Molecular markers of rice amylose content gene ACF1 alleles and primers and methods for its detection

Abstract

The present invention discloses a molecular marker capable of screening the ACF1 gene regulating the low amylose content of soft rice, a primer for amplifying the ACF1 gene molecular marker, and a detection method. The detection method comprises the following steps: (1) extracting the total DNA of rice as a template, adding the above primers for PCR amplification; (2) taking the PCR product for restriction endonuclease MaeII digestion; (3) detecting the enzyme-cut product by gel electrophoresis. The present invention can directly detect the ACF1 gene regulating the low amylose content of rice, laying a foundation for quickly and effectively cultivating new varieties of high-quality soft rice.

Description

Technical Field

[0001] The present invention relates to the field of genetics and plant breeding, and specifically provides a molecular marker for screening low amylose content gene ACF1 allele rice, a primer for amplifying or detecting the molecular marker, and a detection method, which are used to assist conventional breeding to quickly, simply and accurately breed low amylose content rice. Background Art

[0002] Rice is one of the most important food crops. Improving yield and quality have always been the two key goals of rice genetic breeding. In the past few decades, due to the promotion of hybrid rice and the improvement of rice cultivation and management technology, rice yield has been greatly improved, but rice quality improvement has lagged far behind (Zeng et al., 2017, Plant Molecular Biology, 65: 501-509; Zhao et al., 2017, Indian Journal of Genetics and Plant Breeding, 77, 221). At the same time, with the improvement of people's living standards, people's requirements for rice quality are getting higher and higher. Therefore, improving rice quality has become the primary consideration for breeders and consumers. Rice quality mainly includes milling quality, appearance quality, nutritional quality and taste quality, among which taste quality is the core of rice quality (Tian et al., 2009, Proceedings of the National Academy of Sciences of the United States of America, 106 (51): 21760-21765).

[0003] Starch is the main component of rice, accounting for about 90% of the dry weight of polished rice, and is composed of amylose and amylopectin. The ratio of amylose to total starch, also known as amylose content (AC), is the most important factor determining the taste quality of rice (Tian et al., 2009, Proceedings of the National Academy of Sciences of the United States of America, 106 (51): 21760-21765). Soft rice with a lower amylose content has good taste quality, sweet and refreshing, and is fluffy and soft whether hot or cold, and does not regenerate after cooling. The price of soft rice is generally higher than that of non-soft rice fragrant rice varieties. With the improvement of people's living standards, the market demand for soft rice is increasing. These have greatly promoted people's genetic research on the fragrance characteristics of soft rice, and also accelerated the breeding process of new soft rice fragrant rice varieties.

[0004] Rice GBSS I is a key enzyme in the synthesis of rice amylose and is encoded by the Wx gene on chromosome 6. In nature, there are at least 10 different alleles of Wx, including Wx a 、Wx b 、Wx mq 、Wx hp 、Wx mp 、Wx op 、Wx in 、Wx lv 、Wx mw and wx, the amylose content of rice with different genotypes is different (Zhang et al; 2019, Molecular Plants 12, 1157-1166; Zhang et al; 2020, Journal of Integrative Plant Biology. 63: 889-901). mq Rice genotypes with low amylose content have been widely used in the cultivation of high-quality soft rice. However, the only gene related to amylose content reported for high-quality rice cultivation is the Wx gene. Our research group previously cloned a new gene ACF1 that controls amylose content from the high-quality soft rice variety "Yinxiang 38", which encodes a new C2H2 zinc finger protein (CN113637688A, published on the same day).

[0005] Soft rice usually has a low yield and needs to be hybridized with other rice to obtain a higher yield variety. Generally, non-soft rice with higher yield is selected for hybridization with soft rice, and then the hybrid F1 plants are backcrossed with high-yield non-soft rice for several generations. In the past, in the traditional breeding process of soft rice with lower amylose content, breeders mainly distinguished soft rice from non-soft rice by measuring the amylose content. However, this method is time-consuming and the detection cost is very expensive. With the development of various molecular markers, molecular markers of many important agronomic traits and quality traits have been developed for rice breeding.

[0006] Therefore, it is necessary to provide a molecular marker, corresponding primers and detection method to assist in the cultivation and screening of soft rice and non-soft rice in japonica rice. Summary of the invention

[0007] The present invention provides a molecular marker dCAPS (MaeII) for screening the allele type of rice amylose content gene ACF1 and a primer and method for detecting the molecular marker, which can quickly and accurately identify different allele types of the ACF1 gene and can be used to assist in the cultivation and screening of soft rice.

[0008] After gene sequence analysis, it was found that compared with the "Nihonbare" rice, the "Yinxiang 38" soft rice ACF1 has a GA mutation at 655bp in the coding region. This mutation causes the "Yinxiang 38" rice to have the characteristic of low amylose content, that is, the GA variation at 655bp in the ACF1 coding region corresponds to the low amylose content. 655A The allele type japonica rice has a lower content of amylose and is a soft rice, while ACF1 655G The allele type rice has a relatively high content of amylose and is a non-soft rice.

[0009] The technical solution of the present invention:

[0010] A molecular marker of rice low amylose content gene ACF1 (hereinafter referred to as dCAPS (MaeII)), in which the 655bp-658bp portion of the ACF1 gene coding region in soft rice is ACGT; in non-soft rice, the 655bp-658bp portion of the ACF1 gene coding region is GCGT.

[0011] The molecular marker dCAPS (MaeII) is located at 655bp-658bp in the coding region of the amylose content gene ACF1. In the soft rice with low amylose content, the 4 nucleotide sequences "ACGT" at this site can be recognized and cut by the restriction endonuclease MaeII, while the C2H2 zinc finger protein gene ACF1 in the medium amylose content common japonica rice 655G The nucleotide sequence at 655bp-655bp of the coding region is "GCGT", which cannot be recognized and cut by the restriction endonuclease MaeII, thus quickly and accurately distinguishing ACF1 in soft rice and non-soft rice japonica rice varieties. 655A Genotype and ACF1 655G genotype.

[0012] The primers used to amplify and detect the above-mentioned new molecular marker dCAPS (MaeII) or to amplify and detect the rice amylose content gene ACF1 allele have nucleotide sequences including SEQ ID No. 1 and SEQ ID No. 2. More preferably, the nucleotide sequences are as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0013] SEQ ID No.1: 5'-GAATCTCGACGGGTTCAAGT-3'

[0014] SEQ ID No.2: 5'-TCTTATTGTCTGCTCTGACATGG-3'

[0015] The method for detecting the allele of rice amylose content gene ACF1 comprises the following steps:

[0016] (1) Extract rice genomic DNA as a template (CTAB method can be used), add the above two primers for PCR amplification;

[0017] (2) The PCR product was digested with MaeII.

[0018] (3) The digested products were detected by gel electrophoresis.

[0019] Preferably, in step (1), the PCR amplification program is: pre-denaturation at 93-95°C for 4-7 min; denaturation at 93-95°C for 42-50 s, annealing at 51-54°C for 42-50 s, extension at 70-73°C for 25-35 s, for a total of 30-35 cycles; and finally extension at 70-73°C for 9-12 min. More preferably, the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 52°C for 45 s, extension at 72°C for 30 s, for a total of 32 cycles; and finally extension at 72°C for 10 min.

[0020] Preferably, in step (3), electrophoresis detection is performed in 2.5-3.5% agarose gel; more preferably, 3% agarose gel is used for electrophoresis detection.

[0021] The test results showed that ACF1 655A The soft rice alleles all contained two bands with molecular weights of approximately 231 bp and 82 bp, respectively, while the ACF1 655G The non-soft rice alleles all contain a band with a molecular weight of about 333 bp. The above method can be used to screen and identify the ACF1 655A Soft rice allele and ACF1 655G The hybrid progeny of non-soft rice alleles and the progeny of ACF1 655A Soft rice allele and ACF1 655G The self-pollinated progeny of rice with non-soft rice alleles or ACF1 655A Soft rice allele and ACF1 655G The progeny of the backcross of non-soft rice alleles. The rice tested contained ACF1 655A Soft rice allele type rice, containing ACF1 655G Non-soft rice allele rice, containing ACF1 655A Soft rice allele and ACF1 655G Hybrid progenies of non-soft rice allele rice and their self-pollinated or backcrossed progenies.

[0022] If the template total DNA is composed of homozygous ACF1 655AThe soft rice allele rice was extracted, and the digested products contained two bands with molecular weights of approximately 231 bp and 82 bp, respectively. 655G The products extracted from non-soft rice allele rice all contained a band with a molecular weight of about 313 bp after enzyme digestion.

[0023] If the template total DNA is ACF1 655A Soft rice allele and ACF1 655G The heterozygous rice of the hybrid offspring of non-soft rice allele type rice was extracted, and the products after enzyme digestion contained three bands with molecular weights of approximately 313bp, 231bp and 82bp.

[0024] If the template total DNA is ACF1 655A Soft rice allele and ACF1 655G The rice offspring of the hybrid F1 plant of non-soft rice alleles and then self-pollination were extracted. The products after enzyme digestion of different plants should have three types of banding patterns: for homozygous ACF1 655A Soft rice contains two bands with molecular weights of approximately 231bp and 82bp; heterozygous contains three bands with molecular weights of approximately 4313bp, 231bp and 82bp; homozygous ACF1 655G Non-soft rice type rice contains a band with a molecular weight of about 313bp.

[0025] If the template total DNA is ACF1 655A Soft rice allele and ACF1 655G The offspring rice extracted from the hybrid F1 plant of non-soft rice allele rice and backcrossed with non-soft rice allele rice should have two types of bands after enzyme digestion of different plants: homozygous ACF1 655G The non-soft rice allele type rice contains a band with a molecular weight of about 313bp; the heterozygous contains three bands with molecular weights of about 4313bp, 231bp and 82bp.

[0026] The method can be used to identify soft rice, non-soft rice and hybrid varieties of soft rice and non-soft rice.

[0027] The soft rice includes Yinxiang 38 and its hybrid variety with non-soft rice;

[0028] Preferably, the hybrid variety of soft rice and non-soft rice, the non-soft rice parent is homozygous ACF1 655GThe non-soft rice allele type rice is a non-soft rice that shows only one 313bp band after sampling and testing by the above method. The non-soft rice includes but is not limited to: "Nihonbare", "Jiahua No. 1", "Xiushui 134", "Xiushui 123", "Xiushui 114", "Jiahe 218", "Zhongzhikang No. 2", "Guangmingjing No. 3", "Wuxiangjing 14", "Wuyun 2645", "9522", "Shanghai Normal University No. 5", "Zhonghua 11", "Baonong 34", "Fan 14" and "Fan 26".

[0029] The present invention provides a selective molecular marker for the G / A variation at 655bp of the amylose content gene ACF1, which is useful for breeding crops containing ACF1. 655A The soft rice with allele is very practical.

[0030] Using the scheme of the present invention, by identifying ACF1 655A The molecular markers of soft rice alleles were used to detect the ACF1 allele, and the backcross progeny were tested to select the progeny containing ACF1. 655A Plants heterozygous for the soft rice allele were then backcrossed with non-soft rice. Therefore, for molecular markers to identify the soft rice gene, it should be possible to distinguish the ACF1 of the soft rice parent and the soft rice parent. 655A Soft rice allele and ACF1 655G Non-soft rice allele.

[0031] The identification of whether the 655bp site in the coding region of the zinc finger protein gene ACF1 of the non-soft rice is adenine deoxynucleotide (A) can determine which non-soft rice varieties are suitable for being selected as non-soft rice parents when using the soft rice gene ACF1 molecular marker dCAPS (MaeII) of the present invention to perform auxiliary screening for the cultivation of new soft rice varieties.

[0032] The beneficial effect of the present invention is that the provided dCAPS (MaeII) molecular marker and its detection primers and detection method are simple to operate and the results are easy to judge. 655A Alleles and ACF1 655G Allele detection can be easily distinguished by using only about 3% agarose gel, which can not only quickly and conveniently identify soft rice and non-soft rice, but also quickly identify ACF1 in hybrid, backcross and self-cross progenies. 655A Genotype and ACF1 655G Genotype has important application value in the breeding and screening of soft rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 ACF1 in Example 1 655ASoft rice allele type rice, 1 ACF1 655G Identification diagram of ACF1 genotype of non-soft rice allele type rice, hybrid F1 plants and F2 plants produced by self-pollination of F1. M1 is a standard molecular weight DNA of 100bp type; Lane 1 is the enzyme digestion product after PCR amplification of soft rice rice "Yinxiang 38" plant, containing two bands with molecular weights of about 231bp and 82bp; Lane 2 is the enzyme digestion product after PCR amplification of non-soft rice rice "Jiahua No. 1" plant, containing a band with molecular weight of about 313bp; Lane 3 is the enzyme digestion product after PCR amplification of F1 plants hybridized by "Yinxiang 38" and "Jiahua No. 1", containing three bands with molecular weights of about 313bp, 231bp and 82bp respectively; Lanes 4-6 are homozygous ACF1 655A Soft rice allele type rice, containing two bands with molecular weights of about 231bp and 82bp; Lanes 7-9 are F2 populations containing homozygous ACF1 655G Non-soft rice allele type rice contains a band with a molecular weight of about 313bp; Lanes 10-12 are F2 populations containing ACF1 655A ACF1 655G Heterozygous rice contains three bands with molecular weights of approximately 313bp, 231bp and 82bp respectively.

[0034] Figure 2 For Example 2, ACF1 655A Soft rice allele type rice "Yinxiang 38" and 14 ACF1 655G Identification diagram of ACF1 gene in non-soft rice allele type rice. M1 is 250bp type standard molecular weight DNA; Lane 1 is "Yinxiang 38", containing two bands with molecular weights of about 231bp and 82bp; Lanes 2 to 15 are 14 ACF1 655G The enzyme digestion products after PCR amplification of rice varieties with non-soft rice allele type: "Xiushui 134", "Xiushui 123", "Xiushui 114", "Jiahe 218", "Zhongzhikang 2", "Guangmingjing 3", "Wuxiangjing 14", "Wuyun 2645", "9522", "Shanghai Normal University 5", "Zhonghua 11", "Baonong 34", "Fan 14" and "Fan 26" all contain a band with a molecular weight of about 313bp. DETAILED DESCRIPTION

[0035] Example 1 Establishment and detection method of molecular markers capable of identifying the allele type of the amylose content gene ACF1

[0036] The rice "Yinxiang 38" with low amylose content is a soft rice variety, and the rice "Jiahua No. 1" with medium amylose content is an ordinary japonica rice variety.

[0037] The genomic DNA of two rice varieties was extracted, and the sequences of the zinc finger protein gene ACF1 related to the amylose content of rice in these two rice varieties were cloned and sequenced. At the same time, the ACF1 gene sequence (LOC_Os06g48530) of the non-soft rice "Nipponbare" published in Genbank was checked. Compared with the ACF1 sequences of "Nipponbare" and "Jiahua No. 1", the ACF1 coding region of "Yinxiang 38" had a GA mutation at 655bp. The coding region of the ACF1 gene of the soft rice "Yinxiang 38" is ACGT at 655bp-658bp, which can be used as a sequence recognized and cut by the restriction endonuclease MaeII. However, the coding region of the ACF1 gene of the non-soft rice "Nipponbare" and "Jiahua No. 1" is GCGT at 655bp-658bp, which cannot be used as a site recognized by the restriction endonuclease MaeII. Thus, a molecular marker that can identify different alleles of the ACF1 gene was established: dCAPS (MaeII).

[0038] A pair of upstream and downstream primers were designed at both ends of the 655bp-658bp sequence of the rice zinc finger protein ACF1 gene coding region, and the nucleotide sequences were shown in SEQ ID No.1 and SEQ ID No.2 respectively.

[0039] SEQ ID No.1: 5'-GAATCTCGACGGGTTCAAGT-3';

[0040] SEQ ID No. 2: 5'-TCTTATTGTCTGCTCTGACATGG-3'.

[0041] The rice "Yinxiang 38" with low amylose content was used as a soft rice variety, the rice "Jiahua No. 1" with medium amylose content was used as a common japonica rice variety, and the F1 plant hybridized between the soft rice "Yinxiang 38" and the non-soft rice "Jiahua No. 1" was used as the material, the total DNA of the rice genome was extracted as a template, and the above primers were sampled for amplification detection. The method is as follows:

[0042] (1) Extract rice genomic DNA as a template (CTAB method can be used), add the above two primers for PCR amplification;

[0043] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 52°C for 45 s, and extension at 72°C for 30 s, for a total of 32 cycles; and finally extension at 72°C for 10 min.

[0044] (2) PCR products were digested with MaeII;

[0045] The reaction system is: 2ul of 10 fast enzyme reaction buffer (purchased from Fermentas), 14ul of PCR product (≤0.2g), 1ul of MaeII fast enzyme (purchased from Fermentas) (10 enzyme activity units), and 13ul of sterile water. The reaction conditions are: 65℃ water bath for 4 hours;

[0046] (3) The digested product was subjected to electrophoresis in a 3% agarose gel.

[0047] Then have ACF1 655A Genotype of soft rice 'Yinxiang 38' and ACF1 655G The F1 plants of the non-soft rice genotype "Jiahua No. 1" were self-pollinated to obtain the self-pollinated F2 plants, and the total DNA was extracted. PCR amplification, enzyme digestion and electrophoresis detection were performed using the same method.

[0048] The results are as follows Figure 1 As shown, M1 is a 100bp type standard molecular weight DNA; Lane 1 is the enzyme digestion product of soft rice "Yinxiang 38" plant after PCR amplification, containing two bands with molecular weights of about 231bp and 82bp; Lane 2 is the enzyme digestion product of non-soft rice "Jiahua 1" plant after PCR amplification, containing a band with a molecular weight of about 313bp; Lane 3 is the enzyme digestion product of the hybrid F1 plant of "Yinxiang 38" and "Jiahua 1" after PCR amplification, containing three bands with molecular weights of about 313bp, 231bp and 82bp respectively; Lanes 4-6 are the homozygous ACF1 in the F2 population. 655A Soft rice allele type rice, containing two bands with molecular weights of about 231bp and 82bp; Lanes 7-9 are F2 populations containing homozygous ACF1 655G Non-soft rice allele type rice contains a band with a molecular weight of about 313bp; Lanes 10-12 are F2 populations containing ACF1 655A ACF1 655G Heterozygous rice contains three bands with molecular weights of approximately 313bp, 231bp and 82bp respectively.

[0049] The results showed that ACF1 655A The soft rice genotype "Yinxiang 38" contained bands with molecular weights of approximately 231 bp and 82 bp, respectively, with ACF1 655G The non-soft rice genotype "Jiahua 1" contained a band with a molecular weight of about 313 bp, and the hybrid F1 plants all contained bands with molecular weights of about 313 bp, 231 bp and 82 bp, respectively.

[0050] With ACF1 655A Genotype of soft rice 'Yinxiang 38' and ACF1 655GThe F1 plants of the non-soft rice "Jiahua No. 1" genotype were self-pollinated to obtain the self-pollinated F2 plants. The test results showed that the self-pollinated offspring of the two combinations had three plants with different genotypes, among which only two bands with DNA molecular weight of about 231bp and 82bp corresponded to the self-pollinated offspring ACF1. 655A The PCR product of the homozygous plant has three bands of about 313 bp, 231 bp and 82 bp, which correspond to the self-pollinated offspring. The PCR product of the heterozygous plant has only one band of 313 bp, which corresponds to the self-pollinated offspring ACF1. 655G Genotype homozygous non-soft rice plant PCR products (such as Figure 1 ).

[0051] We also used 0.2% KI solution to dye the endosperm of F2 plants. The endosperm of common japonica rice seeds with an amylose content of 15-20% was stained dark blue, while the endosperm of soft rice seeds with an amylose content of about 9% was stained light reddish brown. We took 24 F3 seeds from each F2 plant, cut the endosperm with a sharp blade, added 0.2% KI solution, stained at room temperature for 2-3 minutes, and then observed the endosperm staining one by one. Since the low amylose content soft rice trait is a recessive trait, the endosperm staining results of 24 seeds of homozygous non-soft rice plants with the 313bp band were all dark blue, and the endosperm staining results of 24 seeds of heterozygous plants with the 313bp, 231bp and 82bp bands were 3 / 4 dark blue and 1 / 4 light reddish brown, and the endosperm staining results of 24 seeds of plants with only the 231bp and 82bp bands were all light reddish brown. In other words, the ACF1 655A All homozygous offspring were soft rice.

[0052] The detection results of the two methods using dCAPS (MaeII) molecular markers and 0.2% KI solution are completely consistent with the expected results of self-pollination of a pair of genes in the genetic separation law. This shows that the dCAPS (MaeII) molecular marker and detection method established by the present invention can effectively distinguish soft rice ACF1 655A and non-soft rice ACF1 655G Two genotypes.

[0053] Example 2 has ACF1 655G Identification of dCAPS(MaeII) Molecular Markers for Genotypes of Non-soft Rice

[0054] Since most soft rice varieties have low yields, in the use of molecular markers to assist in the selection of new soft rice varieties, non-soft rice varieties with higher yields are generally selected for hybridization with soft rice varieties. The hybrid F1 plants are then backcrossed with high-yield non-soft rice varieties for several generations. Before each backcross, the ACF1 marker is used to identify the655A The molecular markers of soft rice alleles were used to detect the backcross progeny, and the ACF1 655A Plants heterozygous for the soft rice allele were then backcrossed with non-soft rice. Therefore, for molecular markers to identify the soft rice gene, it should be possible to distinguish the ACF1 of the soft rice parent and the soft rice parent. 655A Soft rice allele and ACF1 655G Non-soft rice allele.

[0055] The identification of whether the 655bp site in the coding region of the zinc finger protein gene ACF1 of the non-soft rice is adenine deoxynucleotide (A) can determine which non-soft rice varieties are suitable for being selected as non-soft rice parents when using the soft rice gene ACF1 molecular marker dCAPS (MaeII) of the present invention to perform auxiliary screening for the cultivation of new soft rice varieties.

[0056] In Example 1, we selected "Jiahua No. 1" as a non-soft rice, and used the dCAPS (MaeII) molecular marker and detection method of the present invention to identify their genomic DNA. As a result, they all showed a 313bp band, indicating that the PCR products of the zinc finger protein gene ACF1 of the non-soft rice "Jiahua No. 1" could not be cut by MaeII, indicating that they were not adenine deoxynucleotides (A) at the 655bp site of the zinc finger protein gene ACF1 coding region, thus also indicating that the dCAPS (MaeII) molecular marker designed by the present invention can be used to identify this "Jiahua No. 1" non-soft rice and ACF1 655A The soft rice of the same genotype is hybridized to breed a new soft rice variety, that is, the non-soft rice variety "Jiahua No. 1" is suitable for being used as a non-soft rice parent to breed a new soft rice variety. In order to explore whether there are more non-soft rice when breeding new soft rice varieties, the dCAPS (MaeII) molecular marker of the present invention is also suitable for detection.

[0057] We also selected 14 important non-soft rice varieties that are currently grown in Shanghai and other surrounding areas: "Xiushui 134", "Xiushui 123", "Xiushui 114", "Jiahe 218", "Zhongzhikang 2", "Guangmingjing 3", "Wuxiangjing 14", "Wuyun 2645", "9522", "Shanghai Normal University 5", "Zhonghua 11", "Baonong 34", "Fan 14" and "Fan 26", and used dCAPS (MaeII) molecular markers to identify these 14 non-soft rice varieties.

[0058] The results are as follows Figure 2As shown, M1 is a 250bp standard molecular weight DNA; Lane 1 is "Yinxiang 38", containing two bands with molecular weights of approximately 231bp and 82bp; Lanes 2 to 15 are 14 types of ACF1 655G The enzyme digestion products after PCR amplification of rice varieties with non-soft rice allele type: "Xiushui 134", "Xiushui 123", "Xiushui 114", "Jiahe 218", "Zhongzhikang 2", "Guangmingjing 3", "Wuxiangjing 14", "Wuyun 2645", "9522", "Shanghai Normal University 5", "Zhonghua 11", "Baonong 34", "Fan 14" and "Fan 26" all contain a band with a molecular weight of about 313bp.

[0059] The above results show that these non-soft rices, like the non-soft rice "Jiahua No. 1" in Example 1, also contain a 313 bp band. That is, the 655th nucleotide of the ACF1 coding region of the non-soft rice is G, which is ACF1 655G Genotype, using the above-mentioned dCAPS (MaeII) molecular marker to identify these non-soft rice is also suitable, and using the above-mentioned rice varieties as non-soft rice parents to breed new soft rice varieties is also suitable. That is to say, the dCAPS (MaeII) molecular marker, primers and method for detecting ACF1 alleles of the present invention are also suitable for using these non-soft rice as non-soft rice parents to breed new soft rice varieties.

[0060] Therefore, we suggest that before using the dCAPS (MaeII) molecular marker of the present invention to assist in the breeding of new soft rice, breeders should preferably use the simple operation method of the present invention to identify the non-soft rice used as the parent in advance. If the identification result shows only one 313bp band, it indicates that the rice variety is suitable for being selected as the non-soft rice parent. Sequence Listing <110> Shanghai Normal University <120> Molecular markers of rice amylose content gene ACF1 alleles and primers and methods for its detection <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 gaatctcgac gggttcaagt 20 <210> 2 <211> twenty three <212> DNA <213> Artificial Sequence <400> 2 tcttattgtc tgctctgaca tgg 23

Claims

1. A rice amylose content gene LOC_Os06g48530 ACF1 A molecular marker for an allele, characterized in that In soft rice with low amylose content, the 655bp-658bp portion of the ACF1 gene coding region is ACGT; in non-soft rice with medium amylose content, the 655bp-658bp portion of the ACF1 gene coding region is GCGT.

2. A method for detecting the low amylose content gene LOC_Os06g48530 in Yinxiang 38 rice ACF1 Allelic method, characterized in that The following steps are involved: (1) Using rice total DNA as a template, PCR amplification was performed using the primers shown in SEQ ID No. 1 and SEQ ID No. 2; (2) The product of step (1) is used Mae Ⅰ enzyme digestion; (3) Detect the digestion product of step (2) by gel electrophoresis.

3. The method according to claim 2, characterized in that: Rice total DNA was derived from ACF1 655A When the soft rice allele was used, the enzyme digestion product contained two bands with molecular weights of approximately 231 bp and 82 bp; Rice total DNA was derived from ACF1 655G In the case of non-soft rice allele type rice, the enzyme digestion product is a band with a molecular weight of about 313 bp; Rice total DNA was derived from ACF1 655G Non-soft rice alleles and ACF1 655A When the hybrid offspring of soft rice allele type rice is heterozygous rice, the enzyme digestion product contains three bands with molecular weights of 313bp, 231bp and 82bp; Rice total DNA was derived from ACF1 655A Soft rice allele and ACF1 655G When the F1 plants of non-soft rice alleles are self-pollinated, the offspring rice is homozygous ACF1 655A The soft rice allele type rice contains two bands with molecular weights of approximately 231bp and 82bp; the heterozygous contains three bands with molecular weights of approximately 313bp, 231bp and 82bp; the homozygous ACF1 655G The non-soft rice allele type rice contained a band with a molecular weight of about 313 bp; Rice total DNA was derived from homozygous ACF1 655A Soft rice allele and homozygous ACF1 655G The hybrid F1 plants of non-soft rice alleles were re-crossed with homozygous ACF1 655G When the offspring of rice with non-soft rice alleles are backcrossed, the homozygous ACF1 655G The non-soft rice allele type rice contains a band with a molecular weight of about 312bp; the heterozygous rice contains three bands with molecular weights of about 313bp, 231bp and 82bp.

4. The method according to claim 2, characterized in that: In step (1), the PCR amplification program is: pre-denaturation at 93-95°C for 4-7 min; denaturation at 93-95°C for 42-50 s, annealing at 51-54°C for 42-50 s, and extension at 70-73°C for 25-35 s, for a total of 30-35 cycles; and finally extension at 70-73°C for 9-12 min.

5. The method according to claim 2, characterized in that: In step (1), the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 52°C for 45 s, and extension at 72°C for 30 s, for a total of 32 cycles; and finally extension at 72°C for 10 min.

6. The method according to claim 2, characterized in that In step (2), the reaction conditions for the enzyme digestion are 65° C. for 3 to 6 hours.

7. The method according to claim 2, characterized in that: In step (3), the gel is 2.5-3.5% agarose gel.

8. Use of the molecular marker according to claim 1 or the method according to any one of claims 2 to 7 in cultivating or screening soft rice.

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