Morphological marker associated with sweet-waxy homograin character of corn and application of morphological marker

Abstract

The invention discloses a morphological marker associated with the sweet-glutinous same-grain character of corn, which is obtained by screening two specific characters that the surfaces of grains in a milk-ripe stage have continuous, dense and winding semitransparent'sugar lines' and the aired grains in a full-ripe stage have non-transparent waxy appearance, the surfaces are not shrunk and the tops of the grains are irregularly recessed. The method can rapidly and accurately breed a sweet-glutinous fresh corn new material, and the accuracy rate is as high as 95%. The method replaces traditional biochemical detection and molecular marker breeding methods, the breeding cost can be reduced by 50% or above, and the breeding period can be shortened by 50% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural technology, in particular to a morphological marker associated with the same-grain trait of waxy corn and application. BACKGROUND

[0002] Fresh corn refers to corn varieties picked at the milk stage for processing or fresh eating, also known as fruit and vegetable corn. According to the composition and flavor characteristics of starch, it is mainly divided into three categories: sweet corn, waxy corn and sweet and waxy corn. Sweet corn is known for its high sugar content, with soluble sugar content ranging from 12-22%, and amylopectin content less than 5%, presenting a sweet and crisp taste; waxy corn has a typical high amylopectin structure, with a content of up to 97%, and a sugar content generally less than 10%, with a unique soft and sticky texture; sweet and waxy corn is a breakthrough in combining the genetic characteristics of sweet corn and waxy corn in the same ear, achieving precise chimerism of sweet and waxy grains, with a stable ratio of 1:3 or 7:9, etc., and both parents' taste advantages. The existing three main types of fresh corn have the following shortcomings: first, sweet corn and waxy corn cannot meet the needs of consumers for both flavors, and there is an urgent need for excellent corn germplasm resources with high sweetness and strong waxy properties; second, the existing sweet and waxy corn has both sweet and waxy grains on the same ear, and the sweet and waxy grains have a certain separation ratio, with the disadvantage of sweet grains not being waxy and waxy grains not being sweet. Third, the existing three types of fresh corn varieties have a generally short harvest period (only 3-5 days), and when extreme weather occurs, it cannot be harvested in the field, causing significant economic losses to farmers and fresh corn planting enterprises, and also causing a large amount of short-term concentrated labor pressure to processing enterprises.

[0003] To solve the above-mentioned practical problems in the development of fresh corn industry, the Fresh Corn Genetics and Breeding Group of the Speciality Research Institute of the Chinese Academy of Agricultural Sciences found a sweet and waxy grain breakthrough germplasm resource (sweet and waxy corn parent B2595T) different from the existing three types of fresh corn (sweet corn, waxy corn, and sweet and waxy corn) through extensive collection, identification and evaluation of fresh corn germplasm resources. All the grains of this new germplasm have high sweetness (more than 15 degrees) and high waxy properties (amylopectin content up to 89%), which is the first time to achieve sweet and waxy grain breakthrough, especially the fresh ear harvest period of the new germplasm can reach more than 15 days, which is a revolutionary new germplasm of fresh corn. However, existing sweet and waxy corn breeding relies on molecular markers (such as InDel) or biochemical detection, which has the defects of high cost (biochemical detection requires laboratory operations such as sample processing and colorimetric detection, with a single sample detection cost of more than 50 yuan), long cycle (from biochemical detection to obtaining results, it takes 1-2 days, delaying field decision-making), and complex operation (requiring professional equipment and technical personnel, limiting large-scale application). Therefore, it is necessary to find a phenotype trait to replace complex detection, achieve simple and rapid screening in the field, and reduce breeding costs. SUMMARY

[0004] The present application provides a morphological marker associated with the waxy kernel trait of Zea mays, which comprises two phenotypic traits as follows: (1) the kernel surface at milk stage has continuous, fine and meandering translucent "sugar line" patterns; (2) the dried kernel at full maturity stage has opaque waxy appearance, no surface shrinkage and irregularly concave top.

[0005] Specifically, the "sugar line" patterns are in the form of fine filament net. The irregularly concave top has a depth of 0.2-0.5 mm.

[0006] More specifically, the kernel surface at milk stage has continuous, fine and meandering translucent "sugar line" patterns as shown in FIG. 1. The dried kernel at full maturity stage has opaque waxy appearance, no surface shrinkage and irregularly concave top as shown in FIG. 2. Figure 1 Figure 2

[0007] The milk stage refers to 20-25 days after pollination. The dried kernel at full maturity stage has a water content of ≤15%.

[0008] The present application also provides an application of the aforementioned morphological marker in the breeding of waxy kernel corn.

[0009] The present application also provides a breeding method of waxy kernel corn based on morphological marker, which comprises the following steps: (1) milk stage screening: observing the kernel surface patterns and selecting the kernels with the aforementioned "sugar line" patterns; (2) full maturity stage screening: detecting the dried kernel seed coat morphology and selecting the kernels with the opaque waxy appearance, no surface shrinkage and irregularly concave top as claimed in any one of claims 1-7; The material that meets the above two traits simultaneously is the waxy kernel corn material.

[0010] Further, the method further comprises further verifying the sweetness and amylopectin content of the material screened by the aforementioned breeding method, and selecting the material with sweetness ≥15% and amylopectin content ≥89%.

[0011] The present application has the following advantages: ​​The morphological marker provided by the application can be used for screening the continuous, fine and meandering translucent "sugar line" on the surface of the milk-ripe grain and the opaque waxy appearance, non-shrinking surface and irregularly concave top of the grain after air-drying at the full-ripe stage, so that the new sweet and waxy same-grain fresh corn material can be quickly and accurately selected, and the accuracy is as high as 95%. The method can replace the traditional biochemical detection and molecular marker selection method, and can reduce the breeding cost by more than 50% and shorten the breeding period by more than 50%. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Schematic diagram of "sugar line" on the surface of sweet and waxy same-grain corn grain at the milk-ripe stage; Figure 2 Schematic diagram of the opaque waxy appearance, non-shrinking surface and irregularly concave top of sweet and waxy same-grain corn grain after air-drying at the full-ripe stage; Figure 3 Phenotype of waxy corn grain at the milk-ripe stage; Figure 4 Phenotype of sweet corn grain at the milk-ripe stage; Figure 5 Phenotype of waxy corn grain after air-drying at the full-ripe stage; Figure 6 Phenotype of sweet corn grain after air-drying at the full-ripe stage; Figure 7 Correlation analysis of sugar line and ear and grain traits. DETAILED DESCRIPTION

[0013] The application will be further described and illustrated below by combining with the examples, but the described examples are only part of the examples of the application, rather than all the examples. Based on the application and the examples, all other applications and examples obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0014] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0015] In the following examples, the materials, reagents and the like used are commercially available unless otherwise specified.

[0016] Example 1, screening of specific traits of sweet and waxy same-grain (1) Selection of phenotypic traits The inventors found that the sweet and waxy same-grain corn grain at the milk-ripe stage will appear a continuous, fine and meandering translucent "sugar line" as shown in Figure 1 The line specifically appears on the surface of the sweet and waxy same-grain corn grain at the milk-ripe stage. Meanwhile, the sweet and waxy same-grain corn grain after air-drying at the full-ripe stage has an opaque waxy appearance, a non-shrinking surface and an irregularly concave top as shown inFigure 2 The image shows irregular indentations 0.2-0.5 mm deep. This feature distinguishes it from the appearance of most other types of corn kernels, such as... Figures 3-4 As shown, neither sweet corn nor glutinous corn exhibits "sugar lines" during the milk stage; the tops of the kernels of glutinous corn, after drying at full maturity, show no indentation (as shown in the image). Figure 5 As shown); after sweet corn kernels are dried at full maturity, they exhibit various characteristics: some show a highly wrinkled, sunken, and shriveled appearance (such as...). Figure 6 As shown in Figure A), some exhibit a moderately shrunken, sunken, translucent appearance (such as...). Figure 6 As shown in Figure B), some exhibit a slightly concave, translucent appearance (such as...). Figure 6 As shown in C), some exhibit a slightly wrinkled, translucent appearance (such as...). Figure 6 (As shown in D).

[0017] Therefore, in this embodiment, two specific traits were used as phenotypic characteristics: the kernels at the milk stage have continuous, fine, and meandering translucent "sugar lines" on their surface, and the kernels at the fully mature stage have an opaque waxy appearance, no wrinkles on the surface, and irregular depressions at the top. This was used to rapidly breed new sweet and waxy corn materials with the same kernel size, and the reliability of the screening was verified by biochemical detection methods.

[0018] (2) Test materials 108 inbred lines of different types of fresh maize, including waxy maize, sweet maize, and sweet-waxy maize of the same kernel, are preserved at the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences. Among them, the original germplasm of sweet-waxy maize, B2595T, was independently created by the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences. The sweet-waxy maize of the same kernel used in this embodiment is an inbred line generated using B2595T as the donor.

[0019] (3) Biochemical testing standards The criteria for judging sweet and glutinous corn are: amylopectin content ≥ 85% of total mass and soluble sugar content ≥ 14% of total mass; the criteria for judging glutinous corn are: amylopectin content ≥ 97% of total mass and soluble sugar content ≤ 10% of total mass; and the criteria for judging sweet corn are: soluble sugar content ≥ 12% of total mass and amylopectin content ≤ 5% of total mass.

[0020] Using these two specific traits alone or simultaneously as technical indicators for breeding sweet and waxy corn varieties, a statistical analysis was conducted on 108 fresh corn varieties. The results are shown in Table 1. Of the 108 fresh corn varieties, 18 varieties showed [specific characteristics] on the surface of the kernels at the milk stage. Figure 1 The "sugar line" pattern shown indicates that the consistency rate between the "sugar line" pattern on the surface of kernels at the milk stage and the biochemical detection of sweet and glutinous kernels is 94.73%. Among the 108 fresh corn samples, the kernels after drying at full maturity have an opaque waxy appearance, no surface wrinkles, and a top that resembles...Figure 2 Of the 23 samples shown, those with irregular depressions of 0.2-0.5 mm depth had an opaque waxy appearance, no wrinkles on the surface, and irregular depressions on the top of the fully matured kernels, with a consistency rate of 82.60% with the biochemically tested sweet and glutinous kernels. At the same time, 18 samples had "sugar line" patterns on the surface of the kernels during the milk stage and had an opaque waxy appearance, no wrinkles on the surface, and irregular depressions on the top of the fully matured kernels, with a consistency rate of 94.73% with the sweet and glutinous kernel phenotype.

[0021] The above results demonstrate that simultaneously screening for two specific traits—continuous, fine, and meandering translucent "sugar lines" on the surface of kernels during the milk stage and opaque, waxy appearance, no wrinkles on the surface, and irregular depressions 0.2-0.5 mm deep at the top of kernels after drying during the fully mature stage—can quickly and accurately screen for new sweet and glutinous fresh corn materials.

[0022] Table 1. Phenotypic and biochemical characteristics of 108 fresh sweet corn samples

[0023]

[0024]

[0025] Example 2: Correlation analysis of sugar lines with kernel and ear traits of maize In this embodiment, the ear diameter (ED), ear length (EL), 100-kernel weight (HKW), number of kernels per row (KNR), number of rows per ear (KRN), corn type (including sweet corn (T), waxy corn (N), sweet and waxy corn with the same kernel (TL)), and presence of sugar line (TX) of 108 fresh corn samples from Example 1 were investigated. The correlation analysis of 9 traits among the 108 fresh corn samples was performed using Pearson correlation coefficient.

[0026] The results are as follows Figure 7 ( Figure 7 The asterisk (*) represents significance. p A value < 0.05 indicates that ** represents p As shown in the value <0.01), correlation analysis revealed a significant positive correlation between the presence of sugar lines and the sweet and glutinous phenotype, with a correlation coefficient of 0.96.

[0027] Example 3: Application and verification of rapid breeding of new sweet and waxy single-kernel fresh corn materials based on two specific phenotypic traits screened in Example 1. The method of step (3) in Example 1 was used to perform biochemical identification on 300 commercially available corn materials (including sweet corn, waxy corn and sweet and waxy corn of the same kernel) in three groups. Then, the two specific phenotypic traits provided in step (1) of Example 1 were used for rapid phenotypic identification.

[0028] Table 2: Statistical results of rapid identification of sweet waxy and same grain fresh corn based on two specific phenotypic traits

[0029] The biochemical and phenotypic identification results are shown in Table 2. As can be seen from Table 2, by using two specific phenotypic traits, new sweet waxy and same grain fresh corn materials are rapidly bred, the results are highly consistent with the biochemical identification results of corn ear, indicating that the two specific traits can be effectively applied to the rapid screening of sweet waxy and same grain corn materials.

Claims

1. A morphological marker associated with a waxy com kernel trait, characterized in that, The morphological marker comprises two phenotypic traits as follows: (1) the kernel surface at milk stage has continuous, fine and meandering translucent "sugar line" pattern; (2) the dried kernel at full ripe stage has opaque waxy appearance, no surface shrinkage and irregular concave top.

2. The morphological marker of claim 1, wherein, The "sugar line" pattern is in the form of fine filament network.

3. The morphological marker of claim 2, wherein, The kernel surface at milk stage has continuous, fine and meandering translucent "sugar line" pattern as shown in Fig.

1.

4. The morphological marker of claim 1, wherein, The irregular concave has a depth of 0.2-0.5 mm.

5. The morphological marker of claim 4, wherein, The dried kernel at full ripe stage has opaque waxy appearance, no surface shrinkage and irregular concave top as shown in Fig.

2.

6. The morphological marker of claim 1, wherein, The milk stage refers to 20-25 days after pollination.

7. The morphological marker of claim 1, wherein, The dried kernel at full ripe stage has water content of ≤15%.

8. Use of the morphological marker of any one of claims 1-7 in the breeding of sweet waxy dent corn.

9. A method for breeding sweet waxy homozygous corn based on morphological markers, characterized by, The method comprises the following steps: (1) milk stage screening: observing the kernel surface pattern and selecting the kernel with "sugar line" pattern of any one of claims 1-7; (2) full ripe stage screening: detecting the dried kernel seed coat morphology and selecting the kernel with opaque waxy appearance, no surface shrinkage and irregular concave top of any one of claims 1-7; The material that meets the above two traits simultaneously is the sweet waxy dent corn material.

10. The method of breeding according to claim 9, wherein, The method further comprises further verifying the sweetness and amylopectin content of the material screened in claim 9 and selecting the material with sweetness ≥15% and amylopectin content ≥89%.

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