Site GAE12 interacting with Wx gene to synergistically regulate and control appearance and taste quality of rice and application of site GAE12

Abstract

The invention discloses a site GAE12 capable of interacting with a Wx gene to synergistically regulate and control the appearance and taste quality of rice and application of the site GAE12, the physical position of the site is located in the 218kb interval of 2.086-2.304 Mb of the 12 # chromosome of the rice, molecular markers are GAE12-1 and GAE12-2, a primer of the GAE12-1 is shown as SEQ ID No.1-SEQ ID No.2, and a primer of the GAE12-2 is shown as SEQ ID No.3-SEQ ID No.4. The invention further discloses a method for preparing the GAE12-1 and the GAE12-2. The invention provides a site GAE12 capable of synergistically improving the appearance and taste quality of rice in different types of rice varieties and a linked molecular marker of the site, and the site is combined with different Wx alleles, so that the synergistic improvement of the appearance and taste quality of the rice can be realized. The method can be used for synergistically improving the appearance and taste quality of indica rice varieties and improving the transparency of soft rice grains with low amylose content. The method provides an important technical route for cultivating a new rice variety with better appearance and taste quality, and has important significance for improvement research on the appearance and taste quality of rice.

Description

Technical Field

[0001] The present invention belongs to the technical field of rice molecular marker-assisted selection breeding, and specifically relates to a breeding method for improving the appearance and taste quality of rice using molecular marker-assisted selection technology. The method is suitable for the synergistic improvement of the appearance and taste quality of varieties with high chalkiness and high amylose content, and for improving the transparency of soft rice grains with low amylose content and dark endosperm. Background Art

[0002] my country's current rice production level is relatively high, but the production of rice with both excellent appearance and excellent flavor ("appearing and delicious") is still far from meeting the demands of the rice industry and the public. Rice quality involves multiple key aspects, including milling, appearance, cooking, flavor, and nutrition. The most important of these are the appearance and flavor qualities (Zhang Changquan et al., Progress in Inheritance of Rice Quality Traits and Cloning of Important Genes in Jiangsu Province [J]. Heredity, 2021, 43, 425–441). Rice appearance quality is an important commercial quality, typically including grain shape, chalkiness, chalkiness rate, and transparency. Clear standards for the degree of chalkiness that defines high-quality rice exist both domestically and internationally. A comprehensive analysis of the quality-of-rice standards achieved by approved rice varieties in my country in recent years shows that the chalkiness rate and chalkiness rate are relatively low, while the other indicators are relatively high. Chalkiness has always been a major factor restricting the improvement of rice quality in my country. Therefore, reducing rice chalkiness has become a pressing issue in improving rice quality (Li Yi-bo et al., Genetic Improvement of Rice Quality Traits and Key Scientific Issues [J]. Life Sciences, 2016, 28, 1168–1179). Furthermore, chalkiness is closely related to rice cooking, flavor, and processing quality. Conventional breeding often aims to reduce chalkiness by selecting narrow-grain varieties. However, the flavor quality of some slender-grain rice varieties with attractive appearances still needs improvement, and the phenomenon of "appearing good but not tasting good" remains common.

[0003] Cooking and eating quality includes cooking characteristics, taste, viscosity and smell, which mainly depends on the physical and chemical properties of storage substances such as starch and protein in rice endosperm. Among them, amylose content (AC) is the most important factor determining the eating quality. Wx Granule-bound starch synthase I (GBSSI) encoded by the gene is catalyzed and synthesized, and there are rich allelic variations among varieties ( Wx lv 、 Wx a 、 Wx in 、 Wx b 、 Wxmw / la 、 Wx op / Wx hp 、 Wx mq 、 Wx mp and wx ), is the most critical site affecting the cooking and eating quality of rice. Wx lv and Wx a Both control alleles with high AC (25% and above), which are mainly found in wild rice and some indica rice varieties. Wx b Controls moderate levels of AC (~15%), mainly found in common japonica rice. Wx mp Controlling low levels of AC (8-12%) is mainly found in japonica soft rice varieties in the Jiangsu and Zhejiang areas, such as the "Nanjing" series of soft rice varieties (Wang Cailin et al., Genetics and breeding of excellent tasting japonica rice in Jiangsu Province [J]. Heredity, 2021, 43, 442-448.).

[0004] Generally, rice with lower AC tends to have better eating quality. However, physical and chemical indicators such as AC that determine rice cooking and eating quality also have a significant impact on other quality traits, such as appearance. Although reducing AC can significantly improve rice eating quality, too low AC can also reduce rice transparency, making the rice translucent (dark endosperm) or opaque (glutinous), seriously affecting its appearance and creating the dilemma of "tasty but not good-looking." For example, soft rice varieties with low amylose content often have excellent eating quality but poor grain transparency. Therefore, under the current premise of high-yield breeding, how to synergistically improve rice appearance and eating quality through genetic regulation is a major challenge. From the perspective of breeding practice, it is urgent to expand new germplasm and new genes that meet breeding needs through the exploration of germplasm resources and new genes, and to develop relevant breeding technologies to accelerate the development of new high-quality rice varieties. Summary of the Invention

[0005] In view of the above-mentioned difficulties in synergistically improving the appearance and taste quality of rice by genetic regulation, the present invention provides a method for Wx Gene interactions coordinately regulate rice appearance and taste quality loci GAE12 And application, it can accurately introduce and improve the high-quality traits of rice, and has important breeding value.

[0006] In order to achieve the above object, the present invention provides a Wx Gene interactions coordinately regulate rice appearance and taste quality loci GAE12The molecular marker of the locus is located in the 218kb interval of 2.086-2.304Mb on rice chromosome 12, which is derived from the allelic type in japonica rice Nipponbare (Nip). GAE12 Nip It can reduce rice chalkiness under conditions of high amylose content, and is derived from the allele type in the indica rice Qingluzhan 11 (Q11). GAE12 Q11 It can increase the amylose content of rice and improve the transparency of rice under conditions of medium and low amylose content without affecting the chalkiness of rice.

[0007] Located at the site GAE12 The adjacent molecular marker GAE12-1, the forward primer sequence is 5'-CGGGTGTATCGGTTCAAGACGA-3' (SEQ ID No. 1), and the reverse primer sequence is 5'-CGAAGATCACGCAAACCCTAGC-3' (SEQ ID No. 2). Located at the site GAE12 The adjacent molecular marker GAE12-2, the forward primer sequence is 5'-TGAATTGGAGCCAGTGGTAG-3' (SEQ ID No. 3), and the reverse primer sequence is 5'-CAAGGCAGCAAGATAAATGA-3' (SEQ ID No. 4).

[0008] The second aspect of the present invention provides the above-mentioned molecular markers in Wx Application of gene interaction in the coordinated regulation of rice appearance and taste quality.

[0009] Using the genomic DNA of rice varieties Nipponbare, indica rice variety Qingluzhan 11, and other rice varieties and lines to be analyzed as templates, PCR amplification was performed using primers for molecular markers GAE12-1 and GAE12-2. Electrophoresis was then used to detect and distinguish the genotypes they carried ( GAE12 Nip , GAE12 Nip / Q11 , GAE12 Q11 ). Among them, Nippon Haru carries GAE12 Nip , Qing Lu accounted for 11 carrying GAE12 Q11 .

[0010] The specific PCR amplification system was configured according to the company's instructions. The PCR amplification system used in this invention consisted of a 20 μL volume, with a 5-minute initial denaturation at 95°C, followed by a three-step amplification process: denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds. After 35 cycles, the amplified product was extended at 72°C for 2 minutes, followed by a 10°C hold. 10 μL of the PCR amplification product was analyzed by 3% agarose gel electrophoresis. The amplified product of the molecular marker GAE12-1 in Nipponbare was 307 bp, while that in Qingluzhan11 was 275 bp. The amplified product of the molecular marker GAE12-2 in Nipponbare was 123 bp, while that in Qingluzhan11 was 145 bp.

[0011] The molecular markers GAE12-1 and GAE12-2 were used to GAE12 Specific allele type ( GAE12 Nip or GAE12 Q11 ) is introduced into the genome of the improved variety through hybridization and backcrossing to achieve the improvement of the target trait. Specifically, in the case of high amylose content (AC>20%) and carrying GAE12 Q11 locus into rice varieties or lines GAE12 Nip It can significantly reduce rice chalkiness and improve rice appearance and taste quality; in low amylose content (AC about 8%~12%) and carrying GAE12 Nip loci were introduced into japonica rice varieties GAE12 Q11 The amylose content of rice can be appropriately increased, significantly improving the transparency of rice grains without affecting the chalkiness of rice.

[0012] The third aspect of the present invention provides a breeding method for improving the appearance and taste quality of high chalky indica rice, which is carried out by conventional hybridization methods with low chalkiness and good taste quality. GAE12 Nip Rice germplasms with high chalkiness, high amylose content and poor eating quality are used as hybrid donors. GAE12 Q11 Rice germplasm of the locus (such as Teqing, Nip-GAE12 Q11 ) and perform molecular marker-assisted selection on the hybrid offspring; the molecular marker-assisted selection is started from the F2 generation, using the above molecular markers to perform PCR amplification and gel electrophoresis detection to select GAE12 Nip The individuals with the same genotype are further backcrossed, and the offspring with good comprehensive agronomic traits and carryingGAE12 Nip The genotyped single plants are grown into lines for plot identification to obtain new lines with stable traits.

[0013] The fourth aspect of the present invention provides a breeding method for improving the dark endosperm phenotype of low amylose content soft rice, which is carried out by conventional hybridization method. GAE12 Q11 The genotype line is used as a hybrid donor and is hybridized with the soft rice variety NG9108 with low amylose content and poor grain transparency, and molecular marker-assisted selection is performed on the hybrid offspring; the molecular marker-assisted selection is performed starting from the F2 generation, using the above molecular markers for PCR amplification and gel electrophoresis detection to select the GAE12 Q11 The individuals with the same genotype are further backcrossed, and the offspring with good comprehensive agronomic traits and carrying GAE12 Q11 The genotyped single plants are grown into lines for plot identification to obtain new lines with stable traits.

[0014] Through the above technical solution, the present invention achieves the following beneficial effects: The present invention provides a locus that can synergistically improve rice appearance and taste quality in different types of rice varieties GAE12 and the linked molecular markers of the site, by linking the site to different Wx Allele combinations can achieve synergistic improvements in rice appearance and taste quality. Therefore, this invention enables the precise introduction and improvement of high-quality rice traits, possessing significant breeding value. This method can be used to synergistically improve the appearance and taste quality of indica rice varieties, as well as improve the transparency of low-amylose soft rice kernels. This provides an important technical route for breeding new rice varieties with superior appearance and taste quality, and is of great significance to research on improving rice appearance and taste quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for GAE12 Identification of the locus and its effect on rice quality. (A): Plant type of chromosome segment substitution line; (B): Rice appearance; (C): Genotype map of CSSL-N8 (blue: Nip genotype, red: Q11 chromosome substitution segment); (D): Genetic analysis of N112 / CSSL-N8 F2 population; (E): Genetic analysis of different chromosome segment substitution lines. GAE12Loci were identified by overlapping mapping; (F): Co-segregation analysis of linked markers (for molecular marker GAE12-1, A: Nip genotype, product 307 bp; B: Q11 genotype, product 275 bp; for molecular marker GAE12-2, A: Nip genotype, product 123 bp; B: Q11 genotype, product 145 bp; lanes 1-5 are high chalkiness plants isolated from the F2 population of the N112 / CSSL-N8 hybrid); (GJ): Comparative data on appearance, chalky kernel rate, chalkiness, and amylose content of rice grains of near-isogenic lines; Figure 2 This is a synergistic improvement of the appearance and taste quality of the highly chalky indica rice variety Teqing. (A): Schematic diagram of the molecular marker-assisted selection process; (B): Linked marker identification, where lanes 1-5 are the carriers of the Teqing background obtained by screening. GAE12 J Homozygous lines of the allele; (CG): Comparison of rice appearance, chalky grain rate, chalkiness, amylose content and taste value of improved lines under the Teqing background; Figure 3 To improve the grain transparency of the soft japonica rice variety with good taste. (A): Schematic diagram of the molecular marker-assisted selection process; (B): Linked marker identification, where lanes 1-5 are the carriers of the Nanjing 9108 background obtained by screening. GAE12 I Homozygous lines for the same allele; (CF): Comparison of rice appearance, grain transparency, amylose content and taste value of improved lines under the Nanjing 9108 background. DETAILED DESCRIPTION

[0016] The following is a detailed description of the specific embodiments of the present invention in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0017] Example 1 GAE12 Identification of loci and their effects on rice quality In research related to rice quality trait gene discovery, the laboratory used the japonica rice variety Nipponbare as the recurrent parent and the indica rice variety Qingluzhan 11 as the donor. Through multiple generations of backcrossing followed by selfing, combined with molecular marker-assisted selection, a total of 83 stable chromosome segment replacement lines were obtained. By investigating the chalkiness of rice in the above CSSLs population, a stable rice line with high chalkiness, CSSL-N8, was identified. Through whole-genome resequencing of rice, it was confirmed that it carries multiple introduced fragments ( Figure 1 In addition to the high chalkiness phenotype, the N8 rice line also has a high amylose content phenotype (carrying Wx lv), in order to exclude the influence of high amylose content, it was compared with the high amylose content (carrying Wx lv ), but the low chalkiness chromosome segment substitution line N112 was hybridized and self-pollinated to obtain an F2 population for genetic analysis of the chalkiness trait. The results showed that among the 251 individual plants surveyed, 186 had high chalkiness and 65 had low chalkiness, with a segregation ratio close to 3:1 for a single gene, indicating the presence of a dominant locus in N8 that controls high chalkiness ( Figure 1 Considering that the above-mentioned segregating populations involved multiple replacement segments, polymorphic molecular markers were designed on rice chromosomes 9, 11, and 12 based on the resequencing results for linkage analysis. The results showed that only lines carrying the introduced segment on the short arm of chromosome 12 exhibited the high chalkiness phenotype. Based on the comparison of chromosome segment overlap, the target site was confined to a 218 kb interval between 2.086 and 2.304 Mb on chromosome 12, and this site was named GAE12 ( Figure 1 To verify the reliability of this locus, molecular markers GAE12-1 and GAE12-2 were designed at both ends of the target segment based on the target segment range and resequencing data. Linkage analysis was performed on high chalky plants in the constructed F2 segregating population, and it was found that all high chalky plants were linked to the molecular markers GAE12-1 and GAE12-2 ( Figure 1 F), description GAE12 Located between two molecular markers, and can be tracked by two molecular markers. In order to clarify the effect of this site on rice quality, the high chalky phenotype will be controlled. GAE12 Named GAE12 Q11 , control low chalkiness, from Nip GAE12 Named GAE12 Nip In the isolated population, two genotypes of Nip ( Wx lv / GAE12 Nip ) and Nip( Wx lv / GAE12 Q11 ) were used to compare the quality of rice. At the same time, considering the effect of amylose content on rice quality, the high chalky white line was compared with its receptor Nip (carrying Wx b ) hybridization, and screening of the segregated progeny to obtain the low and medium amylose content Wx b Nip( Wxb / GAE12 Nip ) and Nip( Wx b / GAE12 Q11 ) Two genotype strains were used for quality analysis. The results showed that GAE12 Q11 exist Wx lv can result in a high chalky phenotype in the background, but Wx b The background does not affect the chalkiness of rice ( Figure 1 The analysis of rice amylose content showed that GAE12 Q11 exist Wx lv The background does not affect rice AC, but Wx b Under the background, it can significantly increase rice AC ( Figure 1 The above results show that GAE12 Can be used with Wx Genetic interactions synergistically improve chalkiness and amylose content in rice.

[0018] Example 2 Synergistic Improvement of Appearance and Taste Quality of Highly Chalky Indica Rice Variety Teqing Teqing (TQ, Teqing) is a super high-yielding conventional indica rice variety developed in my country. Wx a Allelic type, rice AC is high and taste quality is poor, at the same time, the chalkiness of the special green rice is very high. By using the molecular markers GAE12-1 and GAE12-2 in Example 1, it was found that they carry the same locus as Q11 GAE12 Q11 , which shows that through GAE12 Q11 The replacement of the site may improve its poor quality traits. Wx b / GAE12 Nip ) as the donor parent and hybridized and backcrossed with Teqing, respectively using Wx The gene-linked molecular marker QRM190 (SEQ ID No.5: 5'-ATTCCTTCAGTTCTTTGTCTATCTCA-3' and SEQ ID No.6: 5'-TCCTGATGAACAACAGAACAACAC-3', known primers) and GAE12 NipThe progeny of GAE12-1 and GAE12-2 were hybridized and backcrossed by molecular marker assisted selection. Wx b and GAE12 Nip The individual plants of the gene combination were planted into three lines TQ ( Wx b / GAE12 Nip )-1, TQ( Wx b / GAE12 Nip )-2、TQ( Wx b / GAE12 Nip )-3 Comprehensive character identification ( Figure 2 Comparison of quality traits revealed that the chalky grain rate, chalkiness, and amylose content of the improved rice were significantly reduced, while the rice taste value was significantly increased ( Figure 2 Based on the above data, it is shown that the GAE12 Nip joint Wx b The allele can significantly improve the appearance and taste quality of rice in indica rice varieties.

[0019] Example 3: Creation of New Japonica Rice Material with Excellent Taste and Improvement of Rice Transparency Since Nip( Wx b / GAE12 Q11 ) lines have significantly increased rice amylose content, therefore, it is believed that GAE12 Q11 Under the condition of medium-low amylose content, it has the potential to moderately increase the amylose content of rice. For the low amylose content soft rice currently used in production (such as Nanjing 9108), its too low amylose content leads to unfavorable appearance qualities such as "dark endosperm" or "semi-glutinous". However, moderately increasing the amylose content can achieve a synergistic improvement in the "appearance" and "taste" of soft rice to a certain extent. This solution utilizes Nip ( Wx b / GAE12 Q11 ) strain as the donor parent and Nanjing 9108 (9108) as the recurrent parent for hybridization and backcrossing. GAE12 Through 4 generations of backcrossing, the carriers of GAE12-1 and GAE12-2 were selected from BC4F3. GAE12 There are 2 stable strains of the locus, 9108 (GAE12 Q11 )-1 and 9108( GAE12 Q11 )-2Identify the quality characteristics ( Figure 3 Comparison of quality traits revealed that the appearance (transparency) of the improved rice was significantly improved compared to the control parent 9108, and the amylose content of the rice increased by about 2%, while the taste quality showed no significant difference compared to the control parent ( Figure 3 Based on the above data, it is shown that this method can quickly and specifically improve the transparency of low-amylose soft rice (improve the dark endosperm phenotype), thereby obtaining a new excellent strain with both good appearance and taste quality. Based on this, the high-yield soft rice varieties Nanjing 5055 and Nanjing 3908 were used in combination with the above-mentioned 9108 ( GAE12 Q11 ) were further hybridized, and based on molecular marker selection, NYJ22706, Ning 21847, Ning 22614, Ning B9039, NYJ0020, JD925, JD24036, JD2424 and W2675 carrying the gene were selected from their hybrid offspring. Wx mp / GAE12 Q11 Analysis of the amylose content and transparency of the rice of the above-mentioned test materials showed that all the GAE12 Q11 The rice with the locus showed an extremely significant increase in amylose content (Table 1). At the same time, the rice transparency results also showed a similar trend, that is, GAE12 I The overall transparency of rice lines with the locus was significantly improved (Table 1). Based on the above data, it was shown that this molecular marker can accurately identify GAE12-1 and GAE12-2. GAE12 Tracking loci across the rice grains, whether assisting hybrid breeding or selecting superior new lines, can efficiently yield lines with improved target traits. Therefore, in addition to improving the quality of varieties like high-chalkiness indica rice, this breeding method can also selectively increase the amylose content of soft rice and, in turn, improve its transparency (improving the dark endosperm phenotype). More importantly, it can directly assist in the selection of new soft rice varieties, thus possessing significant breeding value.

[0020] Table 1: Comparison of quality traits of new “soft rice” varieties

[0021] Note: GAE12 J and GAE12 I The J in the table indicates that the rice is derived from japonica rice, and the I indicates that the rice is derived from indica rice.

[0022] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0023] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0024] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A Wx Gene interactions coordinately regulate rice appearance and taste quality loci GAE12 A molecular marker characterized by The physical location of this locus is located in the 218 kb interval of 2.086-2.304 Mb on rice chromosome 12. The molecular markers are GAE12-1 and GAE12-2. The primers for GAE12-1 are shown as SEQ ID No.1-SEQ ID No.2, and the primers for GAE12-2 are shown as SEQ ID No.3-SEQ ID No.

4.

2. The molecular marker according to claim 1 Wx Application of gene interaction in the coordinated regulation of rice appearance and taste quality.

3. The use according to claim 2, characterized in that In high amylose content and carrying GAE12 Q11 locus into rice varieties or lines GAE12 Nip It can significantly reduce rice chalkiness and improve rice appearance and taste quality; GAE12 Nip loci were introduced into japonica rice varieties GAE12 Q11 The amylose content of rice can be appropriately increased, significantly improving the transparency of rice grains without affecting the chalkiness of rice.

4. A breeding method for improving the appearance and taste quality of high chalky indica rice, characterized in that: Through conventional hybridization methods, the carrier with low chalkiness and good taste quality GAE12 Nip The rice germplasm of the locus is the hybrid donor, and the rice germplasm with high chalkiness, high amylose content and poor eating quality is GAE12 Q11 The rice germplasm of the locus is hybridized and molecular marker-assisted selection is performed on the hybrid offspring; the molecular marker-assisted selection is started from the F2 generation, PCR amplification and gel electrophoresis detection are performed using the molecular marker of claim 1, and the GAE12 Nip The individuals with the same genotype are further backcrossed, and the offspring with good comprehensive agronomic traits and carrying GAE12 Nip The genotype of a single plant is identified as a line to obtain a new line with stable traits.

5. A breeding method for improving the dark endosperm phenotype of low amylose content soft rice, characterized in that: By conventional hybridization method, GAE12 Q11 The genotype line is used as a hybrid donor, and is hybridized with the soft rice variety NG9108 with low amylose content and poor grain transparency, and molecular marker-assisted selection is performed on the hybrid offspring; the molecular marker-assisted selection is started from the F2 generation, and PCR amplification and gel electrophoresis detection are performed using the molecular markers of claim 1 to select GAE12 Q11 The individuals with the same genotype are further backcrossed, and the offspring with good comprehensive agronomic traits and carrying GAE12 Q11 The genotype of a single plant is identified as a line to obtain a new line with stable traits.

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