阅读说明：本技术 一种从白沙枇杷实生苗筛选三倍体新种质的方法 (Method for screening triploid new germplasm from white loquat seedling ) 是由 王三红 荣志豪 于 2019-10-10 设计创作，主要内容包括：本发明公开了一种从白沙枇杷实生苗筛选三倍体新种质的方法。该方法包含(1)通过形态学观测,根据白沙枇杷三倍的形态学特征从实生苗群体中初筛三倍体,满足以下条件的为疑似白沙枇杷三倍体；(2)通过流式细胞仪进一步鉴定疑似沙枇杷三倍体白的倍性。本发明溶液配制简单,操作步骤易上手筛选成功率高,结果可靠,与目前应用在枇杷流式倍性筛选上的其他方法相比优势明显。与传统的染色体计数技术相比,应用该方法筛选效率可以提高4-5倍。通过本发明方法,能实现白沙枇杷三倍体实生苗的大规模快速鉴定。(The invention discloses a method for screening triploid new germplasm from white loquat seedlings. The method comprises (1) primarily screening triploid from seedling group according to morphological characteristics three times of white sand loquat by morphological observation, wherein suspected white sand loquat triploid satisfies the following conditions; (2) and further identifying the ploidy of the suspected eriobotrya japonica triploid white by a flow cytometer. The method has the advantages of simple solution preparation, easy operation steps, high screening success rate and reliable result, and has obvious advantages compared with other methods applied to loquat flow type ploidy screening at present. Compared with the traditional chromosome counting technology, the screening efficiency can be improved by 4-5 times by applying the method. The method can realize large-scale rapid identification of the triploid seedlings of the white sand loquat.)

1. A method for screening triploid new germplasm from white loquat seedlings is characterized by comprising the following steps:

(1) through morphological observation, triploid is primarily screened from seedling groups according to morphological characteristics three times of white sand loquats, and suspected white sand loquats meet the following conditions: the leaf area of the same-node fully-unfolded leaf of the triploid loquat is more than 1.3 times larger than that of the diploid, the triploid leaf shape index is 2.7 +/-0.25, and the diploid leaf shape index is 3.5 +/-0.3; the included angle between the triploid leaf and the branch is larger than that of the diploid, and the included angle between the triploid leaf and the branch is 70 degrees +/-10 degrees; the included angle between the diploid leaves and the branches is 44 degrees +/-10 degrees; the triploid leaves are darker in color and sunken in veins; the leaf shape index is leaf length/leaf width;

(2) and further identifying the ploidy of the suspected white sand loquat triploid through a flow cytometer.

2. The method of claim 1, wherein said step (2) of further identifying the ploidy of the suspected white loquat leaf triploid by flow cytometry comprises the steps of:

(a) taking clean tender white loquat leaves, shredding the leaves, adding the leaves into a centrifugal tube, and adding clean zirconia beads and 1.5-2 mL of extracting solution A into the centrifugal tube for sample grinding;

(b) after sample grinding is finished, filtering by using a 400-mesh nylon sieve;

(c) centrifuging the filtrate, discarding supernatant, adding extract B into the precipitate, shaking to remove the precipitate, dyeing at low temperature in dark for 5min, and detecting on machine;

(d) detecting the fluorescence intensity of the PI stained sample by a flow cytometer, acquiring data by using random software CellQuestProTM, and counting the CV value of the data by adopting cytExpert of the flow cytometer;

(e) the ploidy calculation method comprises the following steps: the ploidy level of the sample to be detected is multiplied by the ploidy level of the reference sample multiplied by the fluorescence mean value of the peaks G0/G1 of the sample to be detected, namely the fluorescence mean value of the peaks G0/G1 of the reference sample;

wherein the extracting solution A is obtained by adding 1-2% (w/v) polyvinylpyrrolidone PVP into a nuclear separation buffer solution; the extraction solution B is prepared by adding PI powder into a nuclear separation buffer solution to prepare 0.3-0.5 mg/mL^-1Adding 0.03-0.05 mg/mL of PI dye solution^-1The RNase of (1); the formula of the nuclear separation buffer solution is 8-12 mmol.L^-1MgSO₄·7H₂O，45～50mmol·L^-1KCl，5～8mmol·L^-10.25-0.3% (v/v) Triton X-100.

3. The method of claim 2, wherein the nuclear separation buffer formulation is 10 mmol-L^{- 1}MgSO₄·7H₂O，50mmol·L^-1KCl，5mmol·L^-10.25% (v/v) Triton X-100.

4. The method according to claim 3, wherein extract A is obtained by adding 1% (w/v) polyvinylpyrrolidone PVP to the said nuclear separation buffer; the extract B was prepared by adding PI powder to the above nuclear separation buffer solution to give a solution of 0.5 mg/mL^-10.05 mg/mL of the PI dye solution was added^-1The RNase of (1).

5. The method according to any one of claims 1 to 4, wherein in the step (a), young and tender loquat leaves are taken and washed clean by sterilized double distilled water, water is absorbed by absorbent paper, the leaves are shredded and placed into a centrifuge tube, the positions of veins and the like are avoided, the volume of the leaves is about 1/3 of the centrifuge tube, 6-8 clean zirconia beads are added, the extracting solution A is added to a two-milliliter scale line, the amount of the extracting solution is not less than 1.5mL, a sample grinder is adjusted to 50Hz, the sample grinding time is adjusted to 2 minutes, and sample grinding is started.

6. The method according to any one of claims 1 to 4, wherein the centrifugation in step (c) is performed by a low temperature centrifuge at 4 ℃ and 3000rpm for 5 min.

7. The method according to any one of claims 1 to 4, wherein the amount of the extract B added in step (c) is 0.5 mL/tube.

Technical Field

The invention belongs to the field of biological detection, and relates to a method for screening triploid new germplasm from white loquat seedlings.

Background

The white sand loquat is popular with consumers due to the characteristics of fine and smooth meat quality, high sweetness and the like, and Jiangsu Suzhou is one of the main production areas of the white sand loquat. Most of the white sand loquats have many seeds and are large, so that the eating experience of the white sand loquats is influenced. The problem can be effectively solved by cultivating the triploid white loquat, and the method has important significance for promoting the development of the loquat industry. The seedling screening is an important way for breeding new loquat varieties, and in the current main loquat cultivars: white jade (chiffon flood, 2001), Jiefang (Libi lotus, 1999), Dawuxing (Chenguihu et al, 2002) and the like are all obtained by seedling breeding. In several approaches of loquat triploid breeding, seedling screening is an important approach, in recent years, the Beam national Lu professor (2011) of the university in southwest obtains a natural triploid mutant single plant through a 'one-step method', and then 2 plants are preferably selected from 300 plants to obtain a 'Changbai No. 1' Q11, '77-1' K375, a new excellent triploid seedless loquat line. According to the loquat triploid identification, in the past, chromosome counting is mainly carried out under a microscope through chromosome tabletting of root tips, and the method is not only complicated in process, but also time-consuming and labor-consuming and low in screening efficiency. At present, a method capable of screening triploid new germplasm from white sand loquat seedlings in a large scale is urgently needed.

Disclosure of Invention

The invention aims to provide a method for screening triploid new germplasm from white loquat seedlings, aiming at the defects in the prior art.

The purpose of the invention can be realized by the following technical scheme:

a method for screening triploid new germplasm from white loquat seedlings comprises the following steps:

(1) through morphological observation, triploid is primarily screened from seedling groups according to morphological characteristics three times of white sand loquats, and suspected white sand loquats meet the following conditions: the leaf area of the same-node fully-unfolded leaf of the triploid loquat is more than 1.3 times larger than that of the diploid, the triploid leaf shape index is 2.7 +/-0.25, and the diploid leaf shape index is 3.5 +/-0.3; the included angle between the triploid leaf and the branch is larger than that of the diploid, and the included angle between the triploid leaf and the branch is 70 degrees +/-10 degrees; the included angle between the diploid leaves and the branches is 44 degrees +/-10 degrees; the triploid leaves are darker in color and sunken in veins; the leaf shape index is leaf length/leaf width;

(2) and further identifying the ploidy of the suspected white sand loquat triploid through a flow cytometer.

The step (2) of further identifying the ploidy of the suspected white loquat leaf triploid through a flow cytometer preferably comprises the following steps:

(a) taking clean tender white loquat leaves, shredding the leaves, adding the leaves into a centrifugal tube, and adding clean zirconia beads and 1.5-2 mL of extracting solution A into the centrifugal tube for sample grinding;

(b) after sample grinding is finished, filtering by using a 400-mesh nylon sieve;

(c) centrifuging the filtrate, discarding supernatant, adding extract B into the precipitate, shaking to remove the precipitate, dyeing at low temperature in dark for 5min, and detecting on machine;

(d) detecting the fluorescence intensity of the PI stained sample by a flow cytometer, acquiring data by using random software CellQuestProTM, and counting the CV value of the data by adopting cytExpert of the flow cytometer;

(e) the ploidy calculation method comprises the following steps: the ploidy level of the sample to be detected is multiplied by the ploidy level of the reference sample multiplied by the fluorescence mean value of the peaks G0/G1 of the sample to be detected, namely the fluorescence mean value of the peaks G0/G1 of the reference sample;

wherein the extracting solution A is obtained by adding 1-2% (w/v) polyvinylpyrrolidone PVP into a nuclear separation buffer solution; the extraction solution B is prepared by adding PI powder into a nuclear separation buffer solution to prepare 0.3-0.5 mg/mL^-1Adding 0.03-0.05 mg/mL of PI dye solution^-1The RNase of (1); the formula of the nuclear separation buffer solution is 8-12 mmol.L^-1MgSO₄·7H₂O，45～50mmol·L^-1KCl，5～8mmol·L^-10.25-0.3% (v/v) Triton X-100.

The formula of the nuclear separation buffer solution is preferably 10 mmol.L^-1MgSO₄·7H2O，50mmol·L^-1KCl，5mmol·L^-10.25% (v/v) Triton X-100.

The extract A is further preferably obtained by adding 1% (w/v) polyvinylpyrrolidone PVP into the core separation buffer; preferably, the extract B is prepared by adding PI powder to the nuclear separation buffer solution to prepare 0.5 mg/mL^-10.05 mg/mL of the PI dye solution was added^-1The RNase of (1).

Preferably, in the step (1), young and tender loquat leaves are cleaned by using sterilized double distilled water, water is absorbed by absorbent paper, the leaves are shredded and are placed into a centrifugal tube, leaf veins and other positions are avoided, the volume of the leaves approximately accounts for 1/3 of the centrifugal tube, 6-8 clean zirconia beads are added, extracting solution A to 2mL of scale marks are added, a sample grinding machine is adjusted to 50Hz, the sample grinding time is adjusted to 2min, and sample grinding is started.

The centrifugation in the step (3) is preferably performed by a low-temperature centrifuge at 4 ℃ and 3000rpm for 5 min.

The amount of the extract B to be added in the step (3) is preferably 0.5 mL/tube.

Has the advantages that:

the inventor discovers that the diploid and the triploid of the white sand loquat have great difference in morphological parameters such as the included angle of leaves, the size, the shape of leaves, the length of stem internodes and the like through observation, identification and summarization, so that the suspected white sand loquat triploid is screened through morphological observation, the screening range is greatly reduced, the ploidy of the actual loquat is quickly identified through a flow cytometer, particularly, a nuclear separation buffer solution is optimized, the sampling speed of cells is high, 10000 cells can be collected in 2min in each tube of sample, the requirement of quick screening of the actual loquat is met, the average CV value of a target cell mass is 6.46 percent and is less than 8 percent, and the experimental result is basically reliable. The flow-type screening method applied in the invention has the advantages of simple solution preparation, easy operation steps, low requirements on environmental conditions and methods in the operation process, high screening success rate and reliable results, and has obvious advantages compared with other methods applied to loquat flow-type ploidy screening at present. Compared with the traditional chromosome counting technology, the screening efficiency can be improved by 4-5 times by applying the method. The method can realize large-scale rapid identification of the triploid seedlings of the white sand loquat.

Drawings

FIG. 1 flow fluorescence intensity map of different methods

FIG. 2.48 ploidy identification of plant

A, a fluorescent intensity peak of a diploid loquat control group; b: fluorescence intensity peak of loquat No. 48; c: a fluorescence intensity peak of the actual diploid loquats;

a, chromosome counting of a diploid loquat control group; b: chromosome count of loquat 48

FIG. 3 comparison of stomatal density of diploid and triploid loquats

Note: i: diploid loquat; II: triploid loquats; bar is 50 μm.

Detailed Description