Aroma rice is regarded as a treasure of rice because of its pleasant flavor and is loved by consumers. Its sales price is often higher than that of non-aromatic rice, which has important economic value. With the improvement of living standards, the market demand for aroma rice is also increasing. The genetic regulation mechanism of rice (Oryza sativa L.) aroma traits and the breeding of aroma rice varieties have attracted more and more attention from researchers. It was found that aroma traits were mainly controlled by a recessive gene locus on chromosome 8 (Ahn et al., 1992). Bradbury et al. (2005) found that the Badh2 gene encoded on chromosome 8 was directly related to rice aroma. The production of rice aroma was due to the loss-of-function of Badh2, and the first mutant allele badh2-E7 with 8-bp deletion in exon 7 and 3 single nucleotide polymorphisms that caused rice aroma was reported. Since then, a series of aroma mutant alleles have been found. According to statistics, there are 18 known aroma alleles (Amarawathi et al., 2008; Shi et al., 2008; Kovach et al., 2009; Shao et al., 2011; Shao et al., 2013; Ootsuka et al., 2014; Shi et al., 2014; He and Park, 2015). Among these known Badh2 alleles, the sequence of badh2-E2 allele has a 7-bp deletion in the second exon compared to the normal Badh2 gene, which also causes Badh2 to lose function and make rice flavor (Shi et al., 2008). The study on the distribution regions of aroma rice varieties with different Badh2 alleles found that aroma rice varieties with badh2-E2 alleles were only distributed in China (Kovach et al., 2009). Therefore, badh2-E2 is widely used in the breeding of aroma rice varieties in China (Xu et al., 2015).

In traditional breeding, the selection of aroma rice varieties is mainly carried out by grain chewing method (Dhulappanavar, 1976) and KOH soaking method (Sood and Siddiq, 1975). However, these selection methods rely on human senses, and in the breeding process, the selection of aroma phenotypes is often failed due to errors in judgment. The research results of the molecular mechanism of aroma rice formation make it possible to breed new varieties of aroma rice by molecular assisted selection breeding. According to the characteristic of badh2-E2 allele with 7-bp deletion in exon 2, the developed Indel molecular marker could reliably select the lines carrying badh2-E2 allele in the breeding population (Xu et al., 2015), which greatly promotes the breeding of new varieties of aroma rice.

After the discovery of thermostable ligase, LDR was developed to detect SNP loci (Barany, 1991). On this basis, PCR/LDR was developed to detect multiple SNP sites in one reaction system at the same time (Belgrader et al., 1996; Khanna et al., 1999). When PCR/LDR technology was used to detect the genotypes of SNP or Indel sites, the corresponding polymorphic sites need to be amplified by PCR, and then these PCR products were used in LDR reaction. Finally, the LDR reaction products were analyzed by ABI3730 sequencer, and the genotypes of the tested samples were determined according to the location of the fluorescence peak. Current method of PCR/LDR has been widely used in the diagnosis of genetic diseases, pathogenic microorganisms and other fields (Belgrader et al., 1996; Khanna et al., 1999; Favis and Barany, 2000). However, it has not been reported that this technology is used to develop molecular markers in molecular assisted breeding of crops.

KASP (Kompetitive Allele-Specific PCR) is a molecular marker technology developed in recent years that can be used for SNP and Indel site detection, and has been rapidly applied in many agricultural breeding (Neelam et al., 2013; Pariasca et al., 2015; Rasheed et al., 2016; Steele et al., 2018; Yang et al., 2019). However, KASP technology cannot be multiplexed. In the polymerization breeding of multiple target genes, it is necessary to detect the genotypes of multiple gene loci of each individual in the breeding population. PCR/LDR is very suitable for the design of multiple markers. Genotype detection of multiple gene loci in one reaction can greatly save the workload of molecular marker assisted selection (Belgrader et al., 1996; Khanna et al., 1999; Favis and Barany, 2000). Therefore, PCR/LDR technology has broad prospects in molecular marker assisted selection of crops.

In this study, a PCR/LDR-based functional molecular marker of badh2-E2 was developed (Belgrader et al., 1996). This marker does not require agarose or PAGE electrophoresis, and the homozygous or heterozygous badh2-E2 alleles can be identified accurately and quickly by ABI3730 sequencer, which improves the efficiency of aroma rice breeding.

1 Results and Analysis

1.1 Development of PCR/LDR-based badh2-E2 molecular marker

PCR/LDR technology requires a round of PCR and a round of LDR. First, a pair of PCR primers E2-F and E2-R were designed to amplify the DNA fragment containing the polymorphic sites of badh2-E2 and BADH2 sequences (Figure 1). Second, The PCR amplification fragment was used as a template for LDR reaction. There were 3 probes in the LDR reaction system, including a fluorescent labeled probe Probe-FLO, a badh2-E2 allele-specific probe Probe-E2 and a non badh2-E2 allele-specific probes badh2-NON. Probe-FLO probe consists of 20 base and template complementary sequences and 34 oligonucleotide T. FAM fluorescent groups were labeled at the 3’, and phosphorylated at the 5’. Probe-E2 probe consists of 23 template complementary sequences and 34 oligonucleotide T. badh2-NON probe consists of 23 template complementary sequences and 32 oligonucleotide T. The main difference between Probe-E2 and badh2-NON is that the badh2-NON sequence contains badh2-E2 allele with 7-bp deletion, and the length of the oligonucleotide T is different (Figure 1). When the genotype of the sample is badh2-E2, Probe-FLO can connect with Probe-E2 to produce a 111 nt oligonucleotide. When the genotype of the sample is non badh2-E2, Probe-FLO can connect with Probe-NON to produce a 109 nt oligonucleotide. When the sample was hybridized at badh2-E2 site, the products of LDR reaction with 109 nt and 111 nt oligonucleotides. Finally, the LDR product was analyzed by ABI3730. According to the location of FAM fluorescence, the genotype of the detected sample at badh2-E2 site could be accurately determined.

1.2 Validation of PCR/LDR-based molecular marker

In this study, the genotypes of two japonica hybrid restorer lines Shenfan24 and Shenfan26 were detected. The results showed that the LDR product length of Shenfan24 was 111 nt, and the LDR product length of Shenfan26 was 109 nt (Figure 2), indicating that Shenfan24 contained the badh2-E2 allele, and Shenfan26 without badh2-E2 allele, which was completely consistent with the expected results.

In order to verify whether PCR/LDR-based molecular markers were codominant markers, the F1 genotypes of Shenfan24 and Shenfan26 were detected. The results showed that the genotype of F1 plants at the badh2-E2 gene locus was heterozygous, and the corresponding LDR products with 109 nt and 111 nt oligonucleotides. According to the detection map, we can clearly determine whether the genotype of the detected sample at the badh2-E2 gene locus is homozygous or heterozygous, indicating that PCR/LDR molecular markers are codominant markers.

PCR/LDR-based molecular markers were used to identify the genotype of the badh2-E2 locus in 66 strains of the F2 population of Shenfan24 and Shenfan26. The results showed that 16 s/files/upfiles/files/F2-b(2).pngtrains were badh2-E2 homozygous genotype as Shenfan24, and 15 strains did not contain the badh2-E2 allele as Shenfan26, while the other 35 strains showed a heterozygous genotype at the badh2-E2 locus (Figure 2). The results were consistent with the separation ratio of 1:2:1 (χ²=0.273, p=0.873>0.05). This result further indicated that PCR/LDR-based molecular markers could accurately detect the genotype of badh2-E2 locus, which was suitable for marker-assisted selection and breeding of aroma rice varieties containing badh2-E2.

2 Discussion

In the past molecular marker-assisted breeding research using badh2-E2 aroma alleles, the Indel marker designed by the badh2-E2 aroma alleles with 7-bp deletion in exon 2 was used to identify the genotypes of breeding materials. PCR-based Indel molecular markers require agarose or polyacrylamide gel electrophoresis (PAGE) of PCR products for genotyping. This method is time-consuming and laborious and is not suitable for high-throughput detection of a large number of samples. In addition, toxic reagents such as ethyl bromide (EB), acrylamide or silver nitrate in the experiment (Ramkumar et al., 2015).

In this study, PCR/LDR-based functional molecular marker was developed for detecting badh2-E2 aroma alleles. The results showed that this marker could accurately distinguish homozygous and heterozygous lines carrying badh2-E2 gene, providing a new technical means for marker-assisted selection of aroma rice varieties. Genotype analysis of PCR/LDR-based molecular markers did not require agarose or PAGE electrophoresis. ABI3730 sequencer was used to analyze the genotype of PCR/LDR products, and 96 samples were analyzed in about 2 hours, greatly omitting the workload of genotype analysis.

In modern crop breeding, it is often necessary to aggregate important control genes of multiple important traits into a new variety. In traditional molecular assisted breeding, molecular markers associated with multiple traits need to be detected separately, which requires a lot of work. PCR/LDR is very suitable for the development of multiple molecular markers (Belgrader et al., 1996; Khanna et al., 1999; Favis and Barany, 2000), which could complete the analysis of multiple genetic loci genotypes in an experiment. The results of this study laid the foundation for the development of multiple molecular markers in the future breeding of badh2-E2 aroma alleles and other functional alleles.

3 Materials and Methods

3.1 Test materials

In order to verify the reliability of PCR/LDR-based molecular markers, this study selected two japonica hybrid restorer lines Shenfan24 and Shenfan26 genotypes for testing. It is known that Shenfan24 contains badh2-E2 alleles, while Shenfan26 does not contain badh2-E2 alleles (Cheng et al., 2018). The F1 and F2 populations used in this study were derived from the hybridization of Shenfan24 and Shenfan26.

3.2 Design and synthesis of PCR/LDR-based marker for badh2-E2 gene

PCR/LDR-based marker requires two steps of PCR and LDR. The first step is PCR amplification, and the second step is LDR reaction. The probe sequences of PCR primers and LDR reactions, as well as the length of the product were shown in the table (Table 1). And the complementary position of the probe and template DNA of PCR primers and LDR reactions were showed (Figure 1).

3.3 Rice leaf DNA extraction and marker detection

The DNA of rice leaves was extracted by CTAB method and quantified by NanoDrop™ 2000. The PCR reaction system was 25 μL, containing 50 ng genomic DNA, 0.5 pmol each of the upstream and downstream primers, 200 μM each of dATP, dGTP, dCTP and dTTP, 10 mM Tris-HCl (pH=8.8), 1.5 mM MgCl₂, 50 mM KCl, 0.8% (v/v) NP-40 and 5 U Taq DNA polymerase. PCR reaction program was as follows: denaturation at 95°C for 2 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, final, extension at 72°C for 5 min.

The LDR reaction system was 10 μL, containing 4 µL PCR products, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 1 mM NAD⁺, 0.2 pmol each for 3 LDR probes, and 2 U Taq DNA ligase. Reaction program was as follows: denaturation at 95°C for 2 min, then 40 cycles of denaturation at 94°C for 15 s, annealing and linking at 50°C for 25 s. Finally, analyzed the LDR products with ABI3730 DNA sequencer.

Authors’ contributions

CHW is the main executor of this study, completing data analysis. TRJ, ZJH participated in molecular marker detection. NFA, SB, LY, YY, HYW, LZY participated in the management of rice planting in the field. CLM, CC are the designer and director of the project, guiding the experimental design and manuscript revision. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the Construction of Rice Industry Technology System in Shanghai (HNK (2020) No.3), Key R & D Projects of the Ministry of Science and Technology (2016YFD0101106), and Program of Shanghai Technology Research Leader (18XD1424300).

Reference

Ahn S.N., Bollich C.N., and Tanksley S.D., 1992, RFLP tagging of a gene for aroma in rice, Theoretical and Applied Genetics, 84(7): 825-828

https://doi.org/10.1007/BF00227391

Amarawathi Y., Singh R., Singh A.K., Singh V.P., Mohapatra T., Sharma T.R., and Singh N.K., 2008, Mapping of quantitative trait loci for basmati quality traits in rice (Oryza sativa L.), Molecular Breeding, 21(1): 49-65

https://doi.org/10.1007/s11032-007-9108-8

Barany F., 1991, Genetic disease detection and DNA amplification using cloned thermostable ligase, Proc Natl Acad Sci USA, 88(1): 189-193

https://doi.org/10.1073/pnas.88.1.189

Belgrader P., Marino M.M., Lubin M., and Barany F., 1996, A multiplex PCR-ligase detection reaction assay for human identity testing, Genome Science & Technology, 1(2): 77-87

https://doi.org/10.1089/gst.1996.1.77

Bradbury L.M., Fitzgerald T.L., Henry R.J., Jin Q., and Waters D.L., 2005, The gene for fragrance in rice, Plant Biotechnol J, 3(3): 363-370

https://doi.org/10.1111/j.1467-7652.2005.00131.x

Cheng C., Yang J., Zhou J.H., Niu F., Hu X.J., Tu R.J., Luo Z.Y., Wang X.Q., Cao L.M., Chu H.W., 2018, Functional Molecular Marker-based Identification of Fragrant Parents of Japonica Hybrid Rice (Oryza Sativa L.), Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 16(17):5653-5659

Dhulappanavar C.V., 1976, Inheritance of scent in rice, Euphytica, 25(1): 659-662

https://doi.org/10.1007/BF00041603

Favis R., and Barany F., 2000, Mutation detection in K-ras, BRCA1, BRCA2, and p53 using PCR/LDR and a universal DNA microarray, Annals of the New York Academy of Sciences, 906(1): 39-43

https://doi.org/10.1111/j.1749-6632.2000.tb06588.x

He Q., and Park Y., 2015, Discovery of a novel fragrant allele and development of functional markers for fragrance in rice, Molecular Breeding, 35(11): 217

https://doi.org/10.1007/s11032-015-0412-4

Khanna M., Park P., Zirvi M., Cao W., Picon A., Day J., Paty P., and Barany F., 1999, Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors, Oncogene, 18(1): 27-38

https://doi.org/10.1038/sj.onc.1202291

Kovach M.J., Calingacion M.N., Fitzgerald M.A., and Mccouch S.R., 2009, The origin and evolution of fragrance in rice (Oryza sativa L.), Proc Natl Acad Sci USA, 106(34): 14444-14449

https://doi.org/10.1073/pnas.0904077106

Neelam K., Brown G.G., and Huang L., 2013, Development and validation of a breeder-friendly KASPar marker for wheat leaf rust resistance locus Lr21, Molecular Breeding, 31(1): 233-237

https://doi.org/10.1007/s11032-012-9773-0

Ootsuka K., Takahashi I., Tanaka K., Itani T., Tabuchi H., Yoshihashi T., Tonouchi A., and Ishikawa R., 2014, Genetic polymorphisms in Japanese fragrant landraces and novel fragrant allele domesticated in northern Japan, Breed Sci, 64(2): 115-124

https://doi.org/10.1270/jsbbs.64.115

Pariasca T.J., Lorieux M., He C., Mccouch S., Thomson M.J., and Wissuwa M., 2015, Development of a SNP genotyping panel for detecting polymorphisms in Oryza glaberrima/O. sativa interspecific crosses, Euphytica, 201(1): 67-78

https://doi.org/10.1007/s10681-014-1183-4

Ramkumar G., Prahalada G.D., Hechanova S.L., Vinarao R., and Jena K.K., 2015, Development and validation of SNP-based functional codominant markers for two major disease resistance genes in rice (O. sativa L.), Molecular Breeding, 35(6): 129

https://doi.org/10.1007/s11032-015-0323-4

Rasheed A., Wen W., Gao F.M., Zhai S.N., Jin H., Liu J.D., Guo Q., Zhang Y.J., Dreisigacker S., Xia X.C., and He Z.H., 2016, Development and validation of KASP assays for genes underpinning key economic traits in bread wheat, Theoretical and Applied Genetics, 129(10): 1843-1860

https://doi.org/10.1007/s00122-016-2743-x

Shao G.N., Tang S.Q., Chen M.L., Wei X.J., He J.W., Luo J., Jiao G.A., Hu Y.C., Xie L.H., and Hu P.S., 2013, Haplotype variation at Badh2, the gene determining fragrance in rice, Genomics, 101(2): 157-162

https://doi.org/10.1016/j.ygeno.2012.11.010

Shao G.N., Tang A., Tang S.Q., Luo J., Jiao G.A., Wu J.L., and Hu P.S., 2011, A new deletion mutation of fragrant gene and the development of three molecular markers for fragrance in rice, Plant Breeding, 130(2): 172-176

https://doi.org/10.1111/j.1439-0523.2009.01764.x

Shi W.W., Yang Y., Chen S.H., and Xu M.L., 2008, Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties, Molecular Breeding, 22(2): 185-192

https://doi.org/10.1007/s11032-008-9165-7

Shi Y.Q., Zhao G.C., Xu X.L., and Li J.Y., 2014, Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice, Molecular Breeding, 33(3): 701-708

https://doi.org/10.1007/s11032-013-9986-x

Sood B.C., and Siddiq E.A., 1975, A rapid technique for scent determination in rice, Indian Journal of Genetics & Plant Breeding, 38(2): 268-275

Steele K.A., Quinton T.M.J., Amgai R.B., Dhakal R., Khatiwada S.P., Vyas D., Heine M., and Witcombe J.R., 2018, Accelerating public sector rice breeding with high-density KASP markers derived from whole genome sequencing of indica rice, Molecular Breeding, 38(4): 38

https://doi.org/10.1007/s11032-018-0777-2

Xu Y.F., Huang J., Wang Y.C., Wang J., Li J.Y., 2015, Development of Two Molecular Markers used to Detection of badh2-E2 Fragrant Gene in Rice, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 13(11): 2441-2445

Yang G.L., Chen S.P., Chen L.K., Sun K., Huang C.H., Zhou D.H., Huang Y.T., Wang J.F., Liu Y.Z., Wang H., Chen Z.Q., and Guo T., 2019, Development of a core SNP arrays based on the KASP method for molecular breeding of rice, Rice, 12(1): 21

https://doi.org/10.1186/s12284-019-0272-3