Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (1), 12

Development and Validation of Allele-Specific SNP/indel Markers for Eight Yield-Enhancing Genes Using Whole-Genome Sequencing Strategy to Increase Yield Potential of Rice, Oryza Sativa L

Affiliations

Development and Validation of Allele-Specific SNP/indel Markers for Eight Yield-Enhancing Genes Using Whole-Genome Sequencing Strategy to Increase Yield Potential of Rice, Oryza Sativa L

Sung-Ryul Kim et al. Rice (N Y).

Abstract

Background: Rice is one of the major staple foods in the world, especially in the developing countries of Asia. Its consumption as a dietary source is also increasing in Africa. To meet the demand for rice to feed the increasing human population, increasing rice yield is essential. Improving the genetic yield potential of rice is one ideal solution. It is imperative to introduce the identified yield-enhancing gene(s) into modern rice cultivars for the rapid improvement of yield potential through marker-assisted breeding.

Results: We report the development of PCR-gel-based markers for eight yield-related functional genes (Gn1a, OsSPL14, SCM2, Ghd7, DEP1, SPIKE, GS5, and TGW6) to introduce yield-positive alleles from the donor lines. Six rice cultivars, including three each of donor and recipient lines, respectively, were sequenced by next-generation whole-genome sequencing to detect DNA polymorphisms between the genotypes. Additionally, PCR products containing functional nucleotide polymorphism (FNP) or putative FNPs for yield-related genes were sequenced. DNA polymorphisms discriminating yield-positive alleles and non-target alleles for each gene were selected through sequence analysis and the allele-specific PCR-gel-based markers were developed. The markers were validated with our intermediate breeding lines produced from crosses between the donors and 12 elite indica rice cultivars as recipients. Automated capillary electrophoresis was tested and fluorescence-labeled SNP genotyping markers (Fluidigm SNP genotyping platform) for Gn1a, OsSPL14, Ghd7, GS5, and GS3 genes were developed for high-throughput genotyping.

Conclusions: The SNP/indel markers linked to yield related genes functioned properly in our marker-assisted breeding program with identified high yield potential lines. These markers can be utilized in local favorite rice cultivars for yield enhancement. The marker designing strategy using both next generation sequencing and Sanger sequencing methods can be used for suitable marker development of other genes associated with useful agronomic traits.

Keywords: Allele-specific marker; Rice; SNP genotyping; Yield potential; Yield-enhancing genes.

Figures

Fig. 1
Fig. 1
Gn1a markers. a Identification of nucleotide polymorphisms in Gn1a gene for marker development. Gn1a gene consists of four exons (orange boxes) in which translation initiation codon (Start) and stop codon (Stop) are depicted. Each nucleotide variation with corresponding markers (PCR-gel-based markers and Fluidigm genotyping platform (-FD) markers) is mapped on the gene structure. The sequence alignment showed nucleotide polymorphisms in the promoter and 5’UTR regions of Gn1a among 15 varieties, including the reference genome (Nipponbare), two Gn1a donors, and 12 recipients. DNA polymorphisms were highlighted with pink and green color. The number of unrepresented nucleotides (bp) in a sequence was shown in parentheses. Based on the context of the promoter sequences, three Gn1a alleles (Types 1-3: T1-T3) were found. Variety name with asterisk (*) was used as the donor line of target allele. b Agarose gel images analyzed by three Gn1a markers (Gn1a-17SNP, Gn1a-indel3, and Gn1a-indel1 markers) from two donor lines and 12 recipients. Predicted PCR product sizes for yield-positive allele (P), non-target allele (N), and common band (OP) were shown at the right side of the gel image. Primer combination for each marker was shown on the gel images and its sequences were listed in Table 2. M, DNA size marker. c Application of Gn1a markers in the intermediate breeding line. Fourteen BC1F3 plants derived from PR37951 x Habataki cross were genotyped with Gn1a-17SNP and Gn1a-indel3 marker, respectively. Genotyping result was scored for each plant as PP (homozygous for positive allele), NN (homozygous for non-target allele), and PN (heterozygote)
Fig. 2
Fig. 2
OsSPL14 markers. a Identification of donor-specific DNA polymorphisms in OsSPL14 gene. The donor-specific SNPs at the promoter and the third exon were represented through DNA sequence alignment among 15 varieties, including Nipponbare, two different OsSPL14 donors (ST12 and Aikawa1), and 12 recipients. The location of 2.6-kb candidate region of WFP (Miura et al. 2010) was depicted. b Agarose gel images analyzed by the SPL14-04SNP marker and the SPL14-12SNP marker, respectively, from the parental lines. Primer combination for each PCR was shown on the gel images. c Application of SPL14-04SNP marker in the intermediate breeding line, with 12 BC2F2 plants derived from PR38012 x ST12 cross. d Application of SPL14-12SNP marker in the intermediate breeding line. Twelve BC2F2 plants derived from IR04A115 x Aikawa1 cross were genotyped
Fig. 3
Fig. 3
SCM2 marker. a Gene structure of APO1/SCM2. Three regions sequenced by the Sanger method were represented with primer pairs. b Agarose gel image analyzed by SCM2-indel1 marker from the donor line (Habataki), ST12, and 12 recipients. To separate two bands (117 bp and 105 bp) produced by the SCM2-indel1 marker, 4% agarose gel was used. c-d Application of SCM2-indel1 marker in the intermediate breeding line. Thirteen BC3F2 plants derived from Parao x Habataki cross were genotyped by the SCM2-indel1 marker and its PCR products were electrophoresed in 4 % agarose gel (c) and in Fragment Analyzer (d)
Fig. 4
Fig. 4
Ghd7 marker. a Agarose gel image analyzed by Ghd7-05SNP marker from the parental lines. Non-target allele and yield-positive allele (Ghd7-1) were amplified by Ghd7-05SNP-F/AR primer pairs and Ghd7-05SNP- F/TR primer pairs, respectively. Habataki and ST12 were used as the donors of Ghd7-1 allele and Aikawa1 (japonica-type variety) was included as a control of Ghd7-2 allele. b Application of Ghd7-05SNP marker in the intermediate breeding line. Thirteen BC2F2 plants crossed between Parao and Habataki were genotyped by the marker
Fig. 5
Fig. 5
DEP1 marker. a Gene structure of DEP1. FNP (625-bp indel) was shown with the unfilled box on the fifth exon. The location and direction of marker primers (DEP1-indel1-F/R/625F) are mapped on the gene structure. b-c Agarose gel images of PCR with DEP1-indel1 marker (b) and DEP1-indel1P marker (c) from the parents. d-e Application of DEP1-indel1 marker (d) and DEP1-indel1P marker (e) in the intermediate breeding line. Thirteen BC2F2 plants derived from NSIC Rc158 x Osmancik-97 cross were genotyped
Fig. 6
Fig. 6
SPIKE markers. a-c Agarose gel images analyzed by three SPIKE markers from the parental lines. The SPIKE-01SNP marker discriminated G/A SNP located on the third exon using two separated PCRs (SPIKE-01SNP- GF/R primer set and SPIKE-01SNP-AF/R primer set) (a). The SPIKE-03SNP marker detected the G/A SNP on the fifth exon using two separated PCRs (SPIKE-03SNP-F/GR primer set and SPIKE-03SNP-F/AR primer set) (b). The SPIKE-indel3 marker distinguished a 20-bp indel located in the promoter region (c). YP9 was used as a donor line of SPIKE gene. d-f Application of the SPIKE-01SNP marker (d), the SPIKE-03SNP marker (e), and the SPIKE-indel3 marker (f) in the intermediate breeding line. Thirteen BC2F1 plants derived from PSB Rc82 x YP9 cross were genotyped by the three markers, respectively
Fig. 7
Fig. 7
GS5 markers. a The selected DNA polymorphisms in the GS5 promoter region for marker development. The location of the 4-bp indel is 320-bp distance from the translation initiation codon. The C/T SNP was used for designing GS5-03SNP (PCR-gel-based marker) and GS5-03SNP-FD (Fluidigm genotyping marker) markers. b Capillary electrophoresis image from the parental lines analyzed by the GS5-indel1 marker. Aikawa1 and ST6 were used as donors of GS5-WG allele. The WG, MG, and NG alleles of the GS5 gene were designated as W, M, and N, respectively. c Agarose gel image analyzed by the GS5-03SNP marker from the parental lines. d Application of the GS5-03SNP marker in intermediate breeding line. Fourteen BC2F2 plants derived from PR37951 x ST6 cross were tested
Fig. 8
Fig. 8
TGW6 marker. a Agarose gel images analyzed by TGW6-1d marker from the donor line (Kasalath) and 12 recipients. The alleles of the TGW6 gene were determined by TGW6-1d-F/NR primer set detecting the non-target allele and TGW6-1d-F/PR primer set detecting the yield-positive allele. b Application of TGW6-1d marker in intermediate breeding line. Thirteen BC1F2 plants derived from PR38012 x Kasalath cross were tested
Fig. 9
Fig. 9
Genotyping of five yield-related genes from 39 rice accessions using nine Fluidigm SNP genotyping markers. The marker name with the detected-nucleotide polymorphism was shown on the top of each graph. The allele-specific primers were labeled with HEX fluorescence dye (green) for the yield-positive allele and with FAM fluorescence dye (red) for the non-target allele. After PCR on Fluidigm array, the alleles of samples were determined by fluorescent signal intensity. In each marker result, the yield-positive allele of the genes was shown with a green letter. Black spot, non-template control; gray spot, invalid call; blue spot, heterozygote

Similar articles

See all similar articles

Cited by 10 PubMed Central articles

See all "Cited by" articles

References

    1. Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M. Cytokinin oxidase regulates rice grain production. Science. 2005;309(5735):741–745. doi: 10.1126/science.1113373. - DOI - PubMed
    1. Fan C, Xing Y, Mao H, Lu T, Han B, Xu C, Li X, Zhang Q. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor Appl Genet. 2006;112(6):1164–1171. doi: 10.1007/s00122-006-0218-1. - DOI - PubMed
    1. Fan C, Yu S, Wang C, Xing Y. A causal C-A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker. Theor Appl Genet. 2009;118(3):465–472. doi: 10.1007/s00122-008-0913-1. - DOI - PubMed
    1. Fujita D, Trijatmiko KR, Tagle AG, Sapasap MV, Koide Y, Sasaki K, Tsakirpaloglou N, Gannaban RB, Nishimura T, Yanagihara S, Fukuta Y, Koshiba T, Slamet-Loedin IH, Ishimaru T, Kobayashi N. NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars. Proc Natl Acad Sci. 2013;110(51):20431–20436. doi: 10.1073/pnas.1310790110. - DOI - PMC - PubMed
    1. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser. 1999;41:95–98.

LinkOut - more resources

Feedback