necessarily relates to the identification of tree species and the definition of the genetic relationship, and the improvement level of the eucalyptus can be effectively improved only if the identification of each variety is not difficult; in the economic activity of the market, a plurality of excellent artificially cultivated varieties are easy to infringe, and the rapid and effective identification of the varieties can help to judge whether the infringement is formed. About 1000 species (subspecies and varieties) of eucalyptus trees are added, and the number of varieties obtained by artificial breeding is more, so that the variety and the genetic relationship are difficult to identify.
The traditional method for identifying the variety and the genetic relationship of eucalyptus is to observe various physiological organs of eucalyptus, generally by observing leaf type, leaf color, leaf quality, bark grains, bark color, dry type and the like. Because the characteristics of a plurality of varieties are not greatly different, especially some trees with relatively close relationships are very similar in the characteristics, the identification method must depend on very professional background knowledge and years of practical experience, and the professionals are very few, so that the identification method is difficult to effectively utilize.
The other method is to perform identification by a molecular biology means, mainly by a DNA chip, a molecular marker and other methods, the identification method also needs higher specialty, operators have certain molecular biology knowledge background, the previous sample treatment needs higher requirements to obtain more accurate results, and the technical means needs to rely on a large amount of basic data, namely each variety needs genome data or related detailed molecular biology data, and the results cannot be compared if the results are not obtained, so the technical means is not mature at present.
The quantitative analysis method for the structural morphological characteristics of the pollen can accurately and rapidly identify the eucalyptus pollen variety and the relationship of relativity, and solve the problems encountered by the methods.
Disclosure of Invention
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a method for identifying the genetic relationship between eucalyptus tree species and eucalyptus tree species comprises the following steps:
(1) establishing a database: collecting and processing pollen of known tree species, taking a picture of the whole appearance of the pollen by a scanning electron microscope, judging the pattern type of an outer wall, taking a picture of local important characteristics, closing up the shot part comprising an equatorial plane, a polar plane, a germination hole and a germination ditch, measuring the equatorial axial length, the equatorial plane width, the germination ditch length, the germination ditch width and the germination ditch depth of pollen grains, and determining whether an island exists in the middle to obtain pollen morphological characteristic parameters; observing the external characteristics of the pollen and the color of the pollen by using an optical microscope, and observing and measuring the length and the width of a pollen tube by using the optical microscope again after culturing; collecting multiple groups of data on the external wall decoration type, the pollen morphological characteristic parameters, the external characteristics of pollen, the pollen color and the pollen tube length and width of the known tree species, and calculating an average value to obtain a basic database of the known tree species;
(2) the pollen of the target tree species is obtained through the same collection processing as that in the step 1, the outer wall decoration type, the pollen form characteristic parameter, the external characteristic of the pollen, the pollen color, the pollen tube length and the pollen tube width are obtained through a scanning electron microscope and an optical microscope, the comparison is carried out with a basic database of the known tree species, the SAS or SPSS statistical software is used for analyzing the outer wall decoration type, the pollen form characteristic parameter, the external characteristic of the pollen, the pollen color, the correlation coefficient of the pollen tube length and the pollen tube width, the correlation coefficient can be the same tree species within 0.7-0.9, and the type of the target tree species can be judged through the comparison of the shot pollen photo and the original eucalyptus photo;
(3) Measuring the data of the pollen of a plurality of tree species, comparing the data of the plurality of tree species with a basic database of known tree species, analyzing the relevance of the data of the plurality of tree species, analyzing the evolution condition of the data by using SAS software, and analyzing the genetic relationship among the plurality of tree species by combining the outer wall decoration and the difference and the sameness of an equatorial axis, an equatorial plane, a germination ditch and a pollen tube.
Further, the acquisition process comprises: collecting flower branches of tree species by using a high-branch shear sampler, shearing off buds which are ready to be opened but are not opened, uncovering a capsule, shearing off filaments, spreading the filaments in a glass culture dish, putting the culture dish filled with the filaments in a drying dish, filling a silica gel drying agent in the drying dish in advance, and drying for 2 days; after the filaments are dried, sieving the filaments by adopting a 200-mesh sieve, and poking the filaments by using a writing brush during sieving so as to ensure that the pollen is more fully sieved and the sieved pollen is collected.
Furthermore, the method for taking pictures by using the scanning electron microscope comprises the steps of adhering pollen for observation by using the scanning electron microscope on a special copper table for the scanning electron microscope, spraying gold on the scanning electron microscope, adjusting relevant parameters, and observing and measuring.
Further, the culture comprises the step of putting a certain amount of pollen into a culture dish for culture, adding clear water and a small amount of cane sugar into the culture dish, and culturing for 48 hours.
The invention provides a method for identifying the genetic relationship between eucalyptus tree species and eucalyptus tree species, which is used for identifying the genetic relationship between the eucalyptus tree species and eucalyptus tree species through the morphological characteristics of eucalyptus pollen. Eucalyptus is used as a hermaphrodite plant, pollen (male gamete) of the euscaphis is an important physiological organ, is a tissue with stable characters and forms reserved in the plant evolution process, and has obvious characteristics and identification. The pollen is identified by a professional method, the indexes can be accurately measured or described by analyzing the size (equatorial axial length and equatorial plane width) of pollen grains, the length of a germination groove, the width of the germination groove, the depth of the germination groove, the outer wall decoration pattern and the like, the indexes are quantitatively defined, the pollen of a certain eucalyptus variety can be accurately pointed, the pollen is compared with the original eucalyptus pollen data, and mathematical statistical analysis is carried out by adopting professional analysis software, so that the eucalyptus variety can be identified. In addition, since the pollen of the eucalyptus also has relevance evolution in the process of the evolution of one eucalyptus species to another or the evolution of one eucalyptus species into a plurality of eucalyptus species, the genetic relationship between the target tree species and other eucalyptus tree species can be judged and inferred according to the acquaintance and relevance of the measured data of the eucalyptus pollen parameters.
The detailed technical scheme is that eucalyptus pollen is collected firstly, and pollen is collected in a full-bloom stage, so that the pollen is more representative. Then pollen drying is carried out, and the scheme is that silica gel desiccant is used for pollen drying. After drying, the pollen was sieved using a 200 mesh sieve, and the sieved pollen was collected. Then, observing the morphological characteristics of the pollen and measuring data by using a Scanning Electron Microscope (SEM), and measuring the size (equatorial axis length and equatorial plane width), the length of a germination groove, the width of the germination groove, the depth of the germination groove, the decorative pattern of the outer wall and the like of the pollen grains; thereafter, the color and the like of the pollen were observed using an optical microscope, the pollen was cultured for a limited time, and the length of the pollen tube in which the pollen germinated was observed and measured using the optical microscope was continued. And finally, comparing the obtained data with the original data and data, and analyzing by adopting analysis software to determine the tree species or the genetic relationship among the tree species. The method has no technical problem in operation and high accuracy. The species and relationship of eucalyptus trees can be identified through observation and measurement by a scanning electron microscope and an optical microscope, and if the conditions exist, the eucalyptus trees can be further observed by a Transmission Electron Microscope (TEM), so that the judgment result is more convincing.
The invention provides a method for identifying the species and the genetic relationship between the species of a eucalyptus tree, which comprises the following steps: establishing a database, comparing parameters and judging the genetic relationship; the method combines a scanning electron microscope and an optical microscope for use, accurately observes and measures the morphological characteristics of the pollen from different angles, and comprises the important characteristic of pollen germination, so that the pollen identification result is accurate, rapid and effective.
The method has high accuracy in identifying eucalyptus tree species. The pollen of each eucalyptus has the characteristic of fixed character, can accurately point to certain pollen through the definition of pollen technical parameters and the limitation of decorative patterns, and can clearly identify the genetic relationship with similar tree species. The morphological characteristics of the pollen of a certain eucalyptus are stable, the difference of the shape, the color, the crown shape and the like of leaves along with the change of an external environment is avoided, and the higher accuracy can be ensured by identifying eucalyptus tree species through the pollen under different environments. Moreover, experiments prove that the morphological characteristics of eucalyptus pollen can generate certain directional changes along with hybridization, so that the method can identify artificially cultivated varieties.
The method has moderate cost. The cost of pollen collection in the early stage is basically low, and a plurality of small fixed tools are needed. Due to the popularization of the existing scanning electron microscope, the later-stage scanning electron microscope observation and measurement cost is not high, and the measurement of a plurality of samples can be normally finished within one hour. The later data comparison mainly takes time. The method has lower cost compared with molecular biology identification methods such as sequencing or molecular marking.
The method is simple to operate and has low requirement on professional technology. People who do not need to engage in relevant specialties and have abundant experience all the year round can operate the plant cultivation device, people without relevant knowledge can operate the plant cultivation device through simple explanation, and popularization in the industry is facilitated.
The method has short time for identifying tree species. The whole operation process of the method can be controlled within 3-4 days, the pollen collection time is 1 day, the drying time is about 2 days, and the machine can be used for scanning electron microscope observation and measurement after drying; the pollen germination test can be carried out simultaneously, the pollen germination test needs 2 days, and then observation can be carried out immediately, and data comparison can be carried out immediately, and varieties can be identified.
The method has certain learning ability. With the accumulation and the perfection of tree species data, the method can ensure that the identification result is more accurate and easier.
Drawings
FIG. 1 shows the pattern of the outer wall of a conventional pollen. a is granular, b is verrucous/rough, c is rough/regular, d is regular, e is verrucous, f is rough, g is moth-eaten, and h is smooth.
FIG. 2 shows the parameters of eucalyptus pollen to be measured. Germination furrow depth is typically measured at other scan planes. 1, equator axial length, 2, equator surface width, 3, germination furrow length, 4, germination furrow width, 5, germination furrow depth and 6, whether an island exists or not
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Any modifications that can be easily made by a person skilled in the art to the present invention without departing from the technical solutions of the present invention will fall within the scope of the claims of the present invention.
Example 1
A method for identifying the genetic relationship between eucalyptus tree species and eucalyptus tree species comprises the following steps:
(1) establishing a database: acquiring 12 common eucalyptus variety pollens through collection and processing, measuring parameters of the pollens, taking a picture of the whole appearance of the pollens through a scanning electron microscope as shown in table 1, judging the pattern type of an outer wall as shown in figure 1, taking a picture of local important characteristics, wherein the photographed part comprises an equatorial plane, a polar plane, a germination hole and a germination furrow, measuring the axial length of the equator, the width of the equatorial plane, the length of the germination furrow, the width of the germination furrow and the depth of the germination furrow, and determining whether an island exists in the middle to obtain pollen morphological characteristic parameters as shown in figure 2; observing the external characteristics of the pollen and the color of the pollen by using an optical microscope, and observing and measuring the length and the width of a pollen tube by using the optical microscope again after culturing; collecting multiple groups of data on the external wall decoration type, the pollen morphological characteristic parameters, the external characteristics of pollen, the pollen color and the pollen tube length and width of the known tree species, and calculating an average value to obtain a basic database of the known tree species;
(2) The pollen of the target tree species is obtained through the same collection processing as that in the step 1, the outer wall decoration type, the pollen form characteristic parameter, the external characteristic of the pollen, the pollen color, the pollen tube length and the pollen tube width are obtained through a scanning electron microscope and an optical microscope, the comparison is carried out with a basic database of the known tree species, the SAS and SPSS statistical software is used for analyzing the outer wall decoration type, the pollen form characteristic parameter, the external characteristic of the pollen, the pollen color, the correlation coefficient of the pollen tube length and the pollen tube width, the correlation coefficient can reach 0.7-0.9 and is the same tree species, and the type of the target tree species can be judged through the comparison of the shot pollen photo and the original eucalyptus photo;
(3) measuring the data of the pollen of a plurality of tree species, comparing the data of the plurality of tree species with a basic database of known tree species, analyzing the relevance of the data of the plurality of tree species, analyzing the evolution condition of the data by using software such as SAS (serial attached small computer system interface), and analyzing the genetic relationship among the plurality of tree species by combining the outer wall decoration and the similarities and differences of an equatorial axis, an equatorial plane, a germination furrow and a pollen tube.
Further, the acquisition process comprises: collecting flower branches of tree species by using a high-branch shear sampler, shearing off buds which are ready to be opened but are not opened, uncovering a capsule, shearing off filaments, spreading the filaments in a glass culture dish, putting the culture dish filled with the filaments in a drying dish, filling a silica gel drying agent in the drying dish in advance, and drying for 2 days; after the filaments are dried, sieving the filaments by adopting a 200-mesh sieve, and brushing the filaments with a writing brush during sieving so as to ensure that the pollen is sieved more fully, and collecting the sieved pollen.
The method for taking a picture by using the scanning electron microscope comprises the steps of adhering pollen for observing by using the scanning electron microscope on a special copper table for the scanning electron microscope, spraying gold on the scanning electron microscope, adjusting relevant parameters, and observing and measuring.
The culture comprises the steps of taking a certain amount of pollen and placing the pollen in a culture dish for culture, adding clear water and a small amount of cane sugar into the culture dish, and culturing for 48 hours.
TABLE 1 comparison table of pollen parameters of common eucalyptus varieties
Unit: mum of
The above-mentioned embodiments are merely illustrative of the composition and process features of the present system, and are intended to enable those skilled in the art to understand the present invention and implement the present invention, and not to limit the scope of the present invention. The technical solutions formed by the present system or the equivalent substitution method should fall within the protection scope of the claims of the present invention.
Claims (4)
1. A method for identifying the genetic relationship between eucalyptus tree species and eucalyptus tree species is characterized by comprising the following steps:
(1) establishing a database: collecting and processing the pollen of known tree species, taking a picture of the whole appearance of the pollen by a scanning electron microscope, judging the pattern type of the outer wall, taking a picture of local important characteristics, closing up the shot part comprising an equatorial plane, a polar plane, a germination hole and a germination ditch, measuring the equatorial axial length, the equatorial plane width, the germination ditch length, the germination ditch width and the germination ditch depth of pollen grains, and determining whether an island exists in the middle to obtain pollen morphological characteristic parameters; observing the external characteristics and the color of the pollen by using an optical microscope, and observing and measuring the length and the width of the pollen tube by using the optical microscope again after culturing; collecting multiple groups of data to calculate the average value of the external wall decoration type, the pollen morphological characteristic parameters, the external characteristics of the pollen, the pollen color, the pollen tube length and the width of the known tree species, so as to obtain a basic database of the known tree species;
(2) Acquiring pollen of a target tree species through the same acquisition processing as that in the step 1, obtaining an outer wall decoration type, a pollen form characteristic parameter, an external characteristic of the pollen, a pollen color, a pollen tube length and a pollen tube width through a scanning electron microscope and an optical microscope, comparing the obtained pollen with a basic database of a known tree species, and analyzing correlation coefficients of the outer wall decoration type, the pollen form characteristic parameter, the external characteristic of the pollen, the pollen color, the pollen tube length and the pollen tube width by using SAS or SPSS statistical software, wherein the correlation coefficients can be the same eucalyptus tree species between 0.7 and 0.9; then comparing the shot pollen picture with the original picture to judge the type of the target tree species;
(3) measuring the data of the pollen of a plurality of tree species, comparing the data of the plurality of tree species with a basic database of known tree species, analyzing the relevance of the data of the plurality of tree species, analyzing the evolution condition of the data by using SAS software, and analyzing the genetic relationship among the plurality of tree species by combining the difference and the similarity of the outer wall decoration pattern, the equatorial axis, the equatorial plane, the germination ditch and the pollen tube.
2. The method for identifying eucalyptus tree species and genetic relationship between eucalyptus tree species as claimed in claim 1, wherein said collecting process comprises: collecting flower branches of tree species by using a high-branch shear sampler, shearing off buds which are ready to be opened but are not opened, uncovering a capsule, shearing off filaments, spreading the filaments in a glass culture dish, putting the culture dish filled with the filaments in a drying dish, filling a silica gel drying agent in the drying dish in advance, and drying for 2 days; and after the filaments are dried, sieving the filaments by adopting a 200-mesh sieve, poking the filaments with a writing brush during sieving so as to ensure that the pollen is sieved more fully, and collecting the sieved pollen.
3. The method for identifying the genetic relationship between the eucalyptus tree species and the eucalyptus tree species as claimed in claim 1, wherein the method for taking the picture by scanning electron microscope comprises the steps of sticking pollen for observation by scanning electron microscope on a special copper table for electron microscope, spraying gold, then applying the scanning electron microscope, adjusting relevant parameters, and observing and measuring.
4. The method for identifying eucalyptus tree species and species relativity according to claim 1, wherein the cultivation comprises taking pollen and putting the pollen in a culture dish for cultivation, and adding clear water and sucrose into the culture dish for 48 hours.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288606A (en) * | 2011-05-06 | 2011-12-21 | 山东农业大学 | Pollen viability measuring method based on machine vision |
| CN109187673A (en) * | 2018-10-24 | 2019-01-11 | 杭州电子科技大学 | A kind of plant Relationship iden- tification method based on pollen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030134311A1 (en) * | 2000-06-23 | 2003-07-17 | Simon Potter | Nucleic acid-based method for tree phennotype prediction |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288606A (en) * | 2011-05-06 | 2011-12-21 | 山东农业大学 | Pollen viability measuring method based on machine vision |
| CN109187673A (en) * | 2018-10-24 | 2019-01-11 | 杭州电子科技大学 | A kind of plant Relationship iden- tification method based on pollen |
Non-Patent Citations (2)
| Title |
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| 20种桉树及杂交种的花粉形态分析及分类学意义;武海霞 等;《中南林业科技大学学报》;20120331;第32卷(第3期);第29-35页 * |
| 基于SEM分析的６种松属树种花粉特征及亲缘关系研究;解庆 等;《山西农业大学学报》;20181231;第8-13页 * |
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