CN110326503B

CN110326503B - Identification method for elongation capability of hypocotyl in dry direct-seeded rice

Info

Publication number: CN110326503B
Application number: CN201910719624.1A
Authority: CN
Inventors: 刘宏岩; 詹俊辉; 叶国友
Original assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Current assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Priority date: 2019-08-06
Filing date: 2019-08-06
Publication date: 2021-04-13
Anticipated expiration: 2039-08-06
Also published as: CN110326503A

Abstract

The invention discloses a method for identifying the elongation ability of the mesocotyls of dry direct seeding rice. To observe the emergence situation every day, record the order of emergence of different varieties, take samples to measure the length of the mesocotyl on the 3rd day after the emergence of the first variety; take out the seedlings from the plug trays in order, rinse them, and place them flat in order. After taking pictures of all varieties in turn, use Image J software to set the scale according to the scale on the plate to measure the length of the mesocotyls of each variety. The application of the invention in the identification of mesocotyl elongation ability of dry direct seeding rice can significantly shorten the test period, improve the efficiency of mesocotyl phenotype identification, and reduce the test error caused by human factors. At the same time, the reproducibility and stability of the identification results of the mesocotyl elongation ability of dry-seeded rice were greatly improved.

Description

Identification method for elongation capability of hypocotyl in dry direct-seeded rice

Technical Field

The invention relates to the technical field of biology, in particular to a method for identifying the elongation capability of an embryonic axis in dry direct-seeded rice. A

Background

The dry direct seeding rice has a series of advantages of water saving, labor saving, suitability for mechanized cultivation and the like, but in the production of the dry direct seeding rice, because the root cutting depth is insufficient, the lodging phenomenon of the rice at the later growth stage often occurs, and in order to solve the problem, the seeding depth is often required to be increased in the production. Deep sowing is beneficial to the binding of the roots of rice, the absorption capacity of the root systems to water and nutrients is improved, and the lodging resistance of the rice is improved, but the rice is difficult to emerge under the deep sowing condition, the rice emergence rate is reduced, and therefore the problems of insufficient basic seedlings in the rice field and weed prevention and control are caused. And under the condition of deep sowing, the seed germination duration is long, seedlings cannot perform photosynthesis in the period, and the seedling growth is easy to weaken and the young leaves are easy to yellow. After rice germinates in a dark environment, the mesocotyl can eject the new germ out of the soil by extending, photosynthesis is carried out, and the survival rate of rice seedlings is improved. Generally, the long mesocotyl variety has strong soil-bearing capacity and high emergence rate. Therefore, the physiological regulation mechanism of mesocotyl elongation is determined, the gene for regulating mesocotyl elongation is excavated, the corresponding molecular marker is developed, and the screening and creation of long mesocotyl germplasm have important significance for molecular marker assisted breeding (MAS) of the dry direct seeding rice. However, many QTLs for controlling mesocotyl axis elongation have been localized in different studies so far, but the experimental result has poor repeatability due to the significant interaction between mesocotyl axis elongation and environmental conditions.

Disclosure of Invention

In order to solve the problems, the invention provides a method for identifying the elongation capability of the mesocotyl in the dry direct seeding rice, which realizes the stability and repeatability of the identification of the mesocotyl phenotype, thereby providing a stable phenotype for the genetic research of the elongation capability of the mesocotyl in the dry direct seeding rice.

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

a method for identifying the elongation capability of an embryonic axis in dry direct-seeded rice comprises the following steps:

s1, smashing the nutrient soil, sieving the smashed nutrient soil with a 5-mesh sieve, and uniformly stirring the smashed nutrient soil and the sieved nutrient soil for later use;

s2, filling 500g of smashed nutrient soil into the tray, paving and compacting, placing the hole tray into the tray, filling a certain amount of smashed nutrient soil into each hole of the hole tray, and compacting to enable the height difference between the soil surface and the hole opening to be 6 cm;

s3, selecting full and uniform rice seeds with intact glumes, sowing the seeds in sequence, sowing 15 seeds in each hole, and uniformly distributing the 15 seeds at the bottom of each hole;

s4, uniformly covering the smashed nutrient soil after all the materials are sowed until the soil surface is flush with the opening of the hole tray, and weighing the tray, the hole tray and the total mass of the nutrient soil and the seeds in the hole tray as W1;

s5, thoroughly pouring the nutrient soil in the hole tray in a spraying mode, taking the hole tray out of the tray after water leaks out of the holes, pouring the nutrient soil in the tray to saturation with tap water, putting the hole tray back into the tray, controlling the total amount of watering to be 2000g, then, supplementing the height of the nutrient soil in the hole tray to be level to the hole opening with smashed nutrient soil, and weighing the total weight to be W2;

s6, placing the tray and the plug tray together in a dark culture room with the indoor temperature of 35 ℃ and the relative humidity of 50% -60%, spraying once every 24 hours, and controlling the amount of sprayed water to keep the total weight of the sprayed water at the W2 level;

s7, observing the seedling emergence condition every day, recording the seedling emergence sequence of different varieties, and sampling and determining the mesocotyl length on the 3 rd day after the first variety emerges; taking out the seedlings from the plug tray in sequence, washing the seedlings clean, sequentially and flatly placing the seedlings growing uniformly in each material on a flat plate with scales, and taking a picture, wherein the camera position is required to be directly above the flat plate during taking the picture, the height is determined by placing the whole flat plate in a viewing frame, and the camera position is fixed by using a tripod; after all varieties are photographed in sequence, the mesocotyl length of each variety is measured by ImageJ software according to a scale set scale on a flat plate.

By applying the method in the identification of the mesocotyl elongation capability of the dry direct seeding rice, the test period can be obviously shortened, the mesocotyl phenotype identification efficiency can be improved, and the test error caused by human factors can be reduced. Meanwhile, compared with other methods, the method greatly improves the repeatability and stability of the identification result of the elongation capability of the embryonic axis in the dry direct seeding rice. In three other methods for simultaneously evaluating the elongation capacity of the mesocotyl of the dry direct-seeded rice, the correlation coefficient between different batches is up to 0.88 (soil from fields is used), but the method can ensure that the correlation coefficient between the test and the identification results of different batches is up to 0.95, the formula of the commercialized nutrient soil is clear, and different researchers can completely duplicate the method.

Drawings

FIG. 1 shows phenotypic variation of 208 rice varieties in four different culture modes.

FIG. 2 is a graph showing the correlation between the mesocotyl length of 208 rice materials obtained in different culture modes in two replicates.

FIG. 3 is a plot of variation range of mesocotyl length for the MAGIC population over two trials.

FIG. 4 is a graph of the correlation of mesocotyl length in two experimental replicates.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1

The mesocotyl elongation ability of the same rice population (208 rice germplasms introduced by international rice) is respectively identified by a water culture method, a sand culture method, a soil culture method (field loam) and a nutrient soil (Germany K brand-TS 1 type). Each cultivation method was set up for 2 independent replicates. The specifications of the used hole discs are 54cm (length) x 28cm (width) x 11cm (height), 50 holes.

a. In the water culture method, gauze is flatly paved at the bottom of each hole, 15 plump seeds are flatly paved on the gauze, a hole tray is placed in a tray, water is injected into the tray until the water surface of the hole tray is 1cm higher than the seeds, then the hole tray and the tray are sealed and shaded by tinfoil paper, finally the sealed hole tray is placed in a climatic chamber with the temperature of 30 ℃, and the length of a mesocotyl shaft is measured by a ruler after the hole tray is cultured for 7 days.

b. A sand culture method: before sowing, filling each hole in the hole tray with sand to a position 6cm away from the top of the hole tray, and placing the hole tray in a tray filled with sand, wherein the depth of the sand is 3 cm. And (3) selecting 15 full dry seeds, flatly paving the seeds at the bottom of the hole, then covering the seeds in the hole tray with sand until the seeds are flush with the top of the hole tray to ensure that the sowing depth reaches 6cm, and watering the seeds through the hole tray until the tray at the bottom is saturated in water and has no open water. After watering, the weight of each tray and each hole tray is recorded and marked, and water is supplemented once every 24 hours until the marked weight is reached. And finally, placing the plug in a climatic chamber at the temperature of 30 ℃, sampling after the seeds germinate and emerge, and measuring the length of the mesocotyl by using a ruler.

c. The soil culture method comprises filling each hole in the hole tray with soil to a position 6cm away from the top of the hole tray, and placing the hole tray in a tray filled with soil to a depth of 3 cm. And (3) selecting 15 full dry seeds, paving the seeds at the bottom of the hole, covering the seeds in the hole tray with soil until the seeds are flush with the top of the hole tray to ensure that the sowing depth reaches 6cm, and watering the seeds through the hole tray until the tray at the bottom is saturated in water and has no open water. After watering, the weight of each tray and each hole tray is recorded and marked, and water is supplemented once every 24 hours until the marked weight is reached. And finally, placing the plug tray in a climatic chamber at the temperature of 30 ℃, sampling after the seeds germinate and emerge, and measuring the length of the mesocotyl by using a ruler.

d. The nutrient soil culture method comprises filling each hole in the hole tray with nutrient soil to a position 6cm away from the top of the hole tray before sowing, and placing the hole tray in a tray filled with nutrient soil with a depth of 3cm (or 500 g). Similarly, 15 full dry seeds are selected and spread at the bottom of the hole, then the seeds in the hole tray are covered with nutrient soil until the seeds are flush with the top of the hole tray, the sowing depth reaches 6cm, then the hole tray is watered, water leaks out from the hole below the hole, then the hole tray is taken out from the tray, the nutrient soil in the tray is watered to be saturated by tap water, the hole tray is put back into the tray, the total amount of watering is controlled to be 2000g, and then the height of the nutrient soil in the hole tray is supplemented to be flush with the hole opening by the smashed nutrient soil. After watering, the weight of each tray and each hole tray is recorded and marked, and water is supplemented once every 24 hours until the marked weight is reached. Finally, the tray was placed in a dark culture chamber at a temperature of 35 ℃ and a relative humidity of 50% -60%. Observing the rice seedling emergence condition every day, recording the seedling emergence sequence, sampling and photographing on the 3 rd day after the first variety emerges, and rapidly measuring the mesocotyl lengths of different rice varieties in the culture system by using an image fixing method.

And (3) analyzing test results:

the rice mesocotyl phenotype variation under different culture modes.

FIG. 1 shows phenotypic variation of 208 rice varieties in four different culture regimes, with the abscissa representing two independent experimental replicates for each culture regime. As can be seen from FIG. 1, the phenotypic variation of the 208 rice materials was significantly different, and the variation range of the hypocotyls was 0-4.8 cm in the soil culture (FIG. 1A), 0-2.1 cm in the sand culture (FIG. 1B), except for the extreme difference (FIG. 1B), 0-4.0cm in the water culture (FIG. 1C), and the maximum variation range was 0-5.1 cm in the nutrient soil culture (FIG. 1D). The larger phenotypic variation is the basis of genetic analysis, so the nutrient soil culture method proposed in this patent is superior to other methods in view of the variation range of mesocotyl length.

FIG. 2 reflects the correlation between the mesocotyl length of 208 rice materials obtained in different culture modes in two replicates. As can be seen, the correlation between mesocotyl length in two replicates is poor under both sand and hydroponic conditions, R²0.5150 (FIG. 2B) and 0.5966 (FIG. 2C), respectively, are preferred in soil culture, R²0.7858 (FIG. 2A) was reached, whereas R was cultured under nutrient soil conditions²0.9133 (FIG. 2D) was achieved and the slope k was closest to 1, indicating that the nutrient soil culture method proposed in this patent is the best in reproducibility and stability among the four mesocotyl length determination methods.

Example 2

The specific operation steps of detecting the variation of the hypocotyl length in 395 MAGIC rice populations by utilizing the nutrient soil culture method are the same as those of the nutrient soil culture method.

And (3) analyzing test results:

the variation in mesocotyl length of the MAGIC population ranged from 0-4.8 cm, and was evenly distributed and continuous, with results that were essentially identical in both experimental replicates (FIG. 3). At the same time, the mesocotyl length was highly correlated in both experimental replicates (R)²0.8459) (fig. 4), substantially consistent with the correlation results in example 11.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims

1. a method for identifying the mesocotyl elongation ability of dry direct seeding rice, is characterized in that: comprise the steps:

S1. Break the nutrient soil through a 5-mesh sieve, and stir it evenly for later use;

S2. Load 500g of crushed nutrient soil into the tray, flatten and compact it, place the plug tray in the tray, and fill each hole of the plug tray with a certain amount of crushed nutrient soil and compact it so that The height difference between the soil surface and the hole is 6cm;

S3. Select full and even rice seeds with intact glumes, sow them in sequence, one variety per hole, 15 seeds per variety, and evenly distribute 15 seeds at the bottom of the hole;

S4. After the sowing of all materials, the crushed nutrient soil is uniformly covered until the soil surface is flush with the mouth of the plug tray, and the total mass of the tray, plug tray and the nutrient soil and seeds therein is weighed and recorded as W1;

S5. Pour the nutrient soil in the plug tray by spraying, and water leaks out of the following holes, then take the plug tray out of the tray, and pour the nutrient soil in the tray to saturation with tap water. Put the tray back into the tray, and control the total amount of watering to 2000g. After that, use the crushed nutrient soil to fill the nutrient soil in the plug tray to the level of the hole opening, and weigh the total weight as W2;

S6. Place the above-mentioned trays and plug trays together in a dark culture room with a temperature of 35°C and a relative humidity of 50%-60%, spray once every 24 hours, and control the amount of water sprayed so that the total amount of water after spraying The weight remains at the W2 level;

S7. Observe the emergence situation every day, record the order of emergence of different varieties, and take samples to measure the length of the mesocotyl on the 3rd day after the emergence of the first variety; take out the seedlings from the plug tray in order, rinse them, and put each material The seedlings with evenly growing vigor are placed flat on a scaled plate in turn, and take pictures. When taking pictures, it is necessary to pay attention to the position of the camera directly above the plate. The height is based on placing the entire plate in the viewfinder, and use a tripod to fix the position of the camera. ; After taking pictures of all varieties in sequence, use Image J software to set the scale bar according to the scale on the plate to measure the mesocotyl length of each variety.