阅读说明：本技术 一种早中熟苹果杂种苗耐高温着色早期选择方法 (Early-stage selection method for high-temperature-resistant coloring of early-medium-maturing apple seedlings ) 是由 何平 常源升 李林光 王海波 王森 何晓文 于 2021-09-22 设计创作，主要内容包括：本发明公开了一种早中熟苹果杂种苗耐高温着色早期选择方法。属于林业种植技术领域。包括：杂种苗田间选择；实验材料选择和处理；筛选样品中代谢物质增加,激素水平上升,褪黑素合成相关基因、MYB转录因子、类黄酮合成途径中的F3H、DFR基因表达上调的植株作为选择对象。利用果实与叶片间的相关性,进行杂种苗的早期选择和预先选择,淘汰不良类型,减少供选植株的数量,提高育种效率。(The invention discloses a high-temperature-resistant coloring early selection method for early-medium-maturing apple seedlings. Belongs to the technical field of forestry planting. The method comprises the following steps: selecting a hybrid seedling field; selecting and processing experimental materials; screening plants with increased metabolites, increased hormone levels and up-regulated melatonin synthesis related genes, MYB transcription factors, F3H in flavonoid synthesis pathways and DFR gene expression in the samples as selection objects. Early selection and pre-selection of hybrid seedlings are performed by utilizing the correlation between fruits and leaves, bad types are eliminated, the number of selected plants is reduced, and the breeding efficiency is improved.)

1. The early selection method for the high-temperature-resistant coloring of the early-medium-maturing apple seedlings is characterized by comprising the following steps of:

(1) selecting a hybrid seedling field;

(2) selecting and processing experimental materials;

(3) detecting and analyzing, and screening plants with at least 10 indexes of metabolite, hormone level and coloring related gene expression level up-regulated by more than 2 times as selection objects.

2. The early selection method for early-stage high-temperature-resistant and high-color-resistant hybrid apple seedlings according to claim 1, wherein the hybrid seedlings in the step (1) are selected in the field: and (4) carrying out biological morphology investigation on the field hybrid seedlings, removing seedlings with serious diseases and abnormal growth, and selecting strong plants.

3. The early selection method for the high temperature-resistant coloring of the early middle aged apple seedlings according to claim 1, wherein the experimental material selection in the step (2): functional leaves growing on annual branches are selected as test materials.

4. The early selection method for the high temperature-resistant coloring of the early middle aged apple seedlings according to claim 1, wherein the treatment in the step (2): and (3) wrapping the petioles of the selected functional leaves with sterile cotton balls, spraying water for moisturizing, placing the leaves in an illumination incubator at 40 ℃ under 5000LX normal light for treating for 8-12 hours, and storing at room temperature as a control.

5. The early selection method for the high temperature-resistant coloring of the early middle aged apple seedlings according to claim 1, wherein the metabolic substances in the step (3): the photosynthetic pigment content of leaves, catalase CAT, superoxide dismutase SOD, peroxidase POD, total amount of phenols, proline, soluble sugar and soluble protein.

6. The early selection method for high temperature-resistant coloring of early middle aged apple seedlings according to claim 1, wherein the hormone in step (3): melatonin, indoleacetic acid, salicylic acid hormones.

7. The early selection method for the high temperature-resistant coloring of the early middle aged apple seedlings according to claim 1, wherein the coloring-related genes in the step (3): several of the SNAT and HIOMT melatonin synthesis genes, MYB transcription factors, F3H in the flavonoid synthesis pathway, DFR genes.

Technical Field

The invention relates to the technical field of forestry planting, in particular to a high-temperature-resistant coloring early selection method for early-medium-maturing apple seedlings.

Background

The apple is one of the main fruit trees planted in the world, and the output and the cultivation area of the apple in China account for more than half of the total amount of the whole world. Various adversity stresses are often accompanied in the apple cultivation process, and certain influences are caused on the growth, development, yield and quality of apples.

The early-middle-ripe apple fruits are in a high-temperature period in summer in the mature period, so that the color difference and the low quality of the fruits are caused, and the development of the cultivation of the early-middle-ripe apples is disturbed.

The breeding of the early-medium maturing variety with high temperature resistance and excellent coloring has very important significance for the benign sustainable development of the apple industry. But the problems of poor high-temperature coloring, poor quality, intolerance of storage and transportation, fruit cracking and fruit frying in the mature period of the current cultivated early-medium ripe apple varieties and the like.

Therefore, the problem to be solved by those skilled in the art is how to provide a high temperature-resistant coloring early selection method for early-medium-maturing apple seedlings.

Disclosure of Invention

In view of the above, the invention provides an early selection method for high temperature-resistant coloring of early-medium-maturing apple seedlings. Selecting plant leaves from hybrid materials, under the premise of high-temperature treatment, carrying out early selection and pre-selection of hybrid seedlings by utilizing the relativity between fruits and the leaves through comprehensive, multi-angle, scientific and rigorous index detection including photosynthetic pigments, various antioxidant enzymes, organic osmotic adjusting substances, plant hormone levels, related expression genes and the like, eliminating bad types, reducing the number of selected plants and improving the breeding efficiency.

In order to achieve the purpose, the invention adopts the following technical scheme:

a high-temperature-resistant coloring early selection method for early-medium-maturing apple seedlings comprises the following steps:

(1) selecting a hybrid seedling field;

(2) selecting and processing experimental materials;

(3) detecting and analyzing, and screening plants with at least 10 indexes of metabolite, hormone level and coloring related gene expression level up-regulated by more than 2 times as selection objects.

Preferably: step (1), field selection of hybrid seedlings: and (4) carrying out biological morphology investigation on the field hybrid seedlings, removing seedlings with serious diseases and abnormal growth, and selecting strong plants.

Has the advantages that: and abnormal plants are eliminated, and the workload is reduced.

Preferably: step (2), experimental material selection: functional leaves growing on annual branches are selected as test materials.

Has the advantages that: the consistency of the selected materials is ensured, and the experimental error is reduced.

Step (2) processing: and (3) wrapping the petioles of the selected functional leaves with sterile cotton balls, spraying water for moisturizing, placing the leaves in an illumination incubator at 40 ℃ under 5000LX normal light for treating for 8-12 hours, and storing at room temperature as a control.

Has the advantages that: the laboratory simulates the high-temperature environment in the field, and the accuracy of later detection is facilitated.

Preferably: step (3) metabolic substance: the photosynthetic pigment content of leaves, catalase CAT, superoxide dismutase SOD, peroxidase POD, total amount of phenols, proline, soluble sugar and soluble protein.

Preferably: step (3) hormone: melatonin, indoleacetic acid, salicylic acid hormones. Preferably: and (3) coloring related genes: several of the SNAT and HIOMT melatonin synthesis genes, MYB transcription factors, F3H in the flavonoid synthesis pathway, DFR genes.

According to the technical scheme, compared with the prior art, the invention discloses the high-temperature-resistant coloring early selection method for the early-medium-maturing apple hybrid seedlings.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing of a fruit tree provided by the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses an early selection method for high-temperature-resistant coloring of early-medium-maturing apple seedlings.

The detection of each index related in the embodiments is not a conventional method, and is not described herein again, for example:

the index detection method comprises the following steps:

1, measuring the photosynthetic pigment content of leaves:

picrofen, Huanghua pears, Quyuanzu, etc. spectrophotometry measures the appropriate concentration of the extract of chlorophyll content. 1965-1974.

2 Catalase CAT assay:

catalase (CAT) activity was determined by reference to the method of Nakano.

Nakano Y，Asada K.Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts.Plant Cell Physiol，1981：867-880.

3 determination of superoxide dismutase SOD:

the activity of superoxide dismutase (SOD) was measured by nitro blue tetrazolium reduction, as described for plum synbiotic.

Plum symphysis, modern plant physiology 3 edition, beijing: higher education publishers, 2012.

4 peroxidase POD assay:

peroxidase (POD) activity was measured by guaiacol method, according to the method of plum symposium.

Plum symphysis, modern plant physiology 3 edition, beijing: higher education publishers, 2012.

5, total phenolic substance determination:

the phenolic compounds were determined by HPLC.

Plum, small prescription, Zhangzhilian, guidance of plant physiology experiments, 5 th edition, Beijing: higher education publishers, 2016.

6, proline determination:

ninhydrin color-developing spectrophotometry is used for measuring the proline content, and the reference is made to the plum syngamy method.

Plum symphysis, modern plant physiology 3 edition, beijing: higher education publishers, 2012.

7 soluble sugar determination:

the content of soluble sugar is measured by adopting an anthrone colorimetric method.

Liuhai ying, royal hua, treeganghai, etc. improvements of soluble sugar content determination (anthrone method) experiments, laboratory science, 2013, 16 (2): 19-20.

8 soluble protein assay:

coomassie Brilliant blue was used to determine the soluble protein content, in reference to the Living method.

Plum symphysis, modern plant physiology 3 edition, beijing: higher education publishers, 2012.

9 melatonin determination:

melatonin determination was performed by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with reference to the method described by Zhao.

Zhao Y,Tan DX,Lei Q,Chen H,Wang L,Li T,Gao A,Kong J.Melatonin and its potential biological functions in the fruits of sweet cherry.Journal of Pineal Research,2013,55：79-88.

10 Indolylacetic acid determination:

the content of the indoleacetic acid is determined by adopting a high performance liquid chromatography.

11 salicylic acid determination:

the salicylic acid content is determined by high performance liquid chromatography.

12SNAT, himmt, MYB, F3H, DFR gene assay:

the gene expression adopts a fluorescent quantitative PCR (qRT-PCR) method.

Extracting total RNA from apple leaves by CTAB method, and using Prime Script^TMAnd carrying out reverse transcription by using the RT kit. The fluorescent quantitative dye is prepared fromA kit.

The reaction was carried out using a 20. mu.L system comprising 10. mu.L SYBR, 0.5. mu.L forward primer, 0.5. mu.L reverse primer, 8.0. mu.L dd H₂O and 1. mu.L of cDNA template. The MdActin of apple is used as an internal reference gene, the difference multiple of the gene is calculated by 2-delta Ct, each sample is set to repeat the technology for 3 times, and primers used in the test are as follows.

SNAT：

F：TCACGCTGTGGTTGGAGGT；R：CTGCATGCTCCTGAACCAAC；

HIOMT：

F：GAATCACCGTCCACGCTCC；R：GAAATGCTTCCGATGTCCC；

MYB：

F：ATGGAGGGATATAACGAAAACCTG；R：CTATTCTTCTTTTGAATGATTCC；

F3H：

F：GGAAGGAGCTTTTGTGGTCAA；R：GGTTCTGGAATGTGGCTATGGA

DFR：

F：AGGAACTGTGAATGTGGAGGAGC；R：AGGAACTGTGAATGTGGAGGAGC；

MdActin：

F：TGACCGAATGAGCAAGGAAATTACT；R：TACTCAGCTTTGGCAATCCACATC。

The above is only illustrative and is not limiting.

Example 1

A high-temperature-resistant coloring early selection method for early-medium-maturing apple seedlings comprises the following steps:

s1: and (3) selecting a hybrid seedling field, carrying out biological morphology investigation on the field hybrid seedling, mainly removing seedlings with serious diseases and abnormal growth, and selecting a strong plant.

S2: selecting experimental materials, namely selecting functional leaves growing on annual branches as test materials.

S3: and (3) treating experimental materials, namely wrapping the petioles of the selected functional leaves by using sterile cotton balls, spraying water for moisturizing, and treating the leaves for 12 hours in an illumination incubator at 40 ℃ under 5000LX common light.

S4: and (4) detection analysis, namely performing detection analysis on the metabolic substances, hormone levels and coloring related gene expression levels of the treated experimental materials.

S5: early selection, detecting and analyzing the increase of photosynthetic pigment content of leaves, catalase CAT, superoxide dismutase SOD, peroxidase POD, total phenolic substances, proline, soluble sugar, soluble protein, melatonin, indoleacetic acid and salicylic acid in a sample, and detecting plants with the expression of melatonin synthesis related genes SNAT and HIOMT, MYB transcription factors, F3H and DFR genes in a flavonoid synthesis path as a selection object.

In an embodiment, the up-regulation is defined as: the treated group index content/control group index content is > 2.

By adopting the method, 120 parts of high-temperature-resistant, good-coloring and high-quality high-yield superior line and 36 parts of high-yield superior line are selected primarily from more than 8000 Gala and Tanshu I positive and negative crossed seedlings, wherein the Luli, Luyan and other varieties (see figure 1) have acquired the plant variety right of the rural area of the national agriculture, and are demonstrated and popularized for 30 ten thousand mu in major apple producing areas such as Shandong, Shaanxi, Hebei, Liaoning, Shanxi, Henan and the like, thereby obtaining remarkable effect and being expected to become the main cultivated varieties of the early-maturing apples in China.

The technical effects are as follows:

taking 'lury' as an example: the 'vine I' and 'Gala' are parent plants in Shandong province fruit tree research, and a new generation of good early-medium-maturing apple varieties is bred. The fruit is conical, the color of the fruit surface is bright red, the fruit surface is smooth, the fruit point is small, no rust exists, the pulp is light yellow, the pulp is thin, hard and crisp, the juice is more, the sweetness and the sourness are moderate, the fragrance is strong, the quality is high, the fruit development period is about 100 days, and the fruit is early-maturing; strong adaptability, barren soil resistance, early fruit and high yield. The yield of the big seedlings is partial in the second year after field planting, the yield per mu in the third year reaches about 1000 kilograms, and the early fruiting, high yield and stable yield are good.

The bred new variety solves the problems of poor high-temperature coloring, poor quality, intolerance to storage and transportation, fruit cracking, fruit frying, fruit surface cracking and the like in the mature period of the currently cultivated early-medium ripe apple variety, is a new generation of excellent early-medium ripe apple variety, and is expected to become a main cultivated variety of the early-medium ripe apples in China.

Planting the Luli saplings of the second year:

the results of the first selection of Luli are summarized in Table 1

TABLE 1

It should be noted that: in order to achieve a rigorous scientific screening result, at least 10 of the 16 detection indexes are required to be met.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.