CN109539996B - Method for measuring length of rice stigma - Google Patents
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- CN109539996B CN109539996B CN201811380250.7A CN201811380250A CN109539996B CN 109539996 B CN109539996 B CN 109539996B CN 201811380250 A CN201811380250 A CN 201811380250A CN 109539996 B CN109539996 B CN 109539996B
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Abstract
The invention discloses a method for measuring the length of a rice stigma. The method comprises the following steps: the method comprises the following steps of sampling rice glumous flowers in a full-bloom period, correcting the focal length of a measuring tool type microscope and photographing a scale, peeling and photographing a rice stigma, measuring the length of the stigma and processing data. By utilizing the method to investigate the stigma length, the influence of the phenotype data human factor of the stigma length is small, and the accuracy is high. The length character of the stigma investigated by the method can be used for mining, fine positioning, cloning and even functional analysis of the long stigma favorable gene; meanwhile, the developed long stigma gene is utilized to improve the rice sterile line and increase the stigma length of the rice sterile line, thereby improving the rice stigma exposure rate and increasing the seed production yield.
Description
Technical Field
The invention belongs to the field of investigation methods of rice outcrossing related traits, and particularly relates to a method for measuring rice stigma length.
Background
The rice is the most important food crop in China and one of the most important food crops in the world, and more than 65% of people in China use rice as staple food. Under the condition that the population is continuously increased and the cultivated land is reduced year by year at the speed of millions of mu, the food safety of billions of population is ensured, and the yield per unit area is inevitably increased greatly. The yield of the hybrid rice starting in the 70 th 20 th century in China is increased by about 15 percent compared with that of the conventional rice in the same maturity period, and the commercial planting of the hybrid rice greatly improves the rice productivity. Practice proves that the utilization of rice heterosis to improve the yield per unit of rice is one of the most effective ways.
Hybrid rice requires annual production of a first hybrid seed generation. China is the largest country for producing and consuming hybrid rice seeds in the world, the planting area of the hybrid rice is 1400 million hectares each year, and the hybrid rice seeds are required to be about 35 million tons. Research shows that the main factor for limiting the seed production yield of hybrid rice is the low outcrossing seed yield of the sterile line. The exposure of the stigma of the glume flowers of the rice female parent is a determining factor influencing the heterozygosis seed production seed setting rate of the hybrid rice. The stigma exsertion refers to the phenomenon that the pistil stigma is still remained outside the glume after the rice blossoms. The stigma exsertion rate is the percentage of stigma exserted glume flowers in the total glume flower number, and is an important outcrossing character of rice. The opening degree of the exposed stigma is large, the pollen bearing area is increased, the propagation barrier of glumes to exogenous pollen is eliminated, and the pollination chance is still available in the next day of flowering and even in a plurality of days. The research of Zea Junmei et al (1987) finds that the glumes which are not pollinated on the day of flowering of the sterile lines V41A and V20A are freely pollinated in the field by exposed stigmas, and the natural outcrossing rate can reach 35 percent and 37 percent respectively. The higher the stigma exposure rate of the sterile line is, the more beneficial the outcrossing is. The stigma exsertion rate belongs to quantitative trait inheritance, and different researchers use different positioning populations to detect 104 QTLs for controlling the trait on all 12 chromosomes of rice. However, the stigma exposure rate is easily affected by various external environments (such as weather, humidity, temperature and the like in the heading and flowering period), and the character investigation steps are complicated and errors are large, so that subsequent deep research is difficult to perform after initial positioning.
Previous researches show that the rice stigma exposure rate is in obvious positive correlation with pistil length, stigma length and spikelet length, and the positive correlation is mostly caused by multiple effects. Transgenic research of Takano-Kai et al (2011) shows that the length of rice grains can be controlledGS3Alleles are expressed at the base of young stigmas, which increase the length of the stigmas and increase the exposure rate of the stigmas by increasing the number of cells in the non-pinnate segment of the stigmas. The pistil stigma develops and extends in glumous flowers, is slightly influenced by the environment, has high accuracy of phenotype investigation and is easy to explain biological processes, so that the QTL positioning, fine positioning and subsequent gene cloning and function analysis are more reliable and feasible by taking the stigma length as a heterozygosity character index compared with the stigma exposure rate.
However, the current research on the length of the rice stigma is reported less, which probably is because the sampling procedure of the stigma length is complicated and has no unified standard. In 2012, the Chenlan et al takes 5 glumous flowers per spike to investigate the length of stigma when the anther of rice is mature but not pollen is scattered, which is not easy to grasp in time, especially for large population materials; in 2017, Wujian et al carried out the survey of the length of stigma of 5 glumes opened on the same day in the noon or afternoon of sunny days; secondly, as no unified measurement standard exists, Chenlan et al (2012) takes the peeling and measurement under the binocular dissecting mirror, Wujian et al (2017) takes a picture with a camera, and then uses Image J software to measure the length of the stigma. The applicant has investigated the length of the stigma of different periods (just before ear emergence, before flowering, just after flowering and after glume closing) and found that the difference in length of the stigma is large for 4 different periods of the same material. The length of the rice stigma is very small (1.0 mm-3.0 mm), and errors occur in any link in the sampling and measuring processes, so that great differences are brought to phenotype data. The measuring method provided by the invention introduces the sampling period of glumous flowers, subsequent storage, preparation before stigma length survey, peeling of stigma length, photographing, measurement and other links in detail, and the measured stigma length phenotype data is accurate and reliable and has high repeatability. The application of the method has important significance for the discovery of the germplasm resources of the long stigma rice, the breeding of the sterile line with high outcrossing rate, and the excavation, cloning and functional verification of stigma length genes.
Disclosure of Invention
The invention aims to provide a method for measuring the length of a rice stigma, which introduces each link of sampling, stripping, photographing, measuring, data processing and the like in detail, and the measured stigma length phenotype data is accurate and reliable. The length character of the stigma investigated by the method can be used for digging, fine positioning, cloning and function analysis of the long stigma favorable gene, and simultaneously, the dug long stigma gene is utilized to improve the rice sterile line and increase the stigma length of the rice sterile line, thereby improving the rice stigma exposure rate and increasing the seed production yield.
The invention provides a method for measuring the length of a rice stigma. The method comprises the following steps:
(1) sampling: planting the rice test material in a test field, when the test material is in a full-bloom stage, clamping the base part of the glume flower (without touching the glume to avoid damaging the stigma) which is opening from the main stem ear (the highest ear) by using an ophthalmological forceps (with teeth) (shown in figure 1A), taking down and putting the glume flower into a prepared centrifugal tube (shown in figure 2) filled with 1.5ml of clear water, taking 8 glume flowers from each material, and taking the glume flowers back to a laboratory for measurement on the same day;
(2) focusing: opening a body microscope (MZ 11, Guangzhou Ming Mei photoelectricity technology Limited) connected with a computer and provided with a camera (MDX 4, Guangzhou Ming Mei photoelectricity technology Limited) and software thereof (figure 3), placing a scale in the visual field of the body microscope, adjusting the focal distance until scales on the scale are clearly visible, photographing and storing (figure 4), and then, the focal distance is not adjusted;
(3) stripping flowers and taking pictures: taking out 8 glumes stored in a centrifuge tube, placing the 8 glumes on a 2.5 cm-7.5 cm glass slide, pulling out one glume flower, pressing the inner glume edge with VETUS forceps (18 cm) (shown in figure 1C) on the left hand, peeling the glume off (shown in figure 5A) with VETUS forceps (14 cm) (shown in figure 1B) on the right hand to expose the stigma, clipping the stigma and the glume shell with the forceps from the column, pulling out 2 stipes straight with the forceps to form a certain angle (about 150 degrees) with the column (shown in figure 5B), and so on, arranging 16 stipes of 8 flowers on the glass slide in 2 rows, and arranging 4 stipes in each row (shown in figure 5C); placing the glass slide with the stripped column head on the object stage of the body type microscope (figure 6A), and clicking 'photographing-storing' for measurement (figure 6B);
(4) measurement: opening a Mingmei microscopic digital measuring and analyzing system MDX4, clicking a file, opening an image, opening a scale, inserting a unit scale, setting parameters, photographing and storing (figure 7A), clicking a file again, opening an image, measuring, opening a picture of a column head to be measured, inserting line segments, measuring the length of the column head one by one, deriving a document, and storing to finish measurement (figure 7B);
(5) data processing: the mean of the 16 stigma lengths derived in the EXCEL table is the stigma length for this material (fig. 7C).
In the measuring method of the present invention, the sampling time adopted in (1) is unified to be the time when the glumous flower is open and not closed, and the length of the stigma at this time reaches the maximum value and does not change any more.
In the measuring method, in order to facilitate the accuracy of measuring the length of the column cap, after the focal length in (2) is corrected, the lens is not adjusted any more, if the focal length is moved, the focal length needs to be corrected again, the scale takes a picture again, and the updated scale is used for measuring.
In the measuring method, the rice stigma in the step (3) must be straightened after being stripped, and the measured length of the stigma is the linear distance from the top of the stigma to two points of the intersection point of the stigma and the style.
The measuring method of the invention is a method for sampling, stripping, measuring and analyzing the length of the rice stigma.
The structure of the rice flower organ: the rice ear consists of a main shaft, a primary branch, a secondary branch, a spikelet and a spikelet; each spikelet consists of 2 degenerated glumes (minor glumes) peduncles at the base and 3 florets; among the 3 small flowers, 1 at the top grows normally, and the 2 at the bottom all degenerate, only two lemmas (glumes) are seen; the fertile small flower has an external glume, an internal glume, 2 serous sheets, 6 stamens and 1 pistil; anther has 4 anthers, and the stigma has two cracks in feather shape.
Rice stigmas: refers to the pistil of rice, the female reproductive organ of rice, and the rice seeds can be developed through normal fertilization and fructification.
The stigma length refers to the length from the junction of the rice stigmas and the stigma to the top of the stigma.
The measuring method of the invention has the following application in rice genetic breeding:
1. the data of the length of the stigma measured by the method is accurate, and the method can be used for exploring new materials and new resources of the long stigma rice;
2. the data of the stigma length measured by the invention can be used for carrying out fine positioning, cloning and even functional analysis and verification on the gene controlling the character;
3. the long-stigma gene excavated by the invention is used for improving the two-line/three-line sterile line of the rice, and the improved long-stigma sterile line can improve the seed production yield and reduce the seed production cost when the hybrid rice is used for seed production.
The invention has the beneficial effects that:
the invention provides a method for measuring the length of a rice stigma. The whole set of measuring process of the length of the stigma is systematically specified, so that a method capable of accurately measuring the length of the rice stigma is created, the problem that different researchers have different sampling periods and measuring standards in the measuring process of the length of the rice stigma is solved, errors caused by manual operation in the measuring process of the stigma length phenotype data are reduced, the accuracy of the stigma length phenotype data is greatly improved, and the standardized process of the length measurement of the rice stigma is realized.
Drawings
Fig. 1 tweezers used when picking flowers and stigmas: fig. 1A shows that ophthalmic tweezers (with teeth) used for field glume flower sampling are not easy to move after clamping small ear stems, glume flowers are not easy to drop, and glume flowers are prevented from being damaged; FIGS. 1B and 1C show the VETUS forceps used for peeling the stigma, the lengths of which are 14cm and 18cm, respectively, and the tips of the forceps are highly practical for fine operations such as peeling the stigma.
FIG. 2 preservation of glume flowers: 1.5ml centrifuge tubes filled with clear water are arranged in a centrifuge tube box with 100 holes according to the requirement of sampling.
Figure 3 body microscope and its connected computer: in order to obtain a good shooting effect, white paper (purple column head) or gray paper (colorless column head) is padded on the objective table of the body type microscope during shooting.
Scale in fig. 4: before photographing for measuring the length of the column cap, the staff gauge is placed on an objective table, and focusing, photographing (staff gauge) and storing are carried out.
FIG. 5 flow of column cap stripping: FIG. 5A, left hand pressing the inner glume edge with 18cm VETUS forceps and right hand peeling the outer glume off with 14cm VETUS forceps; FIG. 5B, the stigmas are clipped off from the style with tweezers, and then 2 stigmas are straightened with tweezers to form an angle of 150 degrees with the style; in FIG. 5C, 16 stigmas of 8 glumous flowers are arranged on the glass slide in 2 rows, and each row has 4 glumous flowers, so that the arrangement is compact.
Fig. 6 stigmatic photograph: FIG. 6A, when the column heads with the orderly arrangement are placed on an object stage and the water drops on the column heads are observed from a computer to be not agglomerated, a picture is taken; fig. 6B, photograph of column head taken.
Fig. 7 measurement of the stud length: fig. 7A, open the mingmei microscopic digital measurement and analysis system, click "file-open image to measure-open scale-insert unit scale-set parameter-photograph-save"; FIG. 7B, click again "File-open image to measure-open photo of to-be-measured column head-insert line segment-measure column head length one by one-export document-save"; and in the figure 7C, the numerical values of the 16 stigma lengths are exported to an EXCEL table to carry out statistics on the stigma lengths.
The implementation case is as follows: measurement of column head length of 8 different materials
1, rice material:
c fort, R254, Zhendao 11, Malaysia, Huazhan, Strain 1S, C815S, Z913S.
2, test method:
8 parts of the material is planted in the Lu Guo river base of the institute of Paddy, academy of agriculture and sciences of Anhui province in the quarter of 2018, sowed in 13 days in 5 months, and transplanted in 14 days in 6 months. The date of sampling, the tabular values of the column head length, etc. are shown in table 1.
TABLE 18 statistics of sampling date and stigma length data for different test materials
3 results and analysis:
8 parts of test material comprises 4 parts of japonica rice and 4 parts of indica rice (wherein 3 parts of indica type temperature-sensitive genic male sterile lines). From the results, the stigma length of the indica rice material in the experiment is higher than that of the japonica rice material, the stigma length of the indica thermo-sensitive genic male sterile line is higher than that of the indica rice restorer line, and obvious difference exists among the stigma lengths of different materials; from the view of amplitude variation, the lengths of different stigma of the same material have certain difference, but the difference is small, and the standard deviation is between 0.06 and 0.10.
Claims (2)
1. A method for measuring the length of a rice stigma, which is characterized by comprising the following steps:
(1) sampling: planting the rice test material in a test field, when the test material is in a full-bloom stage, clamping the base of an open glume flower from a main stem ear, namely the highest ear by using a pair of toothed ophthalmic tweezers without touching a glume shell so as to avoid damaging a stigma, taking down and putting the glume flower into a pre-prepared centrifugal tube filled with 1.5ml of clear water, taking 8 glume flowers from each material, and taking the glume flowers back to a laboratory for measurement on the same day;
(2) focusing: an MZ11 body type microscope of Mingmei photoelectric technology Limited, Guangzhou city, and a software Mingmei microscope digital measurement and analysis system V1.3.10.8 thereof are connected with a computer, wherein the body type microscope is provided with an MDX4 camera; placing the scale with the scales on an object stage of the body type microscope, adjusting the focal length until the scales on the scale are clearly visible, photographing and storing, and then, not adjusting the focal length;
(3) stripping flowers and taking pictures: taking out 8 glumes stored in a centrifugal tube, placing the 8 glumes on a 2.5 cm-7.5 cm glass slide, pulling out one glume flower, pressing the inner glume edge with a VETUS forceps with the length of 18cm by the left hand, peeling the glume with the VETUS forceps with the length of 14cm by the right hand to expose a stigma, clipping the stigma and a glume shell from a flower column by the forceps, pulling 2 stigma straight by the forceps to form a 150-degree angle between the stigma and the flower column, and so on, arranging 16 stigma of the 8 glumes on the glass slide in 2 rows, wherein 4 stigma is arranged in each row; placing the glass slide with the stripped column cap on an object stage of a body microscope, and clicking 'photographing-storing' for measurement;
(4) measurement: opening a Mingmei microscopic digital measurement and analysis system, clicking a file, opening an image, measuring, opening a scale, inserting a unit scale, setting parameters, photographing and storing, clicking a file again, opening an image, measuring, opening a picture of the length of a column head to be measured, inserting a line segment, measuring the length of the column head one by one, deriving an EXCEL document, storing and finishing measurement;
(5) data processing: the average of 16 stigma lengths derived from the EXCEL table is the stigma length of the material.
2. The method as claimed in claim 1, wherein the open glume is obtained at the same sampling time and the same measurement standard is obtained by calibration.
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| CN110044269A (en) * | 2019-04-08 | 2019-07-23 | 安徽省农业科学院水稻研究所 | A kind of method of the best Proper Sampling Period of rice stigma linear measure longimetry |
| CN112634350A (en) * | 2020-12-26 | 2021-04-09 | 中国水产科学研究院黄海水产研究所 | Intelligent image measurement and analysis system, method, medium, equipment and application |
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| CN102879328B (en) * | 2012-10-17 | 2014-07-23 | 云南省烟草农业科学研究院 | Method for measuring adhesion force between pollen and stigma by using centrifuge method |
| CN103499299B (en) * | 2013-10-24 | 2015-11-18 | 西北农林科技大学 | A kind of very thin thread measurement of length method |
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