A breeding method of long-spike rice with low amylose content

Abstract

This invention discloses a breeding method for long-spike, low-amylose-content rice. The method includes: hybridization using long-spike, large-grain Yiyang Dahe rice as the female parent and low-amylose-content japonica rice as the male parent; backcrossing the F1 generation seeds with the female parent once; planting for three consecutive generations; and performing molecular marker detection on the BC1F4 individual plant lines. Wx This invention involves inserting repetitive sequences into genes, designing dominant marker W1 and / or co-dominant marker W2 to detect the inserted repetitive sequences, and determining whether the corresponding BC1F4 line has a glutinous phenotype. The progeny lines obtained by planting glutinous BC1F4 single plants through multiple generations exhibit non-segregating phenotypes. This invention solves the problem of existing technologies failing to improve the amylose content of Dahu rice and thus being unsuitable for producing high-quality Yiyang rice cakes. By combining traditional hybridization-backcrossing methods with RVA viscosity detection, molecular marker-assisted selection, and phenotypic identification breeding techniques, a new Bai Ruyu Dahu rice variety with long spikes, large grains, disease resistance, and low amylose content can be rapidly and accurately bred.

Description

Technical Field

[0001] This invention relates to a method for breeding rice, specifically a method for breeding long-panicle rice with low amylose content. Background Technology

[0002] Yiyang rice cake has a history of over 1200 years in Yiyang County, Jiangxi Province, and is a traditional local delicacy. Its production process and quality highly depend on the local specialty rice variety, "Yiyang Dahegu." This variety has round grains and a large thousand-grain weight, resulting in rice cakes with a soft and chewy texture, a rich rice aroma, and a unique local flavor. However, with the improvement of people's living standards and the gradual change in dietary habits, the amylose content of the traditional "Yiyang Ma Ke Dahegu" rice is relatively high compared to modern dietary requirements. This results in rice cakes that are harder and more prone to cracking after processing, affecting their appearance, texture, transportation, and storage, thus hindering its industrial upgrading and market competitiveness.

[0003] To address this issue, while preserving the inherent chewy and non-sticky texture of Yiyang rice cakes, the content of amylose is reduced to improve product quality. Existing literature 1 (Chinese invention patent application CN117751839A) discloses a breeding method for a new high-quality long-grain japonica rice variety from southern China. This method includes: selecting a high-quality long-grain indica rice variety from southern China as the male parent and a large-spike japonica rice variety as the female parent for hybridization; backcrossing the F1 generation seeds with japonica rice; planting BC1F1 seeds for three consecutive generations; selecting compact plant types, flag leaf morphology, good color change, low chalkiness, and long grains with large spikes (preferably japonica) for further generations in the field; and performing molecular marker detection on individual plants of the BC1F4 generation. Gn1a, GS3 Two genes, screening for those containing Gn1a The plants were tested again, and the BC1F5 generation of the breeding candidate line was tested again to obtain plants that were homozygous for the target gene.

[0004] Traditionally, Yiyang hemp seed kernels have a moderate amylose content (16%~18%), but modern consumers may prefer a softer, more glutinous texture. However, reference 1 only focuses on genes related to grain shape and number of kernels per ear. Gn1a, GS3 The breeding objectives focused on appearance quality (long grain type) and yield traits (large spike), and the targeted improvement of taste quality traits such as amylose content was not precise enough. Moreover, the variety bred in Literature 1 is a long grain type, not Yiyang Dahegu (oval grain type), and cannot be used to make high-quality Yiyang rice cake. Summary of the Invention

[0005] The purpose of this invention is to provide a breeding method for long-spike, low-amylose-content rice, which solves the problem that existing technologies have not improved the amylose content of large-grain rice and cannot be used to make high-quality Yiyang rice cakes. By combining traditional hybridization-backcrossing methods with RVA viscosity detection, molecular marker-assisted selection and phenotypic identification breeding technology, a new variety of Bai Ruyu large-grain rice with long spikes, large grains, disease resistance and low amylose content can be bred quickly and accurately.

[0006] To achieve the above objectives, the present invention provides a method for breeding long-eared rice with low amylose content, the method comprising: (1) Using long-spike, large-grain Yiyang Dahegu as the female parent and low-amylose content japonica rice as the male parent, the F1 generation seeds were backcrossed with the female parent once to obtain BC1F1; (2) BC1F1 seeds were planted for three consecutive generations to obtain BC1F4; (3) Molecular marker detection of the BC1F4 line Wx Genes, in Wx The second exon of the gene contains a repetitive sequence. A dominant marker W1 and / or a co-dominant marker W2 are designed to detect the inserted repetitive sequence and determine whether the phenotype of the corresponding BC1F4 line is waxy. After inserting repeating sequences Wx The nucleotide sequence of the gene is shown in SEQ ID NO.5; The inserted repeat sequence is the sequence between positions 1388 and 1433 in the nucleotide sequence shown in SEQ ID NO.5; The nucleotide sequence of the dominant marker W1 is shown in SEQ ID NO.1 and SEQ ID NO.2; the nucleotide sequence of the co-dominant marker W2 is shown in SEQ ID NO.3 and SEQ ID NO.4. (4) The BC1F4 single plant line with the glutinous phenotype was planted for a generation to obtain the offspring single plant line. The glutinous phenotype of the offspring single plant line was observed to see if it segregated. If it did not segregate, the genotype of its glutinous phenotype was homozygous, and the corresponding offspring single plant line was a new long-spike type of large rice variety.

[0007] Preferably, the F nucleotide sequence of the dominant marker W1 is as shown in SEQ ID NO.1, which is located at... Wx Within the first intron of the gene, the R nucleotide sequence is as shown in SEQ ID NO.2, located at... Wx The second exon of the gene is inserted into a repetitive sequence.

[0008] Preferably, the F nucleotide sequence of the codominant marker W2 is as shown in SEQ ID NO.3, which is located at... WxWithin the second exon of the gene, the R nucleotide sequence is as shown in SEQ ID NO.4, located at... Wx Downstream of the repetitive sequence inserted into the second exon of the gene.

[0009] Preferably, the long-eared, large-grained Yiyang large-grained millet is Yiyang hemp-hulled large-grained millet.

[0010] Preferably, the japonica rice with low amylose content is Zhe Nuo No. 4.

[0011] Preferably, the average spike length of the long-spike type of new rice variety is more than 26 cm.

[0012] Preferably, the rice of the long-spike type Dahegu new rice variety is milky white in color, and the color of the rice is obtained by RVA analysis of rice stickiness.

[0013] The present invention provides a method for breeding long-spike rice with low amylose content, which solves the problem in existing technologies that fail to improve the amylose content of large rice varieties and cannot be used to make high-quality Yiyang rice cakes. This method has the following advantages: This invention utilizes RVA (Rapid Visco Analyzer) technology to screen for low-AC (amylose content) japonica rice variety Zhe Nuo 4, which meets the viscosity characteristics of Yiyang Dahe Valley rice, as a donor. Improvement is achieved through a first generation of hybridization-backcrossing. Wx The second exon of the gene inserts a repetitive sequence, and in intermediate generations... Wx Gene marker detection and tracking were conducted once. Through panicle length phenotypic identification, resistance identification, and hulling observation in higher generations, a high-yielding, high-quality, well-colored, long-panicleed, large-grained, pure white rice variety with an AC content between 1.5% and 2.0% and a chewy texture, "Bai Ru Yu Da He Gu" (Plant Variety Protection Number: 20251005653), a high-end local specialty rice, was finally bred. This provides a solid foundation for promoting the revitalization and high-quality development of the Yiyang County rice cake industry. The Yiyang rice cake made from the Da He Gu obtained by this invention has the advantages of being crystal clear, pure white, soft, chewy, and non-sticky. Attached Figure Description

[0014] Figure 1 This is an electrophoretic image used in the present invention to detect glutinous and non-glutinous rice varieties.

[0015] Figure 2 This is a phenotypic diagram of the Dahe Valley of the present invention.

[0016] Figure 3 This invention relates to the finished product of Bai Ruyu Dahegu Rice Cake. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1 A method for breeding long-spike rice with low amylose content, the method comprising: (1) Using the local long-spike, large-grained Yiyang Ma Ke Da He Gu as the female parent and Zhe Nuo 4 as the male parent, a cross was performed. The F1 generation was backcrossed with the female parent once to obtain BC1F1. Among them, Zhe Nuo 4 has excellent traits such as japonica straw, moderate resistance to bacterial blight, and low lodging content. Its RVA characteristics are similar to those of Yiyang Da He Gu. It was bred by the Institute of Crop and Nuclear Technology Utilization of Zhejiang Academy of Agricultural Sciences and approved by Zhejiang Province in 2006, with the number: Zhe Shen Dao 2006018. Yiyang Ma Ke Da He Gu was selected and bred by the Yiyang County Agricultural and Rural Industry Development Service Center and approved by Jiangxi Province in 2024, with the number: Gan Shen Dao 20240017. It has the characteristics of long spikes and large grains.

[0019] (2) BC1F1 seeds were planted for three consecutive generations, with no less than 200 individual plants per generation. In the field, plants with good leaf morphology, stems similar to japonica rice, and not easy to lodging were selected and planted with local large rice plants with long ears and large grains and strong disease resistance to obtain BC1F4. (3) Molecular marker detection of BC1F4 single-plant lines Wx Genes, in Wx The second exon of the gene contains a repetitive sequence. Dominant marker W1 and co-dominant marker W2 are designed to detect the inserted repetitive sequence (two 23bp repetitive sequences) and determine whether the phenotype of the corresponding BC1F4 line is waxy.

[0020] After inserting repeating sequences Wx The nucleotide sequence of the gene is shown in SEQ ID NO.5; the inserted repeat sequence is the sequence between positions 1388 and 1433 of the nucleotide sequence shown in SEQ ID NO.5. A dominant marker W1 was designed to attach to the first intron (F) and the position (R) of the inserted repeat sequence. The purpose of the dominant marker W1 is to detect whether the corresponding BC1F4 single plant line is a glutinous or non-glutinous rice variety by agarose gel electrophoresis after detecting the first intron and the inserted 23bp repeat sequence. Since the R nucleotide sequence of W1 attaches to the 23bp repeat sequence, the corresponding BC1F4 single plant line that can be amplified has a glutinous phenotype, while those that cannot be amplified are non-glutinous. The detection results showed that only glutinous varieties showed a single band of 492bp (insertion-specific), while non-glutinous varieties showed no band. Figure 1(a) Wherein, the F nucleotide sequence of W1, as shown in SEQ ID NO.1, GGTGCAACGGCCAGGATA, is located at the first intron position; the R nucleotide sequence of W1, as shown in SEQ ID NO.2, TGGAACCCGTGGGCTTGA, is located at the 23bp position of the double repeat sequence in the second exon.

[0021] The design of co-dominant marker W2 at both ends of the inserted repeat sequence can amplify two band patterns: one with the repeat sequence (207 bp) and one without the repeat sequence (184 bp). Figure 1 (b). Among them, the BC1F4 single-strain line that can amplify the repeat sequence fragment (207bp) is glutinous. The F nucleotide sequence of the codominant marker W2, as shown in SEQ ID NO.3: CTCTCACCACGTCCCAGC, is located in the second exon region and serves as the forward primer binding site; the R nucleotide sequence of the codominant marker W2, as shown in SEQ ID NO.4: ACCGACCGCTGCTGCTTG, is located downstream of the 23bp repeat sequence inserted in the second exon (i.e., the sequence in SEQ ID NO.4 spans the endpoint of the inserted 23bp repeat sequence) and serves as the reverse primer binding site.

[0022] like Figure 1 As shown, the electrophoresis diagrams for detecting glutinous and non-glutinous rice varieties in this invention are as follows: a) Using the W1 dominant molecular marker to detect glutinous and non-glutinous rice varieties, only glutinous varieties show a single 492bp band after agarose gel electrophoresis, while non-glutinous varieties show no band; b) Using W2 as a co-dominant marker to detect glutinous and non-glutinous rice varieties, both show a single bright band after agarose gel electrophoresis, but the amplification band of glutinous varieties shows lag; 2000M, BRY, ZH11, and XB represent the 2000 marker, Bairuyu Dahegu, Zhonghua 11, and Xieqingzao B, respectively. Figure 1 It can be seen that only glutinous varieties will show a single band of 492bp (insertion-specific), while non-glutinous varieties will not show a band. Figure 1 (a) The co-dominant marker W2 is designed at both ends of the inserted repeat sequence, which can amplify two band patterns: one with the repeat sequence (207 bp) and one without the repeat sequence (184 bp). Figure 1 (b) Individual plants that amplified to obtain a fragment of approximately 207 bp were considered glutinous (with duplicate insertions); individual plants that amplified to obtain a fragment of approximately 184 bp were considered non-glutinous (without duplicate insertions).

[0023] The phenotype of glutinous and non-glutinous seeds can be determined visually by drying the seeds and peeling off the shells; pure white seeds are glutinous, while those that are not pure white are non-glutinous.

[0024] (4) The BC1F4 single plant line with the detected glutinous phenotype was planted for a second generation to obtain the BC1F5 single plant line. The phenotype of the BC1F5 single plant line was observed. If the phenotype of the BC1F5 single plant line showed segregation (the color of the grains was uneven after drying and peeling), then its genotype was heterozygous. It was necessary to continue to select for a second generation until the phenotype did not show segregation. If the phenotype of the BC1F5 single plant line did not show segregation, then its genotype was homozygous. The corresponding BC1F5 line was a new high-yielding and high-quality white jade-like rice variety with long spikes, large grains, and white peeled rice. It was resistant to lodging, disease, and high temperature. Plant variety protection number: 20251005653.

[0025] like Figure 2 The diagram shows the phenotypic pattern of the large-grain rice of this invention, where a is the control large-grain rice plant type, b is the white-jade large-grain rice plant type, and c is the white-jade large-grain rice spike type (top) and grain type (bottom). (From...) Figure 2 It can be seen that the present invention results in shorter plant height of the rice, increased milling rate, decreased amylose content (AC value), and longer gel consistency.

[0026] The shapes of the obtained white jade-like large grains and hemp-shell large grains were compared in detail, and the results are shown in Table 1.

[0027] Table 1 Comparison of the characteristics of Dahe Valley The new Dahegu variety contains the nucleotide sequence shown in SEQ ID NO.5, as detailed below:

[0028] The new Bai Ruyu Dahe rice variety obtained by this invention has been tested by the Food Quality Inspection and Testing Center of the Ministry of Agriculture and Rural Affairs (Wuhan) according to the NY / T 593-2021 "Quality of Edible Rice Varieties" standard and meets the quality requirements of a high-quality third-grade edible glutinous rice variety (Test Report: NO.2025QS4176). However, the local Yiyang Ma Ke Dahe rice variety does not meet the quality requirements of a high-quality third-grade edible glutinous rice variety.

[0029] like Figure 3 As shown, the present invention relates to a finished product of Bai Ruyu Dahegu rice cake, wherein a is a rice cake made from Bai Ruyu Dahegu; and b is a rice cake made from local hemp seed cake. Figure 3 It is known that the rice cake made by Bai Ruyu Dahegu is whiter, softer, less prone to cracking, and retains the chewy and non-sticky properties of rice cakes made by local Ma Ke Dahegu.

[0030] The Yiyang rice cake made from the white jade-like grain obtained by this invention is better than the existing Yiyang rice cake, with the advantages of crystal clear color, white as jade, soft texture and chewy and non-sticky texture.

[0031] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for breeding long panicle rice with low amylose content, characterized by, The method includes: (1) Using long-spike, large-grain Yiyang Dahegu as the female parent and low-amylose content japonica rice as the male parent, the F1 generation seeds were backcrossed with the female parent once to obtain BC1F1; (2) BC1F1 seeds were planted for three consecutive generations to obtain BC1F4; (3) Molecular marker detection of the BC1F4 line Wx Genes, in Wx The second exon of the gene contains a repetitive sequence. A dominant marker W1 and / or a co-dominant marker W2 are designed to detect the inserted repetitive sequence and determine whether the phenotype of the corresponding BC1F4 line is waxy. After inserting repeating sequences Wx The nucleotide sequence of the gene is shown in SEQ ID NO.5; The inserted repeat sequence is the sequence between positions 1388 and 1433 in the nucleotide sequence shown in SEQ ID NO.5; The nucleotide sequence of the dominant marker W1 is shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleotide sequence of the codominant marker W2 is shown in SEQ ID NO.3 and SEQ ID NO.4; (4) The BC1F4 single plant line with the glutinous phenotype was planted for a generation to obtain the offspring single plant line. The glutinous phenotype of the offspring single plant line was observed to see if it segregated. If it did not segregate, the genotype of its glutinous phenotype was homozygous, and the corresponding offspring single plant line was a new long-spike type of large rice variety.

2. The breeding method according to claim 1, characterized in that, The F nucleotide sequence of the dominant marker W1 is as shown in SEQ ID NO.1, which is located at... Wx Within the first intron of the gene, the R nucleotide sequence is as shown in SEQ ID NO.2, located at... Wx The second exon of the gene is inserted into a repetitive sequence.

3. The breeding method according to claim 1, characterized in that, The F nucleotide sequence of the codominant marker W2 is as shown in SEQ ID NO.3, and its sequence is located at... Wx Within the second exon of the gene, the R nucleotide sequence is as shown in SEQ ID NO.4, and its sequence is located at... Wx Downstream of the repetitive sequence inserted into the second exon of the gene.

4. The breeding method according to claim 1, characterized in that, The long-eared, large-grained Yiyang Dahegu (Yiyang hemp-shelled Dahegu) is the Yiyang Ma Ke Dahegu.

5. The breeding method according to claim 1, characterized in that, The japonica rice with low amylose content is Zhe Nuo No.

4.

6. The breeding method according to claim 1, characterized in that, The average spike length of the new long-spike type of rice variety is over 26 cm.

7. The breeding method according to claim 1, characterized in that, The rice of the new Dahegu rice variety with long spikelets is milky white in color.

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