MATERIALS AND METHODS

MATERIALS AND METHODS

Rice germplasm

A total of 208 indica P/TGMS lines were selected for analysis from the National Mid-term

Genebank for Rice, Hangzhou, China (Supplemental Table 2), and 100 typical indica and 100

japonica accessions to identify the indica/japonica component of them were also from the

National Mid-term Genebank for Rice, Hangzhou, China (Supplemental Table 5).

DNA extraction and SSR genotyping

Total genomic DNA was extracted from tender shoots following a modified sodium dodecyl

sulphate ‘mini-extraction’ protocol developed by Zheng et al (1995). DNA extracts were

quantified using a spectrophotometer, quality checked by electrophoresing in 1.5% agarose gel

and finally dissolved in 0.1× Tris-EDTA buffer solution prior to storing at -20 ºC. According to

NY/T1433-2014 (2014), a total of 48 randomly distributed SSR loci were chosen from the 12 rice

chromosomes. Detailed information is provided in Supplemental Table 3. Primers were

synthesized by ABI (Applied Biosystems, Foster City, CA, USA). Forward primers were labelled

with blue (FAM), yellow (NED), red (PET) and green (VIC) fluorophores. PCR amplification and

product testing were performed according to Wang et al (2013, 2014). DNA fragment size analysis

and allele calling were performed using GeneScan and GeneMapper software (Applied

Biosystems), followed by manual allele binning. In addition, reference lanes were further verified

using polyacrylamide gel electrophoresis. In the case of no amplification, PCR experiment was

repeated to exclude technical failure prior to recording it as a null allele.

Statistical analysis

PowerMarker (Liu and Muse, 2005) was used to calculate the number of alleles per locus (Na),

Nei’s genetic diversity index (He), as well as the polymorphism information content (PIC).

Genetic distance was calculated using the LogSharedAllele distance, DLS (Ranajit and Li, 1993).

Phylogenetic reconstruction was based on the neighbor-joining (NJ) method implemented in

PowerMarker. The model-based program STRUCTURE (Pritchard et al, 2000; Falush et al, 2003;

Dent et al, 2012) was used to calculate the genetic component for each accession using a burn-in

length of 10 000, run length of 100 000, with the model accommodating admixture and correlated

allele frequencies. To infer the population structure, we used both, the LnP(D) value and Evanno’s

DK (Evanno et al, 2005) based on five independent simulations. We also used NTSYTS-pc

version 2.10e (Rohlf, 1997) to perform the principal coordinate analysis (PCA), which

summarized the major patterns of variation in the multi-loci dataset. To investigate the group

relationships, Fst value was calculated and tested using FSTAT (Goudet, 2001).

Reference:

Dent A. Earl and Bridgett M. vonHoldt. 2012. STRUCTURE HARVESTER: A website and program for

visualizing STRUCTURE output and implementing the Evanno method. Conse Gene Res, 2: 359–361.

Evanno G, Regnaut S, Goudet J. 2005. Detecting the number of clusters of individuals using the software

STRUCTURE: A simulation study. Mol Ecol, 14: 2611–2620.

Falush D, Stephens M, Pritchard J K. 2003. Inference of population structure using multilocus genotype data:

Linked loci and correlated allele frequencies. Genetics, 164: 1567–1587.

Goudet J. 2001. FSTAT, version 2.9.3, A Program to estimate and test gene diversities and fixation indices.

Lausanne University, Lausanne, Switzerland.

Liu K J, Muse S V. 2005. PowerMarker: An integrated analysis environment for genetic marker analysis.

Bioinformatics, 21: 2128–2129.

NY/T1433-2014. 2014. Protocol for identification of rice varieties: SSR marker method. China, Beijing: China

Agriculture Press. (in Chinese)

Pritchard J K, Stephens M, Donnelly P. 2000. Inference of population structure using multilocus genotype data.

Genetics, 155: 945–959.

Ranajit C, Li J. 1993. Determination of relatedness between individuals using DNA fingerprinting. Human Biology,

65: 6.

Rohlf F. 1997. NTSYS-pc: Numerical taxonomy and multivariate analysis system, version 2.00. Exeter Software,

Setauket, New York.

Wang C H, Xu Q, Yu P, Yuan X P, Yu H Y, Wang Y P, Tang S X, Wei X H. 2013. Comparison of Cheng’s index-

and SSR marker-based classification of Asian cultivated rice. Rice Sci, 20(2): 103–110.

Wang C H, Zheng X M, Xu Q, Yuan X P, Huang L, Zhou H F, Wei X H, Ge S. 2014. Genetic diversity and

classification of Oryza sativa with emphasis on Chinese rice germplasm. Heredity, 112(5): 489–496.

Zheng K L, Subudhi P K, Domingo J, Magpantay G, Huang N. 1995. Rapid DNA isolation for marker assisted

selection in rice breeding. Rice Genet Newsl, 12: 255–258.