Primer set for detecting specific molecular markers of genes related to 1000-grain weight of rice and its application

Abstract

The present invention discloses a primer set for detecting specific molecular markers of genes related to the thousand-grain weight of rice and its application. The primer set includes 9 primer pairs, namely: SNAC1-3416 primer pair, qTGW3-3538 primer pair, qTGW3-35390 primer pair, qTGW3-35391 primer pair, qTGW3-35393 primer pair, GW5-5364 primer pair, GW5-5366 primer pair, AET1-2659 primer pair and DHD1-2889 primer pair. The primer set provided by the present invention can accurately identify the haplotypes of SNAC1, qTGW3, GW5, AET1 and DHD1 genes in 137 indica rice varieties with wide sources, can be used for early identification in breeding materials, screen the superior haplotype combinations of the above 5 genes, and improve the breeding selection efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding, and particularly relates to a primer group for detecting specific molecular markers of genes related to 1000-grain weight in rice and its application in aspects such as rice breeding. The related genes include SNAC1, qTGW3, GW5, AET1, and DHD1. Background Art

[0002] Rice is one of the most important food crops in the world, and improving rice yield plays an important role in ensuring food security. The three most important factors constituting rice yield include the number of effective panicles, the number of filled grains per panicle, and the 1000-grain weight. Therefore, cloning the regulatory genes of 1000-grain weight and mining the superior haplotypes of genes related to 1000-grain weight that can be applied in breeding practice is of great significance. Compared with other yield traits, the 1000-grain weight is less affected by the environment and the phenotype is relatively stable. As of 2018, 16 QTLs controlling grain weight and grain shape have been cloned, including OsLG3, OsLG3b / qLGY3, GS3, GL3.1 / qGL3, and qTGW3, etc. (Yu et al. 2017, Yu et al. 2018, Fan et al. 2006, Qi et al 2012, Zhang et al 2012, Hu et al 2018, Ying et al 2018, Xia et al 2018).

[0003] Haplotype breeding mainly focuses on the identification of haplotypes and their application in breeding. Multiple studies have shown that the association analysis method is an effective way to mine excellent haplotypes (Abbai et al. 2019; Sinha et al. 2020). Therefore, using the association analysis method to mine the dominant haplotypes of specific traits and combining molecular breeding methods to apply the identified excellent haplotypes to breeding 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 conducted haplotype analysis on the selected genes, and combined the dominant haplotypes of each gene to report the best haplotype combination affecting the target traits (Abbai et al. 2019). Mishra et al. conducted haplotype analysis on 8 genes in the HTK gene family and first found that two haplotypes, H5 and H1, of the HKT1;5 gene and HKT2;3 in Indian wild rice were significantly correlated with high salt tolerance (Mishra et al. 2016). Zeng et al. analyzed the genetic diversity of genes related to yield, cooking and eating quality, and appearance quality in three rice varieties, Teqing, 9311, and Nipponbare, and carried out reasonable molecular design. Through techniques such as hybridization and backcrossing, multiple dominant alleles were aggregated, and a new variety with yield and quality superior to those of the parents was successfully cultivated in more than 5 years (Zeng et al. 2017).

[0004] PARMS (Penta-primer amplification refractory mutation system) is an 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 can quickly and simply detect the genotypes of SNP alleles. In this system, the Allele 1 and Allele 2 specific amplification primers with two different universal adapter primer sequences bind to the corresponding SNP DNA template after DNA renaturation. The PARMS PCR enzyme and Buffer system can ensure strict allele-specific amplification. Together with the Locus-specific amplification primer, after the first two rounds of PCR, a PCR amplification product with a universal adapter sequence is formed. At this time, a universal probe with a reporter fluorescence and a fluorescence quenching group (without fluorescence signal due to the FRET effect when not amplified) can use the PCR amplification product with a universal adapter sequence as a template for PCR amplification. Once the amplification is successful, the fluorescence quenching group on the fluorescence probe dissociates from the reporter group, and the FRET effect disappears. At this time, fluorescence scanning can be performed to detect the corresponding fluorescence signal, so it can be known whether the corresponding allele exists. Summary of the Invention

[0005] In view of this, the present invention has developed primer pairs for detecting haplotype-specific molecular markers of genes related to 1000-grain weight in rice, namely SNAC1, qTGW3, GW5, AET1, and DHD1, based on the primer amplification-blocked mutation technology, solving the problem that it is impossible to determine the advantageous haplotypes of genes related to 1000-grain weight in rice using traditional technologies. It can be used to quickly distinguish the haplotypes of the above five genes in breeding materials, so as to select their advantageous haplotypes and advantageous haplotype combinations, and achieve rapid and accurate selection of the 1000-grain weight trait in rice.

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

[0007] The present invention provides a primer set for detecting specific molecular markers of genes related to 1000-grain weight in rice. Among them, the related genes are SNAC1, qTGW3, GW5, AET1, and DHD1. The primer set includes 9 primer pairs, specifically: the SNAC1-3416 primer pair with sequences as shown in SEQ ID NO: 1-3, the qTGW3-3538 primer pair with sequences as shown in SEQ ID NO: 4-6, the qTGW3-35390 primer pair with sequences as shown in SEQ ID NO: 7-9, the qTGW3-35391 primer pair with sequences as shown in SEQ ID NO: 10-12, the qTGW3-35393 primer pair with sequences as shown in SEQ ID NO: 13-15, the GW5-5364 primer pair with sequences as shown in SEQ ID NO: 16-18, the GW5-5366 primer pair with sequences as shown in SEQ ID NO: 19-21, the AET1-2659 primer pair with sequences as shown in SEQ ID NO: 22-24, and the DHD1-2889 primer pair with sequences as shown in SEQ ID NO: 25-27.

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

[0009] The above primer set or the above detection kit can be used to identify and screen advantageous haplotype combinations with high 1000-grain weight.

[0010] Specifically, in the above application, the identification method is specifically as follows: Using the genomic DNA of the rice to be detected as a template, PCR amplification is carried out respectively with 9 primer pairs, and a microplate reader is used to read the fluorescence signals of the 9 PCR products to distinguish different bases, so as to identify whether the rice material to be detected contains the advantageous haplotype combination.

[0011] Specifically, in the above application, the advantageous haplotype combination is one of CH1, CH2, CH3, CH4, CH5, CH6, CH7, and CH8, as shown in the following table:

[0012] Haplotype combination Single-gene haplotype CH1 SNAC1-H1, qTGW3-H2, GW5-H1, AET1-H1, DHD1-H1 CH2 SNAC1-H1, qTGW3-H2, GW5-H1, AET1-H1, DHD1-H2 CH3 SNAC1-H1, qTGW3-H1, GW5-H2, AET1-H2, DHD1-H1 CH4 SNAC1-H1, qTGW3-H1, GW5-H2, AET1-H2, DHD1-H2 CH5 SNAC1-H1, qTGW3-H2, GW5-H2, AET1-H1, DHD1-H1 CH6 SNAC1-H1, qTGW3-H2, GW5-H2, AET1-H1, DHD1-H2 CH7 SNAC1-H2, qTGW3-H2, GW5-H2, AET1-H1, DHD1-H1 CH8 SNAC1-H2, qTGW3-H2, GW5-H2, AET1-H1, DHD1-H2

[0013] The situations of each single-gene haplotype described in the above table are specifically shown in the following table:

[0014]

[0015]

[0016] The above primer set or the above detection kit can also be used for rice assisted breeding, specifically: detecting a rice sample, and selecting a rice sample of the CH4 type for breeding.

[0017] The above primer set or the above detection kit can also be used to detect the 1000-grain weight trait of rice.

[0018] Since the 1000-grain weight is an important factor constituting the rice yield, the above primer set or the above detection kit can also be used to identify high-yield rice varieties.

[0019] Compared with the prior art, the advantages of the present invention are specifically as follows: a primer set for detecting specific molecular markers of genes related to the 1000-grain weight of rice, namely SNAC1, qTGW3, GW5, AET1, and DHD1, is provided. Specifically, it is a molecular marker developed for the association sites of the SNAC1, qTGW3, GW5, AET1, and DHD1 genes with the 1000-grain weight of rice. This primer set can identify the haplotypes and their dominant haplotype combinations of the SNAC1, qTGW3, GW5, AET1, and DHD1 genes in 137 widely sourced indica rice germplasms; by simply, quickly, and accurately judging whether the breeding material contains the dominant haplotype combination of the above genes at the early stage of breeding, and selecting breeding materials with high 1000-grain weight, the purpose of improving breeding efficiency is achieved. Description of the Drawings

[0020] Figure 1 It is a microplate reader genotyping map for detecting the genotypes of rice germplasm resources in some primer pairs. Among them, FAM and HEX respectively correspond to the variations corresponding to the designed primers;

[0021] Figure 2 It is a microplate reader genotyping map for detecting the genotypes of rice germplasm resources in the remaining 5 primer pairs. Among them, FAM and HEX respectively correspond to the variations corresponding to the designed primers;

[0022] Figure 3 It is an effect analysis map of the haplotype combination of genes related to the 1000-grain weight of rice on the 1000-grain weight trait. Detailed Embodiments

[0023] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0024] In the following examples, unless otherwise specified, the examples are carried out under conventional experimental conditions or the conditions recommended in the manufacturer's instructions; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

[0025] Example 1

[0026] In the present invention, haplotype analysis was performed on the SNAC1, qTGW3, GW5, AET1, and DHD1 genes, and the superior haplotypes were combined to obtain a superior haplotype combination affecting the 1000-grain weight trait of rice. The specific process is as follows:

[0027] The experimental materials of the present invention were derived from 168 indica rice varieties in the National Key Research and Development Program "Precise Identification and Innovative Utilization of Rice Germplasm Resources in the Central China Rice Region (2016YFD0100101-05)", including 77 varieties, 39 lines, 18 local varieties, 18 restorer lines, and 16 foreign germplasms; however, due to the genotype deletion of individual varieties, 137 of them were selected for analysis, as shown in Table 1 for details.

[0028] All experimental materials were planted in the experimental field of the Jingzhou Academy of Agricultural Sciences, Hubei Province for two consecutive years (2018 and 2019), and the sowing time was May 16th of each year. The planting density was 15 cm × 25 cm, each material was planted in 5 rows, with 10 plants in each row, and the water and fertilizer management was carried out according to the conventional field planting management.

[0029] For each material, 5 representative mature plants were randomly selected in the field, threshed, and dried in the sun. The 1000-grain weight phenotypic values were obtained using the rice digital seed measurement machine (YTS-RICE-04D) of Wuhan Hongxingyang Technology Co., Ltd. (the results are shown in Table 1).

[0030] Targeted capture sequencing was performed on 42 target genes (genes regulating rice yield, quality, salt tolerance, and heat tolerance) of 168 rice materials. The size of the target region was 268132 bp, and variant detection was performed on the target region and 200 bp upstream and downstream of it. Sites with an allele frequency less than 0.05 were filtered out among 168 indica rice subspecies, and a total of 2164 SNPs and 578 Indel mutations were detected.

[0031] Furthermore, through candidate gene association analysis of grain-related traits (analysis of the BLUP values of the traits in 2018, 2019, and the two years, and simultaneously detected with P ≤ 0.01), 6 variant sites of the qTGW3 gene were detected to be associated with the 1000-grain weight, 7 variant sites of the GW5 gene were detected to be associated with the 1000-grain weight, and only 1 variant site of each of the SNAC1, AET1, and DHD1 genes was detected to be associated with the 1000-grain weight, as shown in Table 2 for details.

[0032] Based on the relevant mutation sites in Table 2, the above-mentioned associated genes were divided into different haplotypes and combined haplotypes among different associated genes, and the dominant haplotype combinations were analyzed. The results are shown in Table 1 and Table 3: Based on the associated sites with 1000-grain weight, the genes SNAC1, qTGW3, GW5, AET1, and DHD1 were each divided into only 2 haplotypes among 137 germplasm resources, namely SNAC1-H1 and SNAC1-H2, qTGW3-H1 and qTGW3-H2, GW5-H1 and GW5-H2, AET1-H1 and AET1-H2, DHD1-H1 and DHD1-H2.

[0033] The haplotypes of the above 5 genes could be divided into 8 haplotype combinations among 137 materials, denoted as CH1, CH2, CH3, CH4, CH5, CH6, CH7, and CH8, as shown in Table 4 specifically. The haplotype combinations could explain 22.49% of the phenotypic variation in 1000-grain weight. Among them, the average 1000-grain weight of the haplotype combination CH4 was the largest, being 29.47 g, and it showed significant differences from other haplotype combinations (Table 4, Figure 3 ). The results indicated that the rice germplasm resources with the haplotype combination CH4 of the genes SNAC1, qTGW3, GW5, AET1, and DHD1 had traits such as large 1000-grain weight that were in line with the breeding goals, and the haplotype combination CH4 could be used as the dominant haplotype to be preferentially selected in breeding applications.

[0034] Table 1 Name of experimental materials, haplotypes, and phenotypic values of 1000-grain weight (BLUP)

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] Table 2 Association analysis table of candidate genes for 1000-grain weight

[0041]

[0042] Table 3 Single-gene haplotypes of each associated gene and allelic genotypes of each mutation site

[0043]

[0044]

[0045] Table 4 Haplotype combinations of genes associated with 1000-grain weight, corresponding phenotypic mean values, and explained phenotypic variation

[0046]

[0047] Example 2

[0048] Based on the association sites of the SNAC1, qTGW3, GW5, AET1, and DHD1 genes with 1000-grain weight and their haplotypes in 137 rice germplasm resources, primer sets for detecting the haplotypes of the above genes were designed using the PARMS technique, as shown in Table 5 specifically.

[0049] Table 5 Primer information for haplotype-specific molecular markers of 1000-grain weight-associated genes

[0050]

[0051]

[0052]

[0053] Furthermore, the 137 rice germplasm resources were amplified using the primers in Table 5, and the fluorescence signals of the PCR products were read with a microplate reader to distinguish the bases corresponding to FAM and HEX. The results (as shown in Figure 1 and 2 ) were completely consistent with the capture sequencing results.

[0054] In summary, the haplotype-specific molecular marker primers for the rice 1000-grain weight-related genes SNAC1, qTGW3, GW5, AET1, and DHD1 developed in the present invention can distinguish different haplotypes of the above 5 genes in rice materials, and can be used to screen for superior haplotype combinations of the above 5 genes, thereby improving the breeding efficiency of high 1000-grain weight materials.

[0055] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Use of a primer set or a detection kit containing the primer set in identifying a superior haplotype combination with high 1000-grain weight, characterized in that, The primer set is used to detect specific molecular markers of genes related to the thousand-grain weight of rice, and the related genes are SNAC1 , qTGW3 , GW5 , AET1 and DHD1 . The primer set includes the SNAC1-3416 primer pair with sequences shown in SEQ ID NO: 1-3, the qTGW3-3538 primer pair with sequences shown in SEQ ID NO: 4-6, the qTGW3-35390 primer pair with sequences shown in SEQ ID NO: 7-9, the qTGW3-35391 primer pair with sequences shown in SEQ ID NO: 10-12, the qTGW3-35393 primer pair with sequences shown in SEQ ID NO: 13-15, the GW5-5364 primer pair with sequences shown in SEQ ID NO: 16-18, the GW5-5366 primer pair with sequences shown in SEQ ID NO: 19-21, the AET1-2659 primer pair with sequences shown in SEQ ID NO: 22-24, and the DHD1-2889 primer pair with sequences shown in SEQ ID NO: 25-27; The superior haplotype combination is CH4, as shown in the following table: The specific situations of the single-gene haplotypes in the above table are shown in the following table:

2. The use according to claim 1, characterized in that, Using the genomic DNA of the rice to be detected as a template, PCR amplification is respectively carried out with the primers of the primer set described in claim 1, and a microplate reader is used to read the fluorescence signals of 9 groups of PCR products to distinguish different allelic variations, so as to identify whether the rice material to be detected contains the superior haplotype combination.

3. Use of the primer set according to claim 1 or a detection kit containing the primer set in rice assisted breeding.

4. The use according to claim 3, characterized in that, Detecting a rice sample, and selecting a rice sample with the CH4 haplotype combination described in claim 1 for breeding.

5. Use of the primer set according to claim 1 or a detection kit containing the primer set in identifying high-yield rice varieties.

6. Use of the primer set according to claim 1 or a detection kit containing the primer set in detecting the 1000-grain weight trait of rice varieties.

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