Method for breeding same-grain corn inbred line with dynamic sweet-glutinous transformation characteristic

Abstract

The invention provides a method for breeding a same-grain corn inbred line with a dynamic sweet-glutinous transformation characteristic, and belongs to the technical field of crop genetic breeding. The method comprises the following steps: hybridizing a mutant sh1 homozygous inbred line as a male parent and a waxy wx homozygous inbred line as a female parent to obtain F1, screening and locking a waxy base and a shrinkage form through a stepped phenotype of F2 generation, and carrying out time-phased dynamic detection on F3 generation to screen out a strain of which the soluble sugar content is continuously and obviously higher than that of common waxy corn within 18-35 days after pollination and the waxy property is stably shown. And through backcross reinforcement and high-strength stability screening based on a double-threshold elimination method, finally obtaining the corn inbred line with stable agronomic characters, sweet-waxy dynamic transformation characteristics and a fresh and sweet base taste in a mature period. Molecular marker assistance is not needed, the breeding cost is effectively reduced, and the selection efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of crop genetic breeding technology, and in particular to a method for breeding a same-grain corn inbred line with dynamic sweet-waxy conversion characteristics. BACKGROUND

[0002] Sweet corn and waxy corn, as two main types of fresh corn, are popular among consumers due to their unique flavors and textures. The sweetness of sweet corn mainly comes from the process of sugar conversion to starch in the endosperm being blocked, resulting in high soluble sugar content. The soft and sticky texture of waxy corn comes from the fact that the starch in the endosperm is almost all amylopectin. The fresh corns on the current market are mainly divided into the following categories: Pure sweet corn: its flavor relies on genes such as shrunken2 ( sh2 ), and the sugar content is high (usually > 18%) in the early stage after pollination, but the sugar content decreases rapidly in the later stage, and the taste becomes bland (sugar content is usually less than 3%) at 30 days after pollination, with a short optimal eating window.

[0003] Pure waxy corn: its flavor relies on the waxy ( wx ) gene, and the high-amylopectin content (> 95%) is maintained throughout the pollination period, and the taste is soft and waxy, but the sugar content is always low (usually less than 6%), lacking the sweet and clean taste that consumers love.

[0004] Sweet-waxy separation varieties: by crossing sweet corn and waxy corn, sweet kernels ( sh2 ) and waxy kernels ( wx ) are mixed on the same ear. Although this type of variety has both sweet and waxy flavors, it needs to be manually or mechanically sorted and processed, and the consistency of the products is poor, and the flavor fusion and conversion cannot be achieved in a single kernel.

[0005] The existing fresh corn lines mainly have the following three types: (1) Traditional hybrid mixed sweet-waxy corn By crossing a homozygous sweet corn line (such as su1 a recessive mutant) with a homozygous waxy corn line ( wx a recessive mutant), an F1 seed with a genotype of Su / su ; wx / wx is obtained. This corn has sweet kernels ( su / su ; wx / wx ) and waxy kernels ( Su / -; wx / wx ) on the same ear, with a ratio of 1:3. This type of corn line has the following defects: when consumers eat it, they eat a mixture of sweet and waxy kernels, and the taste is separated and random, and the synergistic effect of sweet and waxy in the same kernel is not experienced.

[0006] (2) Static sweet waxy corn of molecular marker assisted selection By using molecular marker assisted selection technology, sweet gene (such as sh2 ) and waxy gene (such as wx ) are combined into the same inbred line to create a double recessive sweet waxy homogeneous corn with genotype sh2 / sh2 ; wx / wx . The taste of this corn line is "static", lacking dynamic changes. This variety has relatively fixed sweet and waxy proportions during the entire optimal harvest period. Although it realizes the same grain with sweet and waxy, it is only a simple superposition of traits, and the taste level is single. The grain may be extremely sweet in the early stage due to the sh2 gene, but lacks the waxy texture brought by starch accumulation; in the later stage, the sweet taste may be lost too quickly due to sugar degradation, and the continuous and dynamic taste evolution process of "early high sweetness → later waxy dominance and retention of clean sweet aftertaste" cannot be formed.

[0007] Although there are some sweet waxy types using bt1 , bt2 or su1 combined with wx gene in the prior art, they cannot realize "dynamic taste conversion". The taste of the existing varieties will deteriorate rapidly after the harvest period, either becoming hard or losing sweetness. In summary, the fresh corn varieties in the prior art cannot realize the dynamic and continuous taste conversion of "early high sweetness → later waxy dominance and retention of clean sweet aftertaste" in a single corn kernel. In addition, the wrinkling characteristics of dry kernels caused by homozygosity of sh1 gene have traditionally been considered as appearance defects by breeders and have not been effectively utilized in the commercialization process of fresh corn.

[0008] Therefore, there is an urgent need in the art for a new method for breeding a single corn inbred line with dynamic sweet waxy conversion characteristics without relying on expensive molecular marker technology, which can utilize intuitive phenotypic characteristics, and has low cost and high efficiency. SUMMARY

[0009] Therefore, the present application provides a single corn inbred line breeding method with dynamic sweet waxy conversion characteristics to solve the above problems. This method does not require molecular marker assistance, and by using the dry kernel wrinkling phenotype caused by sh1 gene as a morphological marker, combined with a set of strict post-pollination dynamic sugar and waxy change curve screening standards, a corn inbred line that can realize dynamic conversion of sweet and waxy traits in a single kernel and still retain clean sweet aftertaste at the mature stage is directionally bred.

[0010] In order to achieve the above application purposes, the present application provides the following technical solutions: A sweet waxy single corn inbred line breeding method, comprising the following steps: Parental selection and hybridization: Selection of the father: Choose the mutant type sh1 Homozygous inbred lines, which require the soluble sugar content of the grain to be ≥13% 20 days after pollination, and the shrinkage rate of mature dry grain to be ≥95%.

[0011] Selection of the mother: Choose the waxy type wx Homozygous inbred lines, which require the amylopectin content of the grain to be ≥95% 30 days after pollination.

[0012] Hybridize the above-mentioned mother and father to obtain F1 generation seeds, and self-cross the F1 generation to produce F2 separation populations.

[0013] F2 generation stepwise phenotype screening (locking waxy and wrinkled morphology): Mature milk screening: About 22 days after pollination, use iodine staining method (iodine-potassium iodide solution) to detect the grain of single plants in the F2 population. Select single plants with red-brown or brown-red endosperm, which is wx a visual marker of homozygous or heterozygous genes, and preliminarily locks the waxy characteristics.

[0014] Mature screening: Harvest the single plants screened at the mature milk stage and investigate the morphology of the mature dry grain. Select single plants with a dry grain shrinkage rate of ≥90%, which is a visual marker of homozygous genes with a high probability. sh1

[0015] F3 generation dynamic phenotype fine screening (locking sweet waxy dynamic conversion curve): Single seed the single plants screened in the F2 generation to form F3 generation lines.

[0016] Perform dynamic detection of F3 generation lines at different time periods, which must meet the following standards: 18 days after pollination: the soluble sugar content of the grain is ≥12%.

[0017] 22 days after pollination: the soluble sugar content of the grain reaches a peak value, which needs to be ≥18%.

[0018] 28 days after pollination: the soluble sugar content of the grain is ≥10%, and the iodine staining detection is red-brown (waxy stability is stable).

[0019] 35 days after pollination: the soluble sugar content of the grain is ≥8%, and the amylopectin content is ≥90%.

[0020] Retain lines with sugar content significantly higher than ordinary waxy corn (e.g., absolute value higher than 5%) throughout the detection period.

[0021] Backcross reinforcement and stability screening: Backcross the excellent single plants of the F3 generation that meet the standards with the original mother (inbred line) to obtain the backcross generation (BC1F1). sh1 ​​

[0022] High-intensity selection was performed on the backcross progeny population using a "double threshold elimination method": Threshold 1: Individual plants or lines with a peak sugar content of <18% during the milk stage (22 days after pollination) will be directly eliminated.

[0023] Threshold 2: Individual plants or lines with a dry grain shrinkage rate of <95% at maturity will be directly eliminated.

[0024] Through multiple generations of self-pollination and continuous screening according to the above standards, inbred lines with stable and consistent agronomic traits and a 100% qualified rate of dynamic phenotype were obtained.

[0025] By adopting the above technical solution, the present invention has the following beneficial effects: 1. This invention establishes for the first time a selection criterion based on the dynamic sugar content and glutinousness change curves at specific time points after pollination, enabling precise and targeted breeding for continuous transformation of sweet and glutinous traits within a single grain.

[0026] 2. This invention will sh1 The traditional "defect" phenotype of dry grain shrinkage caused by genes has been transformed into a method for indirect and efficient screening. sh1 The visualization morphological markers for homozygous genes have an accuracy rate of over 95%, eliminating the reliance on costly molecular marker technologies.

[0027] 3. By setting a hard target of sugar content ≥8% in the later stage of pollination (35 days), this invention ensures that the inbred lines selected in the final stage can still retain a distinct "sweet base flavor" in the waxy stage. Its sugar content is significantly higher than that of ordinary waxy corn, breaking through the bottleneck of the single flavor of traditional waxy corn and enhancing the market competitiveness of the product. Attached Figure Description

[0028] Figure 1 The target inbred line selected for this invention ( wx wx sh1 sh1 Comparison of kernel size between common sweet corn inbred line 403-11 (mother parent of Jingtian 7) and common waxy corn inbred line "Tongxi 5". Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 (1) Parental selection: Father: Hengbai 522 waxy mutant, which is disclosed in the paper "A novel allele of Sh1 underlies the conversion of waxy corn to wx-sweet corn" (https: / / www.biorxiv.org / content / 10.1101 / 2025.11.05.686684v1), which is sh1 a homozygous line, sh1 sh1 which was determined to have a soluble sugar content of about 16% in the kernels 22 days after pollination and a wrinkled rate of about 95% in the mature dry kernels.

[0031] Mother: Tongxi 5, which is wx a homozygous line, wx wx which was determined to have a amylopectin content of about 95% in the kernels 35 days after pollination.

[0032] (2) Parental cross and F2 generation phenotype screening: ① Parental cross Tongxi 5 was used as the female parent and Hengbai 522 waxy mutant was used as the male parent to cross, obtaining F1 generation seeds. After selfing of F1 generation, about 600 F2 separation population was obtained.

[0033] ② F2 generation ladder phenotype screening criteria (locking waxy basis and wrinkled morphology): Ripening period screening: about 22 days after pollination, iodine staining method (iodine-potassium iodide solution) was used to detect the kernels of single plants in F2 population, and single plants with red-brown or brown-red endosperm were selected, which were wx homozygous or heterozygous, and the waxy characteristics were preliminarily locked.

[0034] Mature period screening: the single plants screened in the ripening period were harvested, and the morphology of the mature dry kernels was investigated. Single plants with dry kernel wrinkled rate ≥ 90% were selected, which was a visual marker of sh1 high probability of homozygosity.

[0035] F2 generation screening results: Iodine staining detection was performed 22 days after pollination, and a total of 458 plants showed waxy quality (red-brown endosperm), accounting for 76.3% of the total population.

[0036] Of these 458 plants, 421 had a dry kernel wrinkled rate of greater than 90%, accounting for 91.9% of the waxy single plants.

[0037] (3) F2 generation seeding and F3 generation phenotype screening: ① The 421 single plants screened from F2 generation were each selfed to form 421 F3 generation lines.

[0038] ②F3 generation dynamic phenotype fine screening criteria (lock sweet glutinous dynamic conversion curve): Each strain of F3 generation is detected at different time periods, and the following criteria must be met: 18 days after pollination: grain soluble sugar content ≥ 12%.

[0039] 22 days after pollination: grain soluble sugar content reaches peak value, ≥ 18%.

[0040] 28 days after pollination: grain soluble sugar content ≥ 10%, and iodine staining detection shows reddish brown (glutinous stable appearance).

[0041] 35 days after pollination: grain soluble sugar content ≥ 8%, and amylopectin content ≥ 90%.

[0042] Strains that retain significantly higher sugar content than ordinary glutinous corn throughout the detection period (e.g., absolute value is 5% or more).

[0043] F3 generation dynamic detection and screening results: There are 266 strains with sugar content ≥ 18% at 22 days after pollination, with a compliance rate of 63.2%.

[0044] Continue to track these 266 strains, and find that there are 79 strains that meet the sugar content ≥ 8% at 35 days after pollination and amylopectin content ≥ 90%, with a key dynamic phenotype compliance rate of 29.7%.

[0045] (4) Backcross stability verification: ① Select the optimal single strain from the 79 strains that meet the standard, and backcross with the female parent "Tongxi 5", and strictly screen the backcross progeny (BC1F3).

[0046] ② Backcross screening criteria: The backcross progeny population is selected by "double threshold elimination method": Threshold one: single plants or strains with sugar content peak value < 18% at the mature stage (22 days after pollination) are directly eliminated.

[0047] Threshold two: single plants or strains with dry seed shrinkage rate < 95% at the mature stage are directly eliminated.

[0048] In the above two screening steps, any individual that does not meet the standard is directly eliminated. The remaining materials are selfed for multiple generations, and the above double threshold criteria are continuously applied for screening until a selfing line with stable and consistent agronomic traits and 100% compliance rate of double threshold phenotype indicators is obtained.

[0049] Ultimately, 12 stable strains with excellent comprehensive traits were successfully selected. The dynamic taste phenotype (sugar and starch indices at each stage after pollination) of these strains reached 100% pass rate, the wrinkling rate of mature dry kernels was stable between 99.2% and 99.8%, the apex was significantly concave, and they exhibited a typical crown shape.

[0050] The target inbred line selected in this invention ( wx wx sh1 sh1 The sugar content, amylopectin content, and mature dry kernel morphology of common sweet corn inbred line 403-11 (mother parent of Jingtian 7) and common waxy corn inbred line "Tongxi 5" were compared. The results are shown in Table 1 and 2. Figure 1 As shown.

[0051] As shown in Table 1, the sugar content of the maize inbred line in Example 1 of this invention is 18-22% 22 days after pollination, 8-10% 35 days after pollination, and 90-93% amylopectin content 35 days after pollination. The maize inbred line bred in this invention achieves a dynamic and continuous taste transformation characteristic of the same-kernel maize inbred line, which transitions from high sweetness in the early stage to a dominant glutinous taste in the later stage while retaining a sweet base flavor.

[0052] Table 1. Characteristics of maize inbred lines

[0053] As can be seen from the above embodiments, the present invention provides a method for breeding single-kernel maize inbred lines with dynamic sweet-waxy conversion characteristics. The maize inbred lines bred by the present invention achieve dynamic conversion of sweet-waxy traits within a single kernel.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for breeding inbred lines of maize with dynamic sweet-waxy conversion characteristics, characterized in that, Includes the following steps: (1) Parental selection and hybridization: Seeds with a soluble sugar content ≥13% and a mature dry seed wrinkling rate ≥95% 20 days after pollination. sh1 Using a homozygous inbred line as the male parent, and with a seed amylopectin content ≥95% 30 days after pollination... wx The homozygous inbred line was used as the female parent to cross and obtain the F1 generation. The F1 generation was then self-crossed to produce the F2 generation segregating population. (2) F2 generation screening: In the F2 population, glutinous plants with reddish-brown endosperm were screened at the milk stage, and plants with a dry grain shrinkage rate of ≥90% were screened from the glutinous plants at the maturity stage. (3) F3 generation screening Individual plants selected from the F2 generation were sown individually to form the F3 generation. The F3 generation was then dynamically monitored at different time points and screened to identify lines that simultaneously met the following conditions: 18 days after pollination: soluble sugar content in the seeds ≥12%; 22 days after pollination: soluble sugar content in the seeds ≥18%; 28 days after pollination: the soluble sugar content of the seeds is ≥10%, and the iodine staining test shows a reddish-brown color; 35 days after pollination: the soluble sugar content of the seeds is ≥8%, and the amylopectin content is ≥90%; Throughout the entire testing period, the sugar content remained more than 5% higher than that of ordinary glutinous corn; (4) Backcrossing and screening The superior F3 generation plants that meet the criteria are backcrossed with the original maternal parent to obtain the first generation backcross. The backcross progeny are then selected based on the following criteria: Eliminate individual plants or lines with a peak sugar content of <18% at the milk stage (22 days after pollination); Eliminate individual plants or lines with a dry grain shrinkage rate of <95% at maturity; Through multiple generations of self-pollination and continuous screening, inbred lines with stable and consistent agronomic traits and a 100% dynamic phenotypic qualification rate were obtained.

2. The method for breeding inbred lines of maize with dynamic sweet-waxy conversion characteristics according to claim 1, characterized in that, The inbred line "Tongxi 5" was used as the female parent and the inbred line "Hengbai 522 sweet glutinous mutant" was used as the male parent.

3. The method for breeding inbred lines of maize with dynamic sweet-waxy conversion characteristics according to claim 1, characterized in that, The milk maturity period is 22 days after pollination.

4. The method for breeding inbred lines of maize with dynamic sweet-waxy conversion characteristics according to claim 1, characterized in that, In step (2), glutinous plants with reddish-brown endosperm were screened using the iodine staining method.

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