CN102768210A - Identification method of interspecific hybrids of Brassica campestris and Brassica napus - Google Patents

Abstract

The invention discloses an identification method of interspecific hybrids of Brassica campestris and Brassica napus, falling into the technical field of plant cytology. The method includes collecting the 4th-7th leaves of a plant in the seedling stage, tearing to collect hypoderm tissues of lower parts of the leaves, staining with iodine-potassium iodide solution, observing stomata under an optical microscope, measuring major/minor axis values of guard cells, calculating perimeter values of the guard cells with an ellipse model while observing and counting chloroplasts, recording 15 data per leaf, and identifying with parents as controls. For Brassica campestris, stoma guard cells have perimeter smaller than 58.90 micrometer and chloroplast count of 9-11; for Brassica napus, stoma guard cells have perimeter greater than 75.83 micrometer and chloroplast count of 19-20; while, for the to-be-identified material, if more than ten stomata simultaneously satisfy that guard cells have perimeter of 58.90-75.83 micrometer and chloroplast count of 14-16, it is determined that the plant is interspecific hybrids F1 of Brassica campestris and Brassica napus, otherwise it is false hybrid.

Description

Translated fromChinese

白菜型油菜与甘蓝型油菜种间杂种鉴定的方法Method for identification of interspecific hybrids between Brassica napus and Brassica napus

技术领域technical field

本发明属于植物细胞学应用技术领域，具体涉及到鉴别白菜型油菜与甘蓝型油菜种间杂种的方法。The invention belongs to the technical field of plant cytology application, and in particular relates to a method for identifying interspecific hybrids of Chinese cabbage-type rape and Brassica napus.

背景技术Background technique

油菜是世界重要的油料作物，油菜发展，品种是关键，而远缘杂交育种鉴定是选育新品种的一个重要途径，为提高远缘杂交育种真杂种育种的选择效率，及时、准确鉴别杂种F1的真伪，具有重要意义。早期种间杂种鉴定常用的传统方法形态学标记鉴定，形态标记指作物特定的肉眼可见的且简单直观的外部特征或借助简单测试即可识别的某些性状。主要包括植物学性状、农艺性状和抗性等，形态学标记虽然简单直观，但它主要是靠田间观察、记载和测量，不仅费时，而且标记数量少，持续时间长，检定周期长，易受外部环境条件、生育期等因素的影响。油菜形态性状复杂多样，真假杂种难以区分，所以单独依靠形态学方法进行杂种研究还远远不能满足育种需要。Rapeseed is an important oil crop in the world. Variety is the key to the development of rapeseed, and the identification of distant hybrid breeding is an important way to select new varieties. In order to improve the selection efficiency of distant hybrid breeding and true hybrid breeding, timely and accurately identify hybrid F1 Authenticity is of great significance. The traditional method commonly used in the identification of early interspecific hybrids is the identification of morphological markers. Morphological markers refer to crop-specific external characteristics that are visible to the naked eye and simple and intuitive, or certain traits that can be identified with simple tests. It mainly includes botanical traits, agronomic traits and resistance, etc. Although morphological markers are simple and intuitive, they mainly rely on field observation, recording and measurement. External environmental conditions, growth period and other factors. The morphological traits of rapeseed are complex and diverse, and it is difficult to distinguish true and false hybrids. Therefore, morphological methods alone for hybrid research are far from meeting the needs of breeding.

随着细胞遗传学的诞生以及显微技术的改进,杂种鉴定过渡到了细胞学标记领域，由于油菜染色体较小、数目多，种属间材料稀少且差异大，从而增加了染色体制片难度，核型分析较难进行,很多研究仅停留在染色体数目变异方面。要制出全套染色体分散好、不丢失、清晰能记数目的片子，需要娴熟的染色体制片技巧，制片十分耗时，不适用于大量种间杂种真伪的鉴别。油菜染色体数量多传递行为复杂，变异幅度大，有时同一材料计数差别达十几条，而且常表现为非整倍体，因此鉴定结果也还不能令人十分信服。近年来国内外流行通过生化标记-同工酶技术鉴定杂种F1真伪，在油菜种间杂种鉴定上多集中于过氧化物酶(PO)和酯酶(AT)。虽然同工酶技术用于血缘关系复杂的亲子关系真实性鉴定有简便、清晰、可靠、酶带易于分析判断等优点，但由于同工酶是基因表达的直接产物，易受环境、栽培条件和植物生长阶段的影响，而且存在着基因位点数目少、多态性水平低等缺点。从而限制了同工酶技术在油菜种间杂种鉴别直接利用。上世纪80年代起，分子标记（RFLP）技术被作为主要手段来鉴定杂种F1，分子标记直接以DNA的形式表现，在植物体的各个组织、各发育时期均可检测到，不受季节、环境限制，不存在表达与否的问题；标记数量极多，遍及整个基因组，多态性高，自然存在着许多等位变异，有许多分子标记表现为共显性(codom inance)能够鉴别出纯合基因型与杂合基因型，提供完整的遗传信息。但该技术存在难度大、耗时、成本高等缺点。因此，急需一种实用于油菜种间杂种真伪鉴别的快速、准确、成本低的方法。With the birth of cytogenetics and the improvement of microscopic technology, the identification of hybrids has transitioned to the field of cytological markers. Due to the small number and large number of chromosomes in rapeseed, the scarcity and large differences between species materials increase the difficulty of chromosome preparation. Type analysis is difficult to carry out, and many studies only stay in the variation of chromosome number. To produce a complete set of chromosomes that are well dispersed, not lost, and clearly numbered requires skilled chromosome production skills, which are time-consuming and not suitable for identifying the authenticity of a large number of interspecific hybrids. Rapeseed rape has a complex transmission behavior with a large number of chromosomes, and the variation range is large. Sometimes the count difference of the same material reaches more than a dozen, and it often shows aneuploidy, so the identification results are not very convincing. In recent years, it has been popular at home and abroad to identify the authenticity of hybrid F1 by biochemical marker-isoenzyme technology, and the identification of interspecific hybrids in rapeseed mostly focuses on peroxidase (PO) and esterase (AT). Although isozyme technology is used in the authenticity identification of parent-child relationship with complex blood relationship, it has the advantages of simplicity, clarity, reliability, and easy analysis and judgment of enzyme bands, but because isozyme is the direct product of gene expression, it is easily affected by the environment, cultivation conditions and The impact of plant growth stage, and there are shortcomings such as the small number of gene loci and the low level of polymorphism. This limits the direct use of isoenzyme technology in the identification of interspecies hybrids in rapeseed. Since the 1980s, molecular marker (RFLP) technology has been used as the main method to identify hybrid F1. Molecular markers are directly expressed in the form of DNA, and can be detected in various tissues and developmental stages of plants, regardless of seasons and environments. Limitation, there is no problem of expression or not; the number of markers is extremely large, throughout the entire genome, polymorphism is high, there are many allelic variations naturally, and there are many molecular markers showing codominance (codom inance) that can identify homozygous Genotype and heterozygous genotype, providing complete genetic information. However, this technology has disadvantages such as difficulty, time-consuming, and high cost. Therefore, there is an urgent need for a fast, accurate, and low-cost method for authenticity identification of rapeseed interspecific hybrids.

据前人研究，叶片气孔保卫细胞长度与叶绿体数与染色体倍性呈正相关关系，刘成洪等(用气孔保卫细胞周长鉴定了甘蓝型油菜植物倍性水平.上海农业学报，2002,18（3）：35-38)测量了甘蓝型油菜品种（2n）和单倍体（n）植物叶片的气孔保卫细胞纵横经长度，采用了公式

计算了保卫细胞周长。他们指出保卫细胞周长值可以作为鉴定甘蓝型油菜染色体倍性水平的指标，张菊平等在辣椒倍性鉴定方面也得到了相似的结论。叶片保卫细胞叶绿体计数法操作简便、结果准确、鉴定工作效率高，已有应用到马铃薯、烟草、花生、葡萄等作物的研究，也有将叶绿体数作为鉴定桑叶和枇杷等作物倍性分界指标的研究．但是至今未见利用叶片气孔保卫细胞周长及叶绿体数来鉴别油菜种间杂种真伪的报道。为了能够快速有效的鉴别油菜种间杂种真伪，加快油菜远缘杂交杂交育种进程，我们以种间杂种植株叶片为材料，研究了白菜型油菜、甘蓝型油菜与其杂种F1保卫细胞周长，叶绿体数之间的相关性，进行了显著差异性分析，找到了亲本及F1细胞周长及叶绿体数的分界域值，建立了将气孔保卫细胞周长及叶绿体数相结合快速鉴别种间杂种F1真伪的技术，使假杂种尽早被剔除，解决了盲目种间杂种选择问题，节约了实验成本及时间，减少了人工及土地等费用，使油菜种间杂种大批量快速有效被鉴别，从而使远缘杂交技术体系在油菜遗传育种中得以有效应用。According to previous studies, the length of leaf stomatal guard cells is positively correlated with the number of chloroplasts and chromosome ploidy, Liu Chenghong et al. : 35-38) measured the longitudinal and transverse lengths of the stomatal guard cells of Brassica napus varieties (2n) and haploid (n) plant leaves, using the formula

Guard cell perimeters were calculated. They pointed out that the guard cell perimeter value can be used as an index to identify the ploidy level of Brassica napus. Zhang Juping also got a similar conclusion in the identification of ploidy in pepper. The leaf guard cell chloroplast counting method is easy to operate, accurate in results, and high in identification efficiency. It has been applied to crops such as potatoes, tobacco, peanuts, and grapes. There are also studies that use the number of chloroplasts as a ploidy dividing index for identifying crops such as mulberry leaves and loquats. Research. But so far, there is no report on using leaf stomatal guard cell perimeter and chloroplast number to identify rapeseed interspecific hybrids. In order to quickly and effectively identify the authenticity of rapeseed interspecific hybrids and speed up the process of distant hybridization and hybridization of rapeseed, we used interspecific hybrid plant leaves as materials to study the guard cell perimeter and chloroplast According to the correlation between the number of stomatal guard cells and the number of chloroplasts, a significant difference analysis was carried out to find the cut-off value of parental and F1 cell perimeter and chloroplast number, and a method was established to quickly identify interspecific F1 true hybrids by combining the stomatal guard cell perimeter and chloroplast number. Pseudo technology, so that false hybrids can be eliminated as soon as possible, solve the problem of blind interspecific hybrid selection, save experimental costs and time, reduce labor and land costs, and quickly and effectively identify large batches of rapeseed interspecific hybrids, so that distant The hybrid technology system has been effectively applied in rapeseed genetics and breeding.

发明内容Contents of the invention

本发明的目的在于避免现有技术的不足，提供一种白菜型油菜与甘蓝型油菜种间杂种鉴定的方法。主要内容为：测量确定亲本材料及种间杂种的叶片气孔保卫细胞周长，记数亲本及种间杂种的叶绿体数目，确定亲本及杂种的叶片气孔保卫细胞周长值范围与叶绿体数目范围，根据两个性状值的测量，计数结果，以其分界域值为参数建立了快速简便大批量鉴别白菜型油菜与甘蓝型油菜种间杂种真伪的有效方法。The purpose of the present invention is to avoid the deficiencies of the prior art and provide a method for identifying interspecific hybrids of Chinese cabbage rape and Brassica napus. The main contents are as follows: measure and determine the perimeter of stomatal guard cells of the parent materials and interspecific hybrids, count the number of chloroplasts of the parents and interspecific hybrids, determine the range of the stomatal guard cell perimeter and the number of chloroplasts of the parents and hybrids, according to The measurement and counting results of the two traits, and the cut-off value as a parameter established an effective method for quickly and easily identifying interspecific hybrids between Brassica napus and Brassica napus in large quantities.

为实现上述目的，本发明采取的技术方案为：一种白菜型油菜与甘蓝型油菜种间杂种鉴定的方法，其特征在于包括以下步骤：In order to achieve the above object, the technical solution adopted by the present invention is: a method for identification of interspecific hybrids of Chinese cabbage-type rape and Brassica napus, which is characterized in that it comprises the following steps:

（1）1%碘-碘化钾染色液的配置；(1) Configuration of 1% iodine-potassium iodide staining solution;

（2）在幼苗期采摘白菜型油菜、甘蓝型油菜以及白菜型油菜与甘蓝型油菜种间杂种F1第4-7叶位处的平展叶片,装入塑料袋中，在5-10min内放入准备好的0°C以下冰盒中，带回，叶片经蒸馏水清洗后，置于通风橱中吹干叶片表面水分，用刀片切叶片下面部位，用镊子撕取下表皮气孔,放在载玻片上，使其平展开，滴1滴1%碘-碘化钾染色液，染色30～60秒，压片；(2) At the seedling stage, pick the flat leaves at the 4th-7th leaf position of Chinese cabbage rape, Brassica napus and interspecific hybrid F1 between Chinese cabbage rape and Brassica napus, put them in plastic bags, and put them in 5-10min Put the leaves in a prepared ice box below 0°C and bring them back. After the leaves are cleaned with distilled water, put them in a fume hood to dry the water on the surface of the leaves. Spread it flat on the slice, drop 1 drop of 1% iodine-potassium iodide staining solution, stain for 30-60 seconds, and press into the slice;

（3）将步骤（2）制好的玻片在光学显微镜下用测微尺测出气孔保卫细胞横径A、纵径B值，并作以记录；同时观察计数叶表皮气孔保卫细胞内的叶绿体数；每张叶片观察14-16个气孔，以4-5张叶片的平均值作为该品种的保卫细胞叶绿体数值；(3) Use a micrometer to measure the transverse diameter A and longitudinal diameter B of the stomatal guard cells on the slide prepared in step (2) under an optical microscope, and record them; at the same time, observe and count the stomatal guard cells in the leaf epidermis Chloroplast number: 14-16 stomata are observed on each leaf, and the average value of 4-5 leaves is used as the guard cell chloroplast value of the variety;

（4）用椭圆模型近似公式

计算出保卫细胞周长，式中：L为保卫细胞周长，A为保卫细胞横径，B为保卫细胞纵径；(4) Approximate formula with ellipse model

Calculate the perimeter of the guard cells, where: L is the perimeter of the guard cells, A is the transverse diameter of the guard cells, and B is the longitudinal diameter of the guard cells;

（5）用气孔保卫细胞周长及叶绿体数量分界域值来判定种间杂种真伪：以其亲本作为对照，若每片叶有10个以上气孔同时满足保卫细胞周长介于58.90um～75.83um，叶绿体个数为14～16个者，判定该植株为白菜型油菜与甘蓝型油菜种间杂种F1。(5) Determine the authenticity of interspecific hybrids by using the perimeter of stomatal guard cells and the number of chloroplasts: using their parents as a control, if each leaf has more than 10 stomata and the guard cell perimeter is between 58.90um and 75.83um, If the number of chloroplasts is 14-16, the plant is judged to be the interspecific hybrid F1 of Brassica napus and Brassica napus.

根据亲本及F1叶片气孔保卫细胞的周长及叶绿体数的相关性，将气孔保卫细胞周长及叶绿体数相结合，通过亲本及种间杂种F1气孔保卫细胞周长及叶绿体数的分界域值鉴定种间杂种。According to the correlation between the stomatal guard cell perimeter and chloroplast number of the parents and F1 leaves, the stomatal guard cell perimeter and chloroplast number were combined to identify the cut-off value of the stomatal guard cell perimeter and chloroplast number of the parent and interspecific F1 hybrids Interspecific hybrids.

本发明的有益效果：Beneficial effects of the present invention:

1.与传统的形态学鉴定相比，本发明明显提高了油菜种间杂种鉴定的准确度，在种间杂种选择时，使用本发明建立的气孔保卫细胞周长及叶绿体数相结合的方法，能够准确的把真杂种鉴别出来，尽早剔除掉假杂种，不需要太多的经验来判定，易操作掌控且不受环境、生育期等因素的影响。1. Compared with the traditional morphological identification, the present invention obviously improves the accuracy of interspecific hybrid identification of rapeseed, and when the interspecific hybrid is selected, the stomatal guard cell perimeter and chloroplast number combination method established by the present invention is used, It can accurately identify the real hybrids and eliminate the false hybrids as soon as possible. It does not require too much experience to judge. It is easy to operate and control and is not affected by factors such as environment and growth period.

2.采用本发明建立的气孔保卫细胞周长及叶绿体数相结合的方法，约6min就可以完成一个植株鉴定，省时，鉴定效率高。2. By adopting the method of combining stomatal guard cell perimeter and chloroplast number established by the present invention, a plant identification can be completed in about 6 minutes, which saves time and has high identification efficiency.

3.本发明建立的气孔保卫细胞周长及叶绿体数相结合的方法技术简便，易操作，不需要娴熟的制片技术，且成本低，比起染色体压片法，每10000株苗就要节约成本8373元。若采用细胞学染色体制片法，则需要相当熟练的制片技术。没有一定细胞学基础的工作人员难以完成本项实验。3. The method for combining the stomatal guard cell perimeter and the number of chloroplasts established by the present invention is simple and easy to operate, does not require skilled sheet-making techniques, and has low cost. Compared with the chromosome compression method, every 10,000 seedlings will save The cost is 8373 yuan. If the cytology chromosome preparation method is used, quite skilled production techniques are required. It is difficult for staff without a certain basis of cytology to complete this experiment.

附图说明Description of drawings

图1白菜型油菜气孔保卫细胞叶绿体数；Figure 1 The number of chloroplasts in stomatal guard cells of Chinese cabbage-type rapeseed;

图2白菜型油菜及甘蓝型油菜种间杂种F1气孔保卫细胞叶绿体数；Figure 2 The number of chloroplasts in stomatal guard cells of F1 interspecific hybrids of Brassica napus and Brassica napus;

图3甘蓝型油菜气孔保卫细胞叶绿体数。Figure 3 The number of chloroplasts in stomatal guard cells of Brassica napus.

具体实施方式Detailed ways

以下对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。The principles and features of the present invention are described below, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

实施例1：一种白菜型油菜与甘蓝型油菜种间杂种鉴定的方法，其包括以下步骤：Embodiment 1: a kind of method for interspecific hybrid identification of Chinese cabbage-type rape and Brassica napus, it comprises the following steps:

（1）1%碘-碘化钾染色液的配置，称取1g碘倒入小烧杯中，加80ml蒸馏水进行溶解后再加2g碘化钾；定容至100ml备用；(1) For the configuration of 1% iodine-potassium iodide staining solution, weigh 1g of iodine and pour it into a small beaker, add 80ml of distilled water to dissolve, then add 2g of potassium iodide; set the volume to 100ml for later use;

（2）在幼苗期采摘白菜型油菜陇油9号、甘蓝型油菜vision以及白菜型油菜陇油9号与甘蓝型油菜vision种间杂种F1第6叶位处的平展叶片,装入塑料袋中，在5-10min内放入准备好的0°C以下冰盒中，带回，叶片经蒸馏水清洗后，置于通风橱中吹干叶片表面水分，用刀片切叶片下面部位，用镊子撕取下表皮气孔,放在载玻片上，使其平展开，滴1滴1%碘-碘化钾染色液，染色30～60秒，压片；(2) Pick the flat leaves at the sixth leaf position of the interspecific hybrid F1 of Chinese cabbage-type rapeseed Longyou No. 9, Brassica napus vision and the interspecific hybrid F1 of Chinese cabbage-type rapeseed No. 9 and Brassica napus at the seedling stage, and put them into plastic bags , put it in a prepared ice box below 0°C within 5-10 minutes, and bring it back. After the leaves are washed with distilled water, put them in a fume hood to dry the water on the surface of the leaves, cut the lower part of the leaves with a blade, and tear them off with tweezers Lower epidermal stomata, put it on a glass slide, spread it flat, drop 1 drop of 1% iodine-potassium iodide staining solution, stain for 30-60 seconds, and press;

（3）将步骤（2）制好的玻片在光学显微镜40倍物镜头下，用测微尺测出气孔保卫细胞横径A、纵径B值，并作以记录；同时观察计数叶表皮气孔保卫细胞内的叶绿体数；每张叶片观察15个气孔，由于叶绿体中的淀粉粒被染液染成蓝黑色，很容易被分辨出来；以5张叶片的平均值作为该品种的保卫细胞叶绿体数值；(3) Measure the transverse diameter A and longitudinal diameter B values of the stomatal guard cells with a micrometer under an optical microscope with a 40-fold objective lens on the slide prepared in step (2), and record them; at the same time, observe and count leaf epidermis The number of chloroplasts in stomatal guard cells; 15 stomata are observed on each leaf, and the starch grains in chloroplasts are easily distinguished because they are stained blue-black by the dye solution; the average value of 5 leaves is used as the guard cell chloroplast of this variety value;

（4）用椭圆模型近似公式

计算出保卫细胞周长，式中：L为保卫细胞周长，A为保卫细胞横径，B为保卫细胞纵径；(4) Approximate formula with ellipse model

Calculate the perimeter of the guard cells, where: L is the perimeter of the guard cells, A is the transverse diameter of the guard cells, and B is the longitudinal diameter of the guard cells;

（5）用气孔保卫细胞周长及叶绿体数量分界域值来判定种间杂种真伪：以其亲本陇油9号与vision作为对照，若每片叶有10个以上气孔同时满足保卫细胞周长介于58.90um～75.83um，叶绿体个数为14～16个者，判定该植株为白菜型油菜与甘蓝型油菜种间杂种F1。(5) Determine the authenticity of interspecific hybrids by using the perimeter of stomatal guard cells and the number of chloroplasts: compared with the parent Longyou No. 9 and vision, if each leaf has more than 10 stomata and the guard cell perimeter is between 58.90um to 75.83um, and the number of chloroplasts is 14 to 16, it is judged that the plant is the interspecific hybrid F1 of Chinese cabbage type rape and Brassica napus.

将步骤（3）所得到的气孔保卫细胞周长及叶绿体数，通过T测验得：亲本与杂种F1有着显著差异，存在着明显的分界；即有70%以上的白菜型油菜陇油9号保卫细胞周长小于58.90um，甘蓝型油菜vision保卫细胞周长大于75.83um，白菜型油菜与甘蓝型油菜杂种F1保卫细胞周长介于58.90-75.83um之间（见表1）；The stomatal guard cell perimeter and the number of chloroplasts obtained in step (3) were tested by T test: there is a significant difference between the parent and the hybrid F1, and there is a clear boundary; that is, more than 70% of the guard cells of the Chinese cabbage type rape Longyou No. 9 The cell perimeter is less than 58.90um, the guard cell perimeter of Brassica napus vision is greater than 75.83um, and the guard cell perimeter of Brassica napus and Brassica napus hybrid F1 is between 58.90-75.83um (see Table 1);

表1白菜型油菜陇油9号、甘蓝型油菜vision与陇油9号×vision种间杂种F1气孔保卫细胞周长值Table 1 The stomatal guard cell perimeter values of F1 interspecific hybrids of Brassica napus Longyou 9, Brassica napus vision and Longyou 9×vision

表1说明：Table 1 explains:

（1）同一品种随机取样5株，sv为变异系数。亲本白菜型油菜陇油9号、甘蓝型油菜vision与其种间杂种F1气孔保卫细胞周长，同一品种不同植株间变异程度（sv）都不大，小于10%.。(1) Five plants of the same variety were randomly sampled, and sv is the coefficient of variation. The stomatal guard cell perimeter of parental Chinese cabbage-type rape Longyou 9, Brassica napus vision and its interspecific hybrid F1, the degree of variation (sv) among different plants of the same variety is not large, less than 10%.

（2）亲本白菜型油菜陇油9号、甘蓝型油菜vision与其种间杂种F1气孔保卫细胞周长在α=0.01水平上极有着相著差异。(2) The stomatal guard cell perimeters of parental Chinese cabbage-type rape Longyou 9, Brassica napus vision and their interspecific hybrids F1 were significantly different at α=0.01 level.

白菜型油菜陇油9号叶绿体数在10-12左右，白菜型油菜与甘蓝型油菜杂种F1叶绿体数在15-16个左右，甘蓝型油菜vision叶绿体数在19-20范围内。亲本与杂种F1之间叶绿体数也达到了显著水平（见表2）。The number of chloroplasts in Chinese cabbage-type rape Longyou No. 9 is about 10-12, the number of chloroplasts in F1 hybrid between Chinese cabbage-type rape and Brassica napus is about 15-16, and the number of chloroplasts in Brassica napus vision is in the range of 19-20. The number of chloroplasts between the parents and the hybrid F1 also reached a significant level (see Table 2).

表2白菜型油菜陇油9号、甘蓝型油菜vision与陇油9号×vision种间杂种F1叶绿体数Table 2 Number of chloroplasts in F1 interspecific hybrids of Chinese cabbage-type rapeseed Longyou 9, Brassica napus vision and Longyou 9×vision

表2说明;Table 2 Description;

（1）同一品种随机取样5株，每株观察100个气孔，统计其叶绿体数。(1) Randomly sample 5 plants of the same variety, observe 100 stomata of each plant, and count the number of chloroplasts.

（2）同一品种不同植株间气孔保卫细胞叶绿体数变异系数十分接近。(2) The coefficient of variation of stomatal guard cell chloroplast number among different plants of the same variety is very close.

（3）同一品种不同植株间没有显著差异(3) There is no significant difference among different plants of the same variety

（4）不同品种间达到了极显著水平。(4) There was a very significant level among different varieties.

见图1白菜型油菜气孔保卫细胞叶绿体数为9个；See Figure 1. The number of chloroplasts in stomatal guard cells of Chinese cabbage type rape is 9;

见图2白菜型油菜及甘蓝型油菜种间杂种F1气孔保卫细胞叶绿体数为15个；See Figure 2. The number of stomatal guard cell chloroplasts in F1 interspecific hybrids of Chinese cabbage-type rape and Brassica napus is 15;

见图3甘蓝型油菜气孔保卫细胞叶绿体数为19个。See Figure 3. There are 19 chloroplasts in stomatal guard cells of Brassica napus.

用分子标记法法将上述已鉴别出来的材料进行进一步验证，达到了97.8.%的吻合率，说明气孔保卫细胞周长及叶绿体数相结合技术可以直接用于鉴别白菜型油菜与甘蓝型油菜种间杂种的真伪。不必再用其它方法进行判定。The above-mentioned identified materials were further verified by molecular marker method, and the coincidence rate reached 97.8%. The authenticity of hybrids. There is no need to use other methods to judge.

实施例2：一种白菜型油菜与甘蓝型油菜种间杂种鉴定的方法，在步骤（2）中，在幼苗期采摘白菜型油菜陇油9号、甘蓝型油菜vision以及白菜型油菜陇油9号与甘蓝型油菜vision种间杂种F1第4片叶位处的平展叶片,装入塑料袋中；其余与实施例1相同。Example 2: A method for identification of interspecific hybrids between Chinese cabbage-type rape and Brassica napus, in step (2), picking Chinese cabbage-type rape Longyou No. 9, Brassica napus vision and Brassica napus Longyou 9 at the seedling stage No. and Brassica napus vision interspecific hybrid F1 The flattened leaves at the 4th leaf position were packed into plastic bags; the rest were the same as in Example 1.

验证例：Verification example:

鉴定方法如实施例1，将现有40粒刚收获的白菜型油菜延油2号与甘蓝型油菜秦川冬甘1的种间杂种，用上述方法对这批种子进行了鉴定。The identification method is as in Example 1. The existing 40 interspecific hybrids of Chinese cabbage-type rape Yanyou 2 and Brassica napus Qinchuan Donggan 1 were identified by the above-mentioned method.

实验步骤：Experimental steps:

将该40粒种子置于培养皿中，喷少量水，让其发芽，待根长长到4mm左右时，移入营养钵，置组培室生长，室温控制在25°C左右。待幼苗期，随机取其第4-7片叶位处的幼叶，按照上述实验步骤进行本次试验。统计结果见表3：This 40 seeds are placed in the petri dish, spray a small amount of water, allow it to germinate, when the root grows to about 4mm, move into the nutrient bowl, put the tissue culture room to grow, room temperature is controlled at about 25 ℃. At the seedling stage, the young leaves at the 4th to 7th leaf positions were randomly selected, and this test was carried out according to the above-mentioned experimental procedures. The statistical results are shown in Table 3:

表3利用白菜型油菜延油2号与甘蓝型油菜秦川冬甘1种间杂种对本发明的验证Table 3 Verification of the present invention by utilizing the interspecific hybrid of Chinese cabbage-type rape Yanyou No. 2 and Brassica napus Qinchuan Donggan

表3说明：Table 3 explains:

(1)+代表种间杂种是真杂种，-代表种间杂种是假杂种；(1) + represents that the interspecific hybrid is a true hybrid, and - represents that the interspecific hybrid is a false hybrid;

(2)同时满足气孔保卫细胞周长在58.0-75.83um范围内，且叶绿体数在14-16个两个条件时，认定为真杂种，只符合其中之一条件时，认定为假杂种。(2) When the perimeter of stomatal guard cells is within the range of 58.0-75.83um and the number of chloroplasts is 14-16, it is considered as a true hybrid, and when only one of the conditions is met, it is considered as a pseudo-hybrid.

（3）在被鉴定的40粒种子中，有37个植株其气孔保卫细胞周长及叶绿体数均落在本发明的界域范围内，即有37个植株是真的种间杂种植株。准确率达到了92.5%。(3) Among the 40 identified seeds, the stomatal guard cell perimeter and the number of chloroplasts of 37 plants fell within the boundaries of the present invention, that is, 37 plants were true interspecific hybrid plants. The accuracy rate reached 92.5%.

比较例：Comparative example:

我们将常规方法与本发明进行了对比，见表4：We have compared conventional method with the present invention, see table 4:

表45种方法鉴定油菜种间杂种真伪比较Table 45 methods to identify the authenticity of interspecific hybrids in rapeseed

其结果明显，本发明操作简单，成本低，单株时间仅为6min/株，准确率高，比起染色体压片法，每10000株苗就要节约成本8373元，不需要娴熟的制片技术，适用于大批量种间杂交材料的鉴定。The results are obvious. The present invention is simple in operation, low in cost, and the single plant time is only 6 minutes per plant, and the accuracy rate is high. Compared with the chromosome pressing method, the cost of every 10,000 seedlings will be saved by 8373 yuan, and no skilled production technology is required. , suitable for the identification of large quantities of interspecific hybrid materials.

以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.

Claims (1)

Translated fromChinese

1.一种白菜型油菜与甘蓝型油菜种间杂种鉴定的方法，其特征在于包括以下步骤：1. a method for interspecific hybrid identification of Chinese cabbage-type rape and Brassica napus, is characterized in that comprising the following steps:（1）1%碘-碘化钾染色液的配置；(1) Configuration of 1% iodine-potassium iodide staining solution;（2）在幼苗期采摘白菜型油菜、甘蓝型油菜以及白菜型油菜与甘蓝型油菜种间杂种F1第4-7叶位处的平展叶片,装入塑料袋中，在5-10min内放入准备好的0°C以下冰盒中，带回，叶片经蒸馏水清洗后，置于通风橱中吹干叶片表面水分，用刀片切叶片下面部位，用镊子撕取下表皮气孔,放在载玻片上，使其平展开，滴1滴1%碘-碘化钾染色液，染色30～60秒，压片；(2) At the seedling stage, pick the flat leaves at the 4th-7th leaf position of Chinese cabbage rape, Brassica napus and interspecific hybrid F1 between Chinese cabbage rape and Brassica napus, put them in plastic bags, and put them in 5-10min Put the leaves in a prepared ice box below 0°C and bring them back. After the leaves are cleaned with distilled water, put them in a fume hood to dry the water on the surface of the leaves. Spread it flat on the slice, drop 1 drop of 1% iodine-potassium iodide staining solution, stain for 30-60 seconds, and press into the slice;（3）将步骤（2）制好的玻片在光学显微镜下用测微尺测出气孔保卫细胞横径A、纵径B值，并作以记录；同时观察计数叶表皮气孔保卫细胞内的叶绿体数；每张叶片观察14-16个气孔，以4-5张叶片的平均值作为该品种的保卫细胞叶绿体数值；(3) Use a micrometer to measure the transverse diameter A and longitudinal diameter B of the stomatal guard cells on the slide prepared in step (2) under an optical microscope, and record them; at the same time, observe and count the stomatal guard cells in the leaf epidermis Chloroplast number: 14-16 stomata are observed on each leaf, and the average value of 4-5 leaves is used as the guard cell chloroplast value of the variety;（4）用椭圆模型近似公式

计算出保卫细胞周长，式中：L为保卫细胞周长，A为保卫细胞横径，B为保卫细胞纵径；(4) Approximate formula with ellipse model

Calculate the perimeter of the guard cells, where: L is the perimeter of the guard cells, A is the transverse diameter of the guard cells, and B is the longitudinal diameter of the guard cells;（5）用气孔保卫细胞周长及叶绿体数量分界域值来判定种间杂种真伪：以其亲本作为对照，若每片叶有10个以上气孔同时满足保卫细胞周长介于58.90um～75.83um，叶绿体个数为14～16个者，判定该植株为白菜型油菜与甘蓝型油菜种间杂种F1。(5) Determine the authenticity of interspecific hybrids by using the perimeter of stomatal guard cells and the number of chloroplasts: using their parents as a control, if each leaf has more than 10 stomata and the guard cell perimeter is between 58.90um and 75.83um, If the number of chloroplasts is 14-16, the plant is judged to be the interspecific hybrid F1 of Brassica napus and Brassica napus.

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