ARMS Molecular Marker for Identifying Long Mesocotyl Genotype gy1Kasa in Rice (Oryza sativa)
LI Hao-Shu1,*, CHANG Yuan2,*, LIU Chun-Mei3, ZHANG Yu-Han1, YANG Yi-Rong1, QIN Guan-Nan3,4,**
1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
2 College of Life Sciences, Capital Normal University, Beijing 100048, China;
3 Key Laboratory of Agro-Ecological Processes in Subtropical Region / Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China;
4 University of Chinese Academy of Sciences, Beijing 100049, China
Abstract：Rice (Oryza sativa), one of the most important food crops, feeds more than 50% of the world's population. Mesocotyl is the first internode of rice seedlings when grown in dark. Rice varieties with longer mesocotyl will come up out of soil easier under direct seeding conditions. In previous studies, 5 quantitative trait loci (QTLs) regulating the length of mesocotyl have been identified from 'Kasalath' (O. sativa ssp. indica cv. Kasalath) with long mesocotyl, one of which has been fine mapped into gaoyao1 gene (GY1) (LOC_Os01g67430). Compared with dominant GY1Nip from 'Nipponbare' (O. sativa ssp. japonica cv. Nipponbare), a G→T mutation occurred at 376 bp of the coding region of recessive gy1Kasa in 'Kasalath', which resulted in the elongation of the mesocotyl. gy1Kasa exists in a variety of natural germplasm resources, all of whom exhibits long mesocotyl phenotype. In order to promote the application of gy1Kasa in breeding of rice suitable for direct seeding, a molecular marker was developed by Amplification Refractory Mutation System (ARMS) to identify the functional nucleotide polymorphism (FNP) of GY1. The molecular marker, denominated as gy1fnp, contained 4 primers located in the conserved flanking region of SNP376. A 470 bp fragment could be amplified by gy1fnpaf and gy1fnpar in both genotypes; a 194 bp fragment could only be amplified in homozygous gy1Kasa (FNP site was T) by gy1fnpbf and gy1fnpar; a 315 bp fragment could only be amplified in GY1Nip (FNP site was G) by gy1fnpaf and gy1fnpbr. All 3 fragments could be amplified in heterozygous genotype. The results of agarose gel electrophoresis showed that different genotypes of GY1 could be distinguished accurately and clearly. Sanger sequencing showed that the difference of banding patterns was caused by SNP376. Investigation of F2 population of 'Kasalath' / 'Nipponbare' showed that homozygous gy1Kasa identified by gy1fnp linked with long mesocotyl phenotype. Taken together, it was concluded that gy1fnp developed in this study could accurately distinguish 2 alleles and heterozygous genotypes of GY1 recognized by SNP276 using only one PCR reaction followed by agarose gel electrophoresis with convenience, rapidness and low cost. Marker gy1fnp could promote the application of gy1Kasa in direct seeding rice breeding by molecular Marker Assisted Selection (MAS). Marker gy1fnp could also identify the genotype of GY1 in rice germplasm resources quickly, which was of great significance for genetic analysis and breeding practice. In addition, the successful development of gy1fnp was helpful to reduce genetic noise caused by GY1 and facilitated map based on cloning of other mesocotyl genes.
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