Primer set for detecting specific molecular markers of rice grain shape genes GS3 and GW5 and its application

Abstract

This invention discloses a primer set for detecting specific molecular markers of rice grain shape genes GS3 and GW5 and its applications. The primer set includes primer pairs GS3-1673, GW5-5364, and GW5-5366. The sequences of the GS3-1673 primer pair are shown in SEQ ID NO:1-3, the sequences of the GW5-5364 primer pair are shown in SEQ ID NO:4-6, and the sequences of the GW5-5366 primer pair are shown in SEQ ID NO:7-9. This primer set for detecting haplotypes of grain shape genes GS3 and GW5 can accurately identify haplotypes of GS3 and GW5 in 168 widely sourced indica rice varieties. It can be used for early identification in breeding materials, screening for dominant haplotype combinations of GS3 and GW5, and improving breeding selection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to a primer set for detecting specific molecular markers of rice grain shape genes GS3 and GW5 and their applications. Background Technology

[0002] Rice grain shape is an important appearance quality trait of rice, including grain length, width, thickness, and length-to-width ratio. It directly determines the weight of the grain, and thus plays an important role in rice yield. Rice grain morphology is a complex genetic trait, a quantitative trait controlled by multiple genes. To date, more than 400 grain morph-related QTLs have been detected (Huang et al. 2013). Researchers have mapped and cloned grain morph QTLs using different methods, including GS3, GW5, GW2, GS5, qGL3, GW8, GS2, GL7, and GLW7 (Fan et al., 2006; Weng et al., 2008; Liu et al., 2017; Song et al., 2007; Li et al., 2011; Qi et al., 2012; Wang et al., 2012; Hu et al., 2015; Duan et al., 2015; Wang et al., 2015; Si et al., 2015). GS3 is the first rice grain shape gene to be map-based and cloned. It significantly affects grain length and thousand-grain weight, and slightly affects grain width and thickness. A C>A mutation in exon 2 of GS3 leads to a change in the cysteine-encoding codon TGC to the stop codon TGA, causing premature termination of protein translation. This results in a missing PEBP-like domain and the absence of the other three functional domains, forming a non-functional GS3 protein. This indicates that the protein encoded by GS3 has a negative regulatory effect on grain weight (Fan et al., 2006). GW5 is a major gene affecting grain width in rice, finely mapped to a 21-kb genomic region containing a 1212-bp deletion. Approximately 5kb downstream of the 1212bp deletion is a gene encoding a calmodulin-binding protein, namely GW5. The 1212bp deletion present in wide-grain varieties regulates grain width by controlling the expression level of GW5. Knocking out the GW5 gene using CRISPR technology can increase grain width and weight in rice varieties that do not have the 1212bp deletion, thus increasing yield (Liu et al. 2017). Therefore, GW5 mainly affects grain width through changes in expression level rather than its coding sequence.

[0003] Haplotype breeding mainly studies the identification of haplotypes and their application in breeding work. Multiple studies have shown that association analysis is an effective method for identifying superior haplotypes (Abbai et al. 2019; Sinha et al. 2020). Therefore, using association analysis to identify dominant haplotypes for specific traits and combining this with molecular breeding methods to apply the identified superior haplotypes to breeding work can effectively improve the breeding process. Abbai et al. used candidate gene association analysis to screen 21 genes from 120 genes related to rice yield and quality, then performed haplotype analysis on the selected genes, and combined the dominant haplotypes of each gene to report the optimal haplotype combination affecting the target trait (Abbai et al. 2019). Mishra et al. performed haplotype analysis on eight genes in the HTK gene family and for the first time discovered that two haplotypes H5 and H1 of the HKT1;5 gene and HKT2;3 in Indian wild rice were significantly associated with high salt tolerance (Mishra et al. 2016). Zeng et al. analyzed the genetic diversity of genes related to yield, cooking quality, and appearance quality in three rice varieties: Teqing, 9311, and Nipponbare. They then conducted a reasonable molecular design and used hybridization and backcrossing techniques to aggregate multiple dominant alleles. Over a period of more than five years, they successfully bred a new variety with yield and quality superior to the parents (Zeng et al. 2017).

[0004] PARMS (Penta-primer amplification refractory mutation system) is a SNP PCR analysis technique that combines a pair of universal fluorescent primers, a pair of SNP allele-specific primers, and a reverse common primer. It allows for rapid and simple SNP allele genotyping. The system uses Allele 1 and Allele 2 specific amplification primers with two different universal adapter primer sequences. After DNA annealing, these primers bind to the corresponding SNP DNA template. The PARMS PCR enzyme and buffer system ensure strictly allele-specific amplification. Combined with Locus-specific amplification primers, after the first two rounds of PCR, a PCR amplification product with the universal adapter sequence is formed. At this point, a universal probe with reporter fluorescence and a fluorescence quencher (which has no fluorescence signal due to the FRET effect when no amplification occurs) can be used as a template for PCR amplification. Once amplification is successful, the fluorescence quencher on the fluorescent probe dissociates from the reporter group, the FRET effect disappears, and a fluorescence scan can detect the corresponding fluorescence signal, thus indicating the presence of the corresponding allele. Summary of the Invention

[0005] In view of this, the present invention has developed primer pairs for detecting haplotype-specific molecular markers of rice grain shape genes GS3 and GW5 based on primer amplification inhibited mutagenesis technology. This solves the problem that traditional techniques cannot determine whether rice contains dominant haplotypes of grain shape genes GS3 and GW5. It can be used to quickly identify haplotypes of GS3 and GW5 in breeding materials, thereby selecting dominant haplotypes and combinations of dominant haplotypes of GS3 and GW5 genes, and achieving rapid and accurate selection of rice grain shape-related traits.

[0006] The specific technical solution of the present invention is as follows:

[0007] This invention provides a primer set for detecting specific molecular markers of rice grain shape genes GS3 and GW5, including the GS3-1673 primer pair, the GW5-5364 primer pair, and the GW5-5366 primer pair; wherein the sequence of the GS3-1673 primer pair is shown in SEQ ID NO:1-3, the sequence of the GW5-5364 primer pair is shown in SEQ ID NO:4-6, and the sequence of the GW5-5366 primer pair is shown in SEQ ID NO:7-9.

[0008] The present invention also provides a detection kit comprising the above-described primer set.

[0009] The primer sets or the detection kits described above can be used to identify and screen dominant haplotype combinations with long grains, wide grains, high aspect ratios, and high thousand-grain weight.

[0010] Specifically, in the above application, the identification method is as follows: using the rice genomic DNA to be tested as a template, PCR amplification is performed using three sets of primer pairs, and the fluorescence signals of the three sets of PCR products are read by an enzyme-linked immunosorbent assay (ELISA) reader to distinguish different bases, thereby identifying whether the rice material to be tested contains the dominant haplotype combination.

[0011] Specifically, in the above applications, the dominant haplotype combination is one of CH1, CH2, CH3 and CH4, where CH1 is a combination of GS3-H1 haplotype and GW5-H1 haplotype, CH2 is a combination of GS3-H1 haplotype and GW5-H2 haplotype, CH3 is a combination of GS3-H2 haplotype and GW5-H1 haplotype, and CH4 is a combination of GS3-H2 haplotype and GW5-H2 haplotype. The preferred dominant haplotype combination is CH1.

[0012] The specific details of haplotypes GS3-H1, GS3-H2, GW5-H1, and GS5-H2 are shown in the table below:

[0013]

[0014]

[0015] The primer set or the detection kit mentioned above can also be used for rice-assisted breeding, specifically: to detect rice samples and select CH1 type rice samples for breeding.

[0016] The primer set or the detection kit described above can also be used to detect rice grain shape traits.

[0017] Since grain shape determines the weight of rice grains, the primer set or the detection kit described above can also be used to identify high-yield rice varieties.

[0018] Compared with the prior art, the advantages of the present invention are as follows: It provides a primer set for detecting specific molecular markers of rice grain shape genes GS3 and GW5, specifically molecular markers developed for the association sites of grain shape genes GS3 and GW5 with grain length, grain width, length-width ratio, and thousand-grain weight. This primer set can identify the dominant haplotypes of GS3 and GW5 in 168 widely sourced indica rice germplasms. By simply, quickly, and accurately determining whether breeding materials contain the dominant haplotypes of GS3 and GW5 in the early stages of breeding, breeding materials with grain length, grain width, large length-width ratio, and high thousand-grain weight can be selected, thereby improving breeding efficiency. Attached Figure Description

[0019] Figure 1 The image shows the genotyping results of three primer pairs for detecting the genotypes of rice germplasm resources using an ELISA reader. FAM and HEX correspond to the variants designed when the primers were created.

[0020] Figure 2 This is a diagram showing the effect of haplotype combinations of genes GS3 and GW5 on grain shape traits. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments thereof. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0022] Unless otherwise specified, the following examples are conducted under conventional experimental conditions or conditions recommended in the manufacturer's instructions; the reagents and materials mentioned are commercially available unless otherwise specified.

[0023] Example 1

[0024] This invention, through haplotype analysis of the GS3 and GS5 genes and combination of dominant haplotypes, obtained dominant haplotype combinations that influence rice grain length, grain width, length-width ratio, and thousand-grain weight traits. The specific process is as follows:

[0025] The experimental materials for this invention were derived from 168 indica rice accessions (see Table 1 for details) from the National Key Research and Development Program "Precise Identification and Innovative Utilization of Rice Germplasm Resources in Central China Rice-Growing Area (2016YFD0100101-05)". These included 77 varieties, 39 lines, 18 local varieties, 18 restorer lines, and 16 foreign germplasm accessions.

[0026] All experimental materials were planted in the experimental field of Jingzhou Academy of Agricultural Sciences, Hubei Province for two consecutive years (2018 and 2019), with sowing on May 16th of each year. The planting density was 15cm × 25cm, with 5 rows of each material and 10 plants per row. Water and fertilizer management were carried out in accordance with conventional field planting management.

[0027] Five representative mature plants were randomly selected from the field for each sample, threshed, and dried. The rice was tested using a digital rice testing machine (YTS-RICE-04D) from Wuhan Hongxing Yang Technology Co., Ltd., and phenotypic values ​​such as grain length, grain width, length-width ratio, and thousand-grain weight were obtained (results are shown in Table 1).

[0028] Targeted capture sequencing was performed on 42 target genes (genes regulating rice yield, quality, salt tolerance, and heat tolerance) from 168 rice materials. The target region was 268,132 bp in size. Variation detection was performed on this target region and 200 bp upstream and downstream. Sites with allele frequencies less than 0.05 were filtered out from the 168 indica rice subspecies, and a total of 2,164 SNPs and 578 Indel mutations were detected.

[0029] Further, through candidate gene association analysis of grain-related traits (BLUP value analysis of traits in 2018, 2019 and two years, with simultaneous detection and P≤0.01), 31 variant sites of GS3 were detected that were associated with grain length, among which the SNP variant site located at position 16733441 was a functional site, while 7 variant sites of GW5 were associated with grain length, length-width ratio and thousand-grain weight, as shown in Table 2.

[0030] Based on the relevant variant sites in Table 2, the aforementioned associated genes were divided into different haplotypes and haplotype combinations among different associated genes, and the dominant haplotypes were analyzed. The results are shown in Tables 1 and 3: It was found that the GS3 gene, based on the genotype of 31 variant sites, can be divided into GS3-H1 haplotype and GS3-H2 haplotype in 168 germplasm resources; the GW5 gene, based on the genotype of 7 variant sites, can also be divided into 2 haplotypes in 168 germplasm resources, namely GW5-H1 haplotype and GW5-H2 haplotype; the combination of the two genes GS3 and GW5 can be divided into 4 haplotype combinations, denoted as CH1, CH2, CH3 and CH4, where CH1 is GS3-H1 / GW5-H1, CH2 is GS3-H1 / GW5-H2, CH3 is GS3-H2 / GW5-H1, and CH4 is GS3-H2 / GW5-H2.

[0031] The haplotype combinations explained phenotypic variations in grain length, grain width, length-to-width ratio, and thousand-grain weight by 30.37%, 23.73%, 34.64%, and 12.26%, respectively. Haplotype CH1 had the largest average grain length (9.35 mm), the second largest average grain width (3.07 mm), the second largest average length-to-width ratio (3.09), and the largest average thousand-grain weight (30.15 g). (See Table 4 for details.) Figure 2 In summary, the rice germplasm resource with haplotype CH1 containing the GS3 and GW5 genes has traits such as long grains and large thousand-grain weight that are consistent with the breeding objectives. Haplotype CH1 can be selected as the preferred dominant haplotype in breeding applications.

[0032] Table 1. Names of test materials, haplotypes, and particle type phenotypic values ​​(BLUP)

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] Table 2 Association analysis of genes GS3 and GW5 with candidate genes for grain shape-related traits.

[0039]

[0040]

[0041]

[0042] Table 3. Haplotypes of genes GS3 and GW5 and allelic genotypes at each variant site.

[0043]

[0044]

[0045] Table 4. Haplotype combinations of genes GS3 and GW5, corresponding phenotypic mean, multiple comparisons, and explanation of phenotypic variation.

[0046]

[0047] Example 2

[0048] Based on the association sites of genes GS3 and GW5 with grain shape traits and their haplotypes in 168 rice germplasm resources, primer sets for detecting haplotypes of genes GS3 and GW5 were designed using PARMS technology, as shown in Table 5.

[0049] Table 5. Primer information for haplotype-specific molecular markers of genes GS3 and GW5.

[0050]

[0051]

[0052] Further amplification of 168 rice germplasm resources was performed using the primer sets in Table 5. The fluorescence signals of the PCR products were read using a microplate reader to distinguish the bases corresponding to FAM and HEX. Results (e.g.) Figure 1 (As shown) is completely consistent with the captured sequencing results.

[0053] In summary, the rice grain shape-related genes GS3 and GW5 haplotype-specific molecular marker primers developed in this invention can distinguish different haplotypes of GS3 and GW5 in rice materials and can be used to screen for dominant haplotype combinations of GS3 and GW5.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting rice grain shape genes GS3 and GW5 The application of primer sets with specific molecular markers or detection kits containing said primer sets in identifying and screening dominant haplotype combinations with long grains, wide grains, high aspect ratios, and high thousand-grain weight, wherein said primer sets include the GS3-1673 primer pair with sequences as shown in SEQ ID NO:1-3, the GW5-5364 primer pair with sequences as shown in SEQ ID NO:4-6, and the GW5-5366 primer pair with sequences as shown in SEQ ID NO:7-9, wherein the rice is indica rice. GS3 The gene ID is Os03g0407400, which is... GW5 The gene ID is LOC_Os05g09520, the dominant haplotype combination is CH1, and CH1 is a combination of GS3-H1 and GW5-H1. The specific details of GS3-H1 and GW5-H1 are shown in the table below: 。 2. The application according to claim 1, characterized in that, Using the rice genomic DNA to be tested as a template, PCR amplification was performed using the three primer pairs described in claim 1. The fluorescence signals of the three PCR products were read using an enzyme-linked immunosorbent assay (ELISA) reader to distinguish different allelic variations, thereby identifying whether the dominant haplotype combination is present in the rice material to be tested.

3. A method for detecting rice grain shape genes GS3 and GW5 The application of primer sets containing specific molecular markers or detection kits containing said primer sets in assisted breeding of indica rice, wherein, The primer set includes the GS3-1673 primer pair with sequences as shown in SEQ ID NO:1-3, the GW5-5364 primer pair with sequences as shown in SEQ ID NO:4-6, and the GW5-5366 primer pair with sequences as shown in SEQ ID NO:7-9. The specific application is as follows: testing indica rice samples and selecting CH1 type indica rice samples for breeding. CH1 is a combination of GS3-H1 and GW5-H1. The specific details of GS3-H1 and GW5-H1 are shown in the table below. 。 4. A method for detecting rice grain shape genes GS3 and GW5 The application of primer sets with specific molecular markers or detection kits containing said primer sets in the detection of grain shape traits in indica rice, wherein, The primer set includes the GS3-1673 primer pair with sequences as shown in SEQ ID NO:1-3, the GW5-5364 primer pair with sequences as shown in SEQ ID NO:4-6, and the GW5-5366 primer pair with sequences as shown in SEQ ID NO:7-9. When the indica rice is of the CH1 type, it exhibits long grains and a large thousand-grain weight. CH1 is a combination of GS3-H1 and GW5-H1. The specific details of GS3-H1 and GW5-H1 are shown in the table below. 。 5. A method for detecting rice grain shape genes GS3 and GW5 The application of primer sets containing specific molecular markers or detection kits containing said primer sets in the identification of high-yielding indica rice varieties, wherein, The primer set includes the GS3-1673 primer pair with sequences as shown in SEQ ID NO:1-3, the GW5-5364 primer pair with sequences as shown in SEQ ID NO:4-6, and the GW5-5366 primer pair with sequences as shown in SEQ ID NO:7-9. When the indica rice is of type CH1, it is a high-yielding indica rice variety. CH1 is a combination of GS3-H1 and GW5-H1. The specific details of GS3-H1 and GW5-H1 are shown in the table below: 。

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