A multiplex PCR system for detection of wide compatibility allele S5-n and erect panicle allele dep1 in rice

Abstract

Wide compatibility and erect panicles are important traits for heterosis and ideal plant type breeding in rice and accordingly are targets for rice yield improvement. In this study, a multiplex PCR system based on functional markers was developed to simultaneously identify genotypes of the wide compatibility allele S5-n and the erect panicle allele dep1. Forty-nine rice varieties in the Huang-Huai-Hai region of China were identified using this system. Thirty-three varieties contained the erect panicle allele dep1 and two varieties contained the wide compatibility allele S5-n. The frequency of dep1 was obviously higher than that of S5-n. The multiplex PCR method was also tested using an F₂ segregating population and was found to be simple, efficient, and reliable. It will be useful in marker-assisted selection based on the wide compatibility allele S5-n and erect panicle allele dep1 to breed super rice varieties using indica and japonica heterosis.

Key words:
Functional marker; S5-n; DEP1; multiplex PCR

INTRODUCTION

Rice is one of the most important food crops worldwide. The continuous improvement of rice yield is an important way to ensure food security (Liang et al. 2014). According to Chen et al. (2001), rice yield improvement in the future will depend on super high-yield rice breeding that combines ideal plant type and heterosis. Indica and japonica, two subspecies of Asian cultivated rice (Oryza sativa L.), show strong F₁ heterosis, and accordingly have the potential to produce super hybrid rice plants (Konishi et al. 2006, Qian et al. 2016). However, owing to the high genetic distance between indica and japonica, the F₁ hybrids generally have low fertility, which is a major obstacle in the utilization of inter-subspecific heterosis. Accordingly, overcoming the problem of hybrid sterility between indica and japonica to improve yield is an important topic in rice breeding research.

Ikehashi and Araki (1986) proposed the wide compatibility theory and mapped the S5 locus for indica-japonica hybrid sterility to chromosome 6. There are three alleles at the S5 locus, i.e., the indica allele S5-i, the japonica allele S5-j, and the neutral allele S5-n (referred to as the wide-compatibility allele). Plants with the S5-n/S5-j (or S5-i) genotype are fully fertile, while plants with the S5-i/S5-j genotype have low fertility. Chen et al. (2008) cloned the S5 gene from the wide-compatibility variety 02428 using a map-based cloning approach and characterized its function. The S5 of indica and japonica differ by two nucleotides, resulting in an amino acid difference and thus leading to hybrid F₁ sterility. A 136-bp deletion in S5 including the translation start site in the wide-compatibility variety results in the functional loss of aspartic protease, leading to a lack of a reproductive barrier between indica and japonica. The successful cloning of the wide compatibility allele S5-n facilitates gene exchange and the utilization of indica and japonica heterosis.

An erect panicle is an ideal plant type trait in high-yield rice (Qian et al. 2016, Zhao et al. 2016). Erect panicle varieties with short, dense panicles, straight leaves, and a small leaf angle could improve the rice population structure and thus increase the efficiency of photosynthesis and the accumulation of photosynthetic products (Xu et al. 1995, Cheng et al. 2011). Huang et al. (2009) cloned the DEP1 from the super rice variety Shennong 265 using a map-based cloning method. The DEP1, including 5 exons and 4 introns, encodes amino acids that are functionally similar to the phospholipid diethanolamide binding protein. The erect panicle allele dep1 has a 625-bp deletion in the fifth exon of DEP1 resulting in the premature termination of transcription. The mutated dep1 allele could enhance rice yield by improving panicle density, branch number, grain number per panicle, and nitrogen uptake and metabolism.

DNA marker-assisted breeding combines molecular markers and crop breeding. The technique has many advantages, such as its insensitivity to environmental conditions, high reliability, and time-efficiency (Dudley et al. 1993, Ribaut et al. 1998, Schuster 2011, Xu 2013). A functional marker is designed according to polymorphic sites within gene sequences affecting phenotypic variation (Andersen and Lübberstedt et al. 2003). Compared with the traditional marker-assisted selection, functional markers have apparent advantages for the detection of the target trait allele in both artificial and natural selection populations (Andersen and Lübberstedt 2003, Varshney et al. 2005). The multiplex PCR functional marker system has various benefits, including its favorable accuracy, repeatability, and efficiency (Varshney et al. 2005, Ramkumar et al. 2010, Salgotra et al. 2011). In this study, a functional marker-based multiplex PCR assay for the simultaneous detection of S5-n and dep1 was developed and was used to detect 49 rice varieties in the Huang-Huai-Hai region of China and a F₂ segregating population derived from a cross between indica and japonica. The results indicated that the system could be applied to the rapid detection of S5-n and dep1 in rice germplasm resources and to molecular marker-assisted selection in indica-japonica hybrid breeding.

MATERIAL AND METHODS

Plant material

Forty-nine japonica varieties in the Huang-Huai-Hai region of China and one F₂ population derived from a cross between japonica Huaidao 6 and indica 9311 were used to evaluate the multiplex PCR system. Information about the 49 varieties is provided in Online Resource 1 and additional information can be found at the China Rice Data Center (http://www.ricedata.cn/index.htm). The wide-compatibility variety 02428 was used as the positive control for S5-n and Nipponbare, an incompatible japonica variety with the S5-i genotype, was used as the negative control for S5-n. Wuyunjing 8, an erect and dense panicle variety, was used as the positive control for dep1 and Nipponbare, a non-erect and non-dense panicle variety, was used as the negative control. From fresh leaves of 3-week-old seedlings, DNA was extracted using the modified CTAB method (Rogers et al. 1989).

Online Table 1
Name, approval date, and pedigree of 49 japonica rice varieties

Development of functional markers

One deletion of 136 bp near the upstream and downstream regions of the translation initiation site (ATG) differentiates the wide compatibility allele S5-n and the incompatibility alleles S5-j or S5-i and leads to a loss of function of the S5 (Os06g0213100). The primer pair S5-1 for S5 was designed according to the flanking sequence of the 136-bp deletion (Figure 1). For DEP1 (Os09g0441900), a 625-bp deletion in the fifth exon distinguishes the erect panicle variety from the non-erect panicle variety. The primer pair DEP1-1 for DEP1 was designed according to the sequences upstream and downstream of the fifth exon (Figure 2). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Figure 1
Location of the primers and deletion sequence in S5-n. The dashed line indicates the deletion in S5-n; arrows indicate the primers; red letters indicate the translation start site; S5 indicates Os06g0213100.

Figure 2
Location of the primers and deletion sequence in DEP1. The dashed line indicates the deletion in DEP1; dots indicate common bases between the two varieties, which were omitted; arrows indicate the primers; DEP1 indicates Os09g0441900.

Multiplex PCR amplification and detection

The multiple PCR reaction system for S5-n and dep1 used a reaction volume of 20 μL, containing 10 μL of 2×Es Taq Master Mix (including 0.1 U μL^-1 Es Taq DNA polymerase, 2×Es PCR Buffer, 3 mM MgCl₂, and 0.4 mM dNTP mix, provided by Beijing ComWin Biotech Co., Ltd.), 2 μL (40-60 ng μL^-1) of template DNA, 1 μL (0.4 μmo1 L^-1) of forward primer for each gene, 1 μL (0.4 μmo1 L^-1) of reverse primer for each gene, and 4 μL of ddH₂O. The PCR was performed using a T100 thermal cycler (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, 30 s; 72 °C for 8 min. PCR products were then separated on 1% agarose gel containing DNA green in 1× TAE buffer at 120 V for 30 min. The resulting bands were visualized under ultraviolet light and recorded using Gel Doc^TM EZ imager (BIO-RAD).

RESULTS AND DISCUSSION

Based on the comprehensive consideration of factors that influence PCR for the two genes, e.g., PCR product length, annealing temperature, and PCR byproducts, sequence polymorphisms were used to design functional markers for S5-n and dep1. The primer pair S5-1 was designed according to the 136-bp deletion in S5 of the wide-compatibility variety compared to the incompatible variety. The primers amplified a 321-bp fragment in the wide-compatibility variety and a 457-bp fragment in the incompatible variety (Figure 1). The primer pair DEP1-1 was designed according to the 625-bp deletion in DEP1 in the erect panicle variety compared to the non-erect panicle variety. The primers amplified a 1235-bp fragment in the erect panicle variety and a 1860-bp fragment in the non-erect panicle variety (Figure 2). Finally, two pairs of primers, S5-1 and DEP1-1, yielding clear and differential PCR products with similar annealing temperatures were obtained.

By the continuous optimization of the annealing temperature, number of reaction cycles, and DNA template concentration, the optimum PCR reaction conditions were obtained, i.e., an annealing temperature of 56 °C, 35 cycles, and a template concentration of 4-6 ng μL^-1 . Using agarose gel electrophoresis, four amplification products of 321, 457, 1235, and 1860 bp for S5-n in the wide-compatibility variety, S5-(i or j) in the incompatible variety, dep1 in the erect panicle variety, and DEP1 in the non-erect panicle variety were clearly observed.

The multiple PCR system was used to detect the distribution of S5-n and dep1 genotypes in 49 rice varieties recently grown in the Huang-Huai-Hai region of China (Figure 3). Huaidao 6 and Handao 277 had the same 321-bp band as that observed for the wide compatibility control variety 02428, indicating that 4.08% were wide-compatibility varieties. With respect to dep1, 33 of 49 varieties had the same 1235-bp band as the erect panicle control variety Wuyunjing 8, indicating that 67.3% were erect panicle varieties. Only Huaidao 6 had both wide compatibility allele S5-n and erect panicle allele dep1 (Table 1).

Figure 3
Pattern of PCR amplification obtained using the multiplex PCR system for 49 rice varieties. M indicates the DNA marker; ck1 indicates the positive control for S5-n; ck2 indicates the incompatible japonica variety with the S5-i allele as the negative control for S5-n; ck3 indicates the non-erect and non-dense panicle variety with the DEP1 allele as the negative control for dep1; ck4 indicates the erect and dense panicle variety with the dep1 allele as the positive control. Samples 1-49 are the 49 rice varieties described in Supplemental Table S1; S5 indicates Os06g0213100; DEP1 indicates Os09g0441900.

Table 1
Name, sequences, and expected fragment size for functional markers

Table 2
The detection results for the multiplex PCR system for 49 rice varieties. “+” indicates the positive detection of the wide-compatibility allele S5-n or the erect panicle allele dep1, “-” indicates otherwise. S5 indicates Os06g0213100; DEP1 indicates Os09g0441900

The multiplex PCR system was applied to an F₂ population derived from Huaidao 6 and 9311. Twenty individual plants were randomly selected from the F₂ population for detection (Figure 4). Two individuals (2 and 6) were homozygous for S5-n and dep1. Three individuals (10, 12, and 20) were homozygous for S5-i and DEP1. Six individuals (1, 5, 8, 15, 16, and 19) were heterozygous for S5 and DEP1. Other individuals had one homozygous gene and one heterozygous gene. The results indicated that the multiplex PCR system can be used to detect different S5 and dep1 genotypes in F₂ segregating populations.

Figure 4
Pattern of PCR amplification obtained using the multiplex PCR system for an F₂ population derived from a cross between japonica Huaidao 6 and indica 9311. M indicates the DNA marker; ck1 indicate the positive control for S5-n; ck2 indicate the incompatible japonica variety with the S5-i allele as the negative control for S5-n; ck3 indicate the erect and dense panicle variety with the dep1 allele as the positive control; ck4 indicate the non-erect and non-dense panicle variety with DEP1 allele as the negative control for dep1. Samples 1-20 are the 20 individual plants that were randomly selected from the F₂ population; S5 indicates Os06g0213100; DEP1 indicates Os09g0441900.

Using molecular marker technology, a large number of functional alleles controlling important agronomic traits in rice have been successfully located or cloned, providing an impetus for super rice breeding. The combination of heterosis and ideal plant type is an important breakthrough in super rice breeding. The wide compatibility allele S5-n and erect panicle allele dep1 are important for heterosis and ideal plant type breeding in rice. Conventional breeding methods to select plants based on S5-n and dep1 require tedious, time-consuming, and inefficient work related to phenotyping and determining offspring fertility. Thus, a simple and efficient detection system for S5-n and dep1 is required for super rice breeding. Multiplex PCR technology can be used to construct an effective diagnostic system (Chen et al. 2015). Recently, multiplex PCR was successful applied to detect several genes in rice, such as xa13, Xa21, and fgr (Salgotra et al. 2011), fgr and Wx (Cheng et al. 2015), and Xa4, xa5, Xa7, xa13, and Xa21 (Yap et al. 2016). Here, the multiplex PCR system based on the functional markers was a reliable and sensitive method for the simultaneous detection of S5-n and dep1. The system could precisely detect plants with S5-n and dep1 by one-time conventional PCR amplification and agarose gel electrophoresis. All results demonstrated that the method is rapid, has high repeatability, and is efficient for identifying S5-n and dep1 in germplasm and for gene-based selection in rice breeding programs, irrespective of plant growth stage, tissue type, and variety.

Compared with conventional PCR, the establishment of a multiplex PCR system is more challenging; it requires a comprehensive analysis and repeated optimization (Chen et al. 2015). Based on the characteristics of the target gene and PCR product, it is essential to design appropriate primer combinations and establish a suitable PCR reaction system. In our opinion, primer design is the most critical step. The annealing temperature should be the same or similar and the length differences between the PCR product of multiplex primers should be distinct. Other factors, such as PCR cycles, template DNA concentration, Taq DNA polymerase, and dNTP concentrations should also be repeatedly tested and modified. In this study, after the repeated optimization procedure, four specific bands of 321, 457, 1235, and 1860 bp for S5-n in the wide-compatibility variety, S5-(i or j) in the incompatible variety, dep1 in the erect panicle variety, and DEP1 in the non-erect panicle variety were clearly observed. The results demonstrated the feasibility of our method for multiplex PCR design.

In this study, the erect panicle allele dep1 was substantially more frequent than the wide-compatibility allele S5-n in 49 varieties from the Huang-Huai-Hai region of China. Thirty-three varieties had the erect panicle allele dep1, two varieties had the wide-compatibility allele S5-n, and only one variety had both alleles. These results indicated that DEP1 is widely used in the Huang-Huai-Hai region of China and most rice varieties have erect panicles, as observed in the field. In contrast, wide-compatibility varieties were very rare and S5-n is seldom used in rice breeding in the Huang-Huai-Hai region of China, limiting the utilization of japonica and indica heterosis. S5 is a key gene involved in indica-japonica hybrid sterility. Wide-compatibility varieties with S5-n are able to overcome reproductive barriers. Thus, they produce highly fertile hybrids when crossed with indica or japonica varieties, which promotes gene flow and heterosis. The DEP1 gene is pleiotropic; it is involved in erect panicles, number of grains per panicle, and nitrogen uptake and metabolism (Huang et al. 2009), and could control the efficiency of photosynthesis and nitrogen-use efficiency (Sun et al. 2014). The dep1 alleles have effects on vascular bundle- and panicle-related traits of rice in both indica and japonica genetic backgrounds (Xu et al. 2015). According to Li et al. (2016), a mutation of DEP1 could increase panicle density in japonica Zhonghua 11, using the CRISPR/Cas9 system. An elite indica restorer line with dense and erect panicles was selected by marker-assisted selection of dep1 alleles from backcross populations (Cheng et al. 2011). Therefore, using S5-n and DEP1 to breed a super rice variety based on heterosis and ideal plant type is highly significant. Based on the distributions of S5-n and dep1 in the Huang-Huai-Hai region of China, the utilization of S5-n needs to increase to develop more wide-compatibility lines. Selection based on S5-n and dep1 to breed elite rice lines with wide compatibility and erect panicle traits may also be useful for super rice breeding by the combined application of heterosis and ideotype.

CONCLUSIONS

A multiplex PCR system for simultaneously detecting genotypes of the wide compatibility allele S5-n and the erect panicle allele dep1 was developed. This method was found to be simple, efficient, and reliable for 49 rice varieties in the Huang-Huai-Hai region of China and one F₂ segregating population. The dep1 allele has already been widely exploited in japonica rice breeding, unlike the rather scarce allele S5-n, which needs to be included more frequently in breeding, to obtain more incompatible varieties in the Huang-Huai-Hai region of China.

ACKNOWLEDGEMENTS

This work was supported by the Major Science and Technology Project of Henan Province (141100110600), the Science and Technology Research Project of the Education Department of Henan Province (14A210009), the Scientific and Technological Innovative Talents Supporting Project of the Universities of Henan Province (16HASTIT016), and the Project of Construction Key Laboratory of Zhengzhou City (121PYFZX185).

REFERENCES

• Andersen JR & Lübberstedt T (2003) Functional markers in plants. Trends in Plant Science 8: 554-560.
• Chen JJ, Ding JH, Ouyang YD, Du HY, Yang JY, Cheng K, Zhao J, Qiu SQ, Zhang XL, Yao JL, Liu KD, Wang L, Xu CG, Li XH, Xue YB, Xia M, Ji Q, Lu JF, Xu ML and Zhang QF (2008) A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice. Proceedings of the National Academy of Sciences of UAS 105: 11436-11441.
• Chen WF, Xu ZJ, Zhang WZ, Zhang LB and Yang SR (2001) Creation of new plant type and breeding rice for super high yield. Acta Agronomica Sinica 27: 665-672.
• Chen S, Cao YY, Li TY and Wu XX (2015) Simultaneous detection of three wheat pathogenic fungal species by multiplex PCR. Phytoparasitica 43: 449-460.
• Cheng ZP, Liu CD, Yang DW, Lu LB, Ye L, Zheng XH and Ye XF (2011) Molecular markers-assisted selection of restorer line of dense and erect panicle. Molecular Plant Breeding 5: 561-566.
• Cheng A, Massawe F, Ismail I, Osman M and Hashim H (2015) High resolution agarose-based system for single-tube genotyping of fgr and Waxy genes in rice: MAGE to displace PAGE? Plant Omics 8: 348-352.
• Dudley JW (1993) Molecular markers in plant improvement: manipulation of genes affecting quantitative traits. Crop Science 33: 660-668.
• Huang XZ, Qian Q, Liu ZB, Sun HY, He SY, Luo D, Xia GM, Chu CC, Li JY and Fu XD (2009) Natural variation at the DEP1 locus enhances grain yield in rice. Nature Genetics 41: 494-497.
• Ikehashi H & Araki H (1986) Genetics of F₁ sterility in remote crosses of rice. In Rice Genetics. Proceedings International Rice Genetics Symposium. IRRI, Manila, pp. 119-130.
• Konishi S, Izawa T, Lin SY, Ebana K, Fukuta Y, Sasaki T and Yano M (2006) An SNP caused loss of seed shattering during rice domestication. Science 312: 1392-1396.
• Li MR, Li XX, Zhou ZJ, Wu PZ, Fang MC, Pan XP, Lin QP, Luo WB, Wu GJ and Li HQ (2016) Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science 7: 377.
• Liang WH, Shang F, Lin QT, Lou C and Zhang J (2014) Tillering and panicle branching genes in rice. Gene 537: 1-5.
• Qian Q, Guo LB, Smith SM and Li JY (2016) Breeding high-yield superior-quality hybrid super-rice by rational design. National Science Review 3: 283-294.
• Ramkumar G, Sivaranjani AKP, Pandey MK, Sakthivel K, Rani NS, Sudarshan I, Prasad GSV, Neeraja CN, Sundaram RM, Viraktamath BC and Madhav MS (2010) Development of a PCR-based SNP marker system for effective selection of kernel length and kernel elongation in rice. Molecular Breeding 26: 735-740.
• Ribaut JM & Hoisington D (1998) Marker-assisted selection: new tools and strategies. Trends in Plant Science 3: 236-239.
• Rogers SO & Bendich AJ (1989) Extraction of DNA from plant tissue. Plant molecular biology manual. Springer, Netherlands, p. 73-83.
• Salgotra RK, Millwood RJ, Agarwal S and Stewart CN (2011) High-throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.). Electrophoresis 32: 2216-2222.
• Schuster I (2011) Marker-assisted selection for quantitative traits. Crop Breeding and Applied Biotechnology S1: 50-55.
  » S1
• Sun HY, Qian Q, Wu K, Luo JJ, Wang SS, Zhang CW, Ma YF, Liu Q, Huang XZ, Yuan QB, Han RX, Zhao M, Dong GJ, Guo LB, Zhu XD, Gou ZH, Wang W, Wu YJ, Lin HX and Fu XD (2014) Heterotrimeric G proteins regulate nitrogen-use efficiency in rice. Nature Genetics 46: 652-656.
• Varshney RK, Graner A and Sorrells ME (2005) Genomics-assisted breeding for crop improvement. Trends in Plant Science 10: 621-630.
• Xu J (2013) Pyramiding of two BPH resistance genes and_^Stv-bi gene using marker-assisted selection in japonica rice. Crop Breeding and Applied Biotechnology 13: 99-106.
• Xu Q, Liu TS, Bi WJ, Wang YZ, Xu H, Tang L, Sun J and Xu ZJ (2015) Different effects of DEP1 on vascular bundle-and panicle-related traits under indica and japonica genetic backgrounds. Molecular Breeding 35: 173.
• Xu Z, Chen W, Zhang L and Yang S (1995) Advance in estimation and utilization of rice erect panicle. Journal of Shenyang Agricultural University 26: 335-341.
• Yap RS, Hsu YC, Wu YP, Lin YR and Kuo CW (2016) Multiplex PCR genotyping for five bacterial blight resistance genes applied to marker-assisted selection in rice (Oryza sativa). Plant Breeding 135: 309-317.
• Zhao MZ, Sun J, Xiao ZQ, Cheng F, Xu H, Tang L, Chen WF, Xu ZJ and Xu Q (2016) Variations in DENSE AND ERECT PANICLE 1 (DEP1) contribute to the diversity of the panicle trait in high-yielding japonica rice varieties in northern China. Breeding Science doi:10.1270/jsbbs.16058.
  » https://doi.org/10.1270/jsbbs.16058

Publication Dates

• Publication in this collection
  Sept 2017

History

• Received
  07 Dec 2016
• Accepted
  02 Mar 2017