A rapid identification method for homologous tetraploids of lily of Lanzhou

Abstract

The present invention discloses a method for rapid identification of homologous tetraploids of Lanzhou lily, and belongs to the technical field of plant ploidy identification. The method comprises the steps of identifying the ploidy of Lanzhou lily according to leaf density and the number of flower buds per branch, and specifically comprises dividing Lanzhou lily into two categories of high leaf density and low leaf density according to the leaf density value, wherein the Lanzhou lily with high leaf density is diploid, and the Lanzhou lily with low leaf density is homologous tetraploid; when the high leaf density and the low leaf density cannot be distinguished according to the leaf density value, the lily with single branch flower bud number is identified according to the number of flower buds per branch of Lanzhou lily, and the number of flower buds per branch ≥3 is diploid, and the number of flower buds per branch ≤2 is homologous tetraploid. The present invention screens diploids and homologous tetraploids according to the leaf density and the number of flower buds per branch, and compared with the existing method, has the advantages of simple method, no dependence on any instrument and equipment, and strong field practicality.

Description

Technical Field

[0001] The invention relates to the technical field of plant ploidy identification, and in particular to a rapid identification method for the homologous tetraploid of Lanzhou lily. Background Art

[0002] Lanzhou lily (Liliumdavidii var.unicolor) is a variant of wild Sichuan lily of the genus Lilium in the Liliaceae family. It is the only edible sweet lily in my country. It has a unique flavor and health benefits. It is a geographical indication product in Gansu Province.

[0003] Lanzhou lily is a diploid plant with a chromosome number of 2n=24. It is an asexually propagated crop that harvests the vegetative body (lily bulb), and is very suitable for breeding high-yield and stress-resistant new varieties using polyploid breeding technology. It is reported that the homologous tetraploid Lanzhou lily has higher yield, better stress resistance and higher quality than the diploid plant, but in field production, it is easy to cause the bulbs of different ploidy to mix. How to quickly and efficiently identify autotetraploid and diploid plants to maintain the purity of polyploid varieties has not been reported yet.

[0004] The root tip chromosome pressing method and flow cytometry method are the most direct and accurate methods for identifying plant ploidy, but they have the disadvantages of high difficulty in operation and strong instrument dependence. Guard cell size and chloroplast number are also often used for polyploid identification, but they must be observed under a microscope, and the number of chloroplasts in Lanzhou lily is large and inconvenient to count, so they are not very applicable in the identification of ploidy in Lanzhou lily. Yu Wenjin et al. from Guangxi University used morphology such as plant leaf color, leaf shape, internode distance, leaf size and thickness to screen tetraploid eggplant rootstocks; Wang Chenggang et al. from Anhui Agricultural University first screened Wucai tetraploids with external morphological indicators such as growth potential, leaf color, leaf thickness and flower organ enlargement, and then combined photosynthetic measurement and flow cytometry for identification. Although tetraploids and diploids do differ in leaf color and leaf thickness traits, these two phenotypes are easily affected by the environment, and leaf color judgment is highly subjective. The difference in leaf thickness is not easy to be measured by ordinary measuring rulers, and can only be clearly seen when the anatomical structure is compared, and the practicality of field is not high.

[0005] Based on the above situation, developing a rapid identification method for tetraploid homologues of Lanzhou lily that is fast, efficient, independent of instruments and equipment, and practical for field use is an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide a method for quickly identifying the homologous tetraploid of Lanzhou lily to solve the problems existing in the above-mentioned prior art. The present invention distinguishes diploids from autotetraploids by comparing the leaf density and the number of flower buds per branch. The method is simple, does not rely on any instrument or equipment, and has practicality in the field.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides application of leaf density and single-branch flower bud number as identification indexes in rapid identification of Lanzhou lily homotetraploid.

[0009] The invention also provides a method for quickly identifying the homotetraploid of Lanzhou lily, comprising the steps of identifying the ploidy of Lanzhou lily according to the leaf density and the number of flower buds per branch.

[0010] Furthermore, the ploidy includes diploid and autotetraploid.

[0011] Furthermore, the Lanzhou lilies are Lanzhou lilies grown at the same altitude.

[0012] Further, the following steps are included:

[0013] The Lanzhou lily is divided into two categories, high leaf density and low leaf density, according to the leaf density value, wherein the Lanzhou lily with high leaf density is diploid, and the Lanzhou lily with low leaf density is autotetraploid; when the high leaf density and the low leaf density cannot be distinguished according to the leaf density value, the Lanzhou lily is identified according to the number of flower buds per branch, and the number of flower buds per branch ≥3 is diploid, and the number of flower buds per branch ≤2 is autotetraploid;

[0014] When the altitude is 1500-1700 meters, a leaf density value ≥3 is a high leaf density, and a leaf density value ≤2 is a low leaf density; when the altitude is 1800-2600 meters, a leaf density value ≥6 is a high leaf density, and a leaf density value ≤5 is a low leaf density.

[0015] Furthermore, the Lanzhou lily is a Lanzhou lily that is planted for the first year.

[0016] The present invention discloses the following technical effects:

[0017] The present invention selects Lanzhou lily planting sites at different altitudes, and performs statistics on leaf density and the number of flower buds per branch for Lanzhou lilies planted at the same altitude. Through analysis and comparison, it is found that the diploid and autotetraploid Lanzhou lilies can be distinguished according to the leaf density. When the leaf density is difficult to judge based on the leaf density alone, the ploidy of the Lanzhou lily can be accurately distinguished by further combining the number of flower buds per branch.

[0018] The invention screens diploids and autotetraploids according to leaf density and the number of flower buds per branch. Compared with the existing method, the invention has the advantages of being simple, not relying on any instrument or equipment, and being highly practical in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a comparison of leaf density of diploid and autotetraploid Lanzhou lily plants in the budding stage of the first year of planting (planting site altitude: 2179 meters);

[0021] Figure 2 This is a comparison of leaf density and number of flower buds per branch of diploid and autotetraploid Lanzhou lily plants in full bloom in the first year of planting (planting site altitude: 2179 meters);

[0022] Figure 3 The statistical diagram of leaf density and number of flower buds per branch of diploid and autotetraploid plants in the first year of planting (planting site altitude: 2179 meters);

[0023] Figure 4 This is a comparison of leaf density of diploid and autotetraploid Lanzhou lily plants in the budding stage of the first year of planting (planting site altitude: 1530 meters);

[0024] Figure 5 This is a comparison of leaf density and number of flower buds per branch of diploid and autotetraploid Lanzhou lilies in full bloom in the first year of planting (planting site altitude: 1530 meters);

[0025] Figure 6 Statistical chart of diploid and autotetraploid leaf density and number of flower buds per branch in the first year of planting (planting site altitude: 1530 meters). DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example

[0032] 1. Seed selection, disinfection and graded sowing

[0033] Select the bulbs of Lanzhou lily without diseases, insects, rot, mechanical damage and intact base as bulbs, dilute 500 times with 50% carbendazim wettable powder, soak for 30 minutes, and dry in the shade. Sow according to the size of bulbs (first-grade bulbs 20-30g, second-grade bulbs 12-20g, third-grade bulbs less than 12g), and select the planting sites at altitudes of 2179 meters and 1530 meters respectively.

[0034] 2. Statistics of leaf density and number of flower buds per branch

[0035] At the peak flowering stage, according to the description specifications of lily germplasm resources, the plant height (unit: cm) was measured, the number of leaves (unit: pieces) and the number of flower buds per branch (unit: pieces) were counted, and the leaf density was calculated. According to the grading of the bulbs when they were sown, the bulbs of the same grade were counted as the same group.

[0036] Method for measuring plant height: During the flowering period, when the plant is in its natural state, the vertical distance from the base of the ground to the highest point of the plant is the natural height.

[0037] Leaf number counting method: During the flowering period, count the number of leaves on the upright stems of the plant.

[0038] Statistical method for the number of flower buds per branch: During the peak flowering period, count the number of flower buds per plant.

[0039] The calculation formula for leaf density is: leaf density = number of leaves / plant height, unit: leaf / cm.

[0040] 3. Statistical analysis

[0041] (1) Comparison within the same planting area at the same altitude

[0042] In the field planting area at an altitude of 2179 meters, the leaf density phenotypes of diploid and autotetraploid plants of Lanzhou lily at the budding stage and the flowering stage were compared as follows: Figure 1 and Figure 2 As shown by Figure 1 and Figure 2 It can be seen that judging from the phenotype, the leaf density of diploid Lanzhou lily is significantly higher than that of autotetraploid, whether in the budding stage or the flowering stage.

[0043] Combined with the measured data, the leaf density and number of flower buds per branch of diploid and autotetraploid Lanzhou lilies grown in the field at an altitude of 2179 meters were statistically analyzed. Figure 3 As shown. Figure 3 It can be seen that the average leaf density of diploid Lanzhou lily is 6.62 pieces / cm, and the average number of flower buds per branch is 3.80; the average leaf density of autotetraploid Lanzhou lily is 4.68 pieces / cm, and the average number of flower buds per branch is 1.10. The statistical results are consistent with the results judged based on phenotype.

[0044] In the field planting area at an altitude of 1530 meters, the leaf density phenotypes of diploid and autotetraploid plants of Lanzhou lily at the budding stage and the flowering stage were compared as follows: Figure 4 and Figure 5 As shown by Figure 4 and Figure 5 It can be seen that judging from the phenotype, the leaf density of diploid Lanzhou lily is significantly higher than that of autotetraploid, whether in the budding stage or the flowering stage.

[0045] Combined with the measured data, the leaf density and number of flower buds per branch of diploid and autotetraploid Lanzhou lilies grown in the field at an altitude of 1530 meters were statistically analyzed. Figure 6 As shown. Figure 6 It can be seen that the average leaf density of diploid Lanzhou lily is 3.07 pieces / cm, and the average number of flower buds per branch is 3.83; the average leaf density of autotetraploid Lanzhou lily is 1.60 pieces / cm, and the average number of flower buds per branch is 1.33. The statistical results are consistent with the results judged based on phenotype.

[0046] (2) Comparison between planting sites at different altitudes

[0047] The leaf density and number of flower buds per branch of annual Lanzhou lily diploid and autotetraploid were statistically analyzed at different planting sites at different altitudes, as shown in Table 1.

[0048] Table 1 Leaf density and number of flower buds per branch of diploid and autotetraploid lilies of Lanzhou lily at different altitudes

[0049]

[0050] From the data in Table 1, we can see that since the height of lily plants is greatly affected by the altitude of the planting site, the leaf density values ​​of diploid and autotetraploid Lanzhou lily vary greatly between different planting sites at different altitudes. Specifically, when the altitude is high (2179 meters above sea level), the leaf density of diploid Lanzhou lily in the first year of planting is ≥6 leaves / cm, and the leaf density of homotetraploid Lanzhou lily is ≤5 leaves / cm; when the altitude is low (1530 meters above sea level), the leaf density of diploid Lanzhou lily is ≥3 leaves / cm, and the leaf density of homotetraploid Lanzhou lily is ≤2 leaves / cm, but the overall trend of leaf density between different altitudes is consistent with the trend of diploid Lanzhou lily > homotetraploid Lanzhou lily. The number of flower buds per branch of diploid Lanzhou lily and autotetraploid Lanzhou lily is not much different between planting sites at different altitudes, and the overall performance is that the number of flower buds per branch of diploid Lanzhou lily is ≥3, and the number of flower buds per branch of homotetraploid Lanzhou lily is ≤2.

[0051] 4. Construct identification method

[0052] Based on the above statistical analysis, Lanzhou lilies planted at the same altitude are divided into two categories according to the leaf density value: high and low. When the altitude is between 1500 and 1700 meters, a leaf density value ≥3 is defined as high leaf density, and a leaf density value ≤2 is defined as low leaf density; when the altitude is between 1800 and 2600 meters, a leaf density value ≥6 is defined as high leaf density, and a leaf density value ≤5 is defined as low leaf density.

[0053] The ones with high leaf density are diploid, and the ones with low leaf density are autotetraploid.

[0054] For suspected plants that are difficult to distinguish based on leaf density alone, the ploidy of the plant is distinguished based on the number of flower buds per branch. If the number of flower buds per branch is ≥3, it is a diploid, and if the number of flower buds per branch is ≤2, it is an autologous tetraploid.

[0055] 5. Practical application verification of identification methods

[0056] In order to verify the feasibility and accuracy of the identification method constructed by the present invention, the identification method constructed by the present invention was used to verify the ploidy in two Lanzhou lily planting fields at planting altitudes of 2200 meters (high) and 1610 meters (low), respectively. At the same time, the flow cytometer determination method was used as a control.

[0057] The results are shown in Table 2.

[0058] Table 2 Comparison of the consistency of homologous tetraploid rates of Lily of Lanzhou determined by different ploidy identification methods

[0059]

[0060] Note: Tetraploid rate in the table = (number of tetraploid plants / number of tested plants) × 100%; consistency rate = 1 - the difference in tetraploid rate between the two determination methods.

[0061] The above shows that the identification method constructed by the present invention can quickly and efficiently identify diploid and autotetraploid Lanzhou lily with an accuracy of 98% to 100%. Compared with the flow cytometer determination method, it is simple to operate, does not require expensive instruments and specific reagents, is low in cost, and is more practical in the field.

[0062] The above embodiments are only verified by taking the planting site altitude in the range of 1500-1700 meters and 1800-2600 meters as examples. When the ecological environment of the planting site changes, even if the values ​​are not within this range, the identification method based on leaf density and the number of flower buds per branch of the present invention is still effective.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of leaf density and number of flower buds per branch as identification indicators in the rapid identification of homotetraploid Lanzhou lily.

2. A rapid identification method for the homologous tetraploid of Lanzhou lily, characterized in that: The invention comprises the steps of identifying the ploidy of Lanzhou lily according to the leaf density and the number of flower buds per branch.

3. The method according to claim 2, characterized in that The ploidy includes diploid and autotetraploid.

4. The method according to claim 3, characterized in that The Lanzhou lilies are Lanzhou lilies grown at the same altitude.

5. The method according to claim 4, characterized in that The following steps are involved: The Lanzhou lily is divided into two categories, high leaf density and low leaf density, according to the leaf density value, wherein the Lanzhou lily with high leaf density is diploid, and the Lanzhou lily with low leaf density is autotetraploid; when the high leaf density and the low leaf density cannot be distinguished according to the leaf density value, the Lanzhou lily is identified according to the number of flower buds per branch, and the number of flower buds per branch ≥3 is diploid, and the number of flower buds per branch ≤2 is autotetraploid; When the altitude is 1500-1700 meters, a leaf density value ≥3 is a high leaf density, and a leaf density value ≤2 is a low leaf density; when the altitude is 1800-2600 meters, a leaf density value ≥6 is a high leaf density, and a leaf density value ≤5 is a low leaf density.

6. The rapid identification method according to claim 2, characterized in that: The Lanzhou lily is a Lanzhou lily that is planted for the first year.

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