Two BPH populations collected in Thailand were used in this study: (1) Kalasin-2013 (KLS13) was collected in Kalasin province, northeastern Thailand in 2013 and (2) Krabi-2013 (KBI13) was collected in Krabi province, southern Thailand in 2013 (Figure 1). Both populations have been maintained in laboratory conditions for approximately 40 generations. A red-eyed mutant colony was established from a red-eyed female adult found in the KLS13 population. KBI13 was selected as a wild-type colony to cross with the red-eyed mutant colony to generate an F₂ mapping population. All BPH strains were reared on rice seedlings of cv. “TN1” and maintained in the laboratory at 26˚C ± 2˚C under a 14 L:10 D photoperiod. To generate the mapping population, a pair of insects from two different colonies was selected and crossed using single-pair mating. An F₂ population was generated from a cross of KLS13 (♀) × KBI13 (♂). The crosses were performed by placing a virgin female and a male from each colony on “TN1” seedlings

in a glass tube. After mating and oviposition, the parental insects were collected and stored in 95% ethanol. Seedlings with eggs were kept until the eggs hatched. Newly hatched nymphs of an F₁ progeny were transferred into a plastic 800 mL container containing 80 to 100 seedlings and reared to the fifth-in star nymph stage. An individual fifth-instar nymph of each sex was selected and reared in a separate glass tube to obtain unmated adults. The F₁ progenies were then allowed to inbreed to generate the F₂ generation. All offspring were then collected and stored in 95% ethanol.

Genomic DNA of the preserved samples was isolated individually using a modified version of the potassium acetate procedure of Dellaporta et al. [13] . To avoid viability problems that may occur at later stages, second- and third-instar nymph stages of F₂ offspring were collected for DNA isolation. The whole-genome amplification technique [14] was used to produce large amounts of DNA from individual insects for large-scale genotyping.

BSA with SSR markers was used to tag a red-eye mutant locus. Based on the presence of eye color of the BPH, we generated two groups of 29 brown-eyed (B) and 29 red-eyed (R) progenies from the F₂ population. A total of 387 SSR markers from Jairin et al. [9] and Jing et al. [10] covering 14 chromosomes of the BPH were selected to identify the R and B groups. The genetic linkage map was calculated by Join Map 4 [15] using genotype data from 95 F₂ individuals derived from crosses of KLS13/KBI13. Grouping of markers was performed using a minimum-independence LOD (logarithm of odds) threshold of 5.0. Groups were converted into linkage maps using the regression algorithm provided by Join Map with a recombination frequency of less than 0.45, and then Kosambi’s mapping function [16] was used to calculate map distances. Linkage maps were drawn using the Map Chart software for Windows [17] .

A red-eyed female adult BPH was isolated from a field population collected from a farmer’s rice field in KLS to establish a red-eyed mutant colony. The colony was used as genetic material for mapping the red gene. The mutant individuals could be easily discriminated from the brown-eyed individuals (Figure 2). Individuals in the mutant colony had the red eye phenotype at all developmental stages. We observed the red eye phenotype in both macropterous and brachypterous adults, as well as adults with both brown and black body colors. Except for eye color, no other external differences were observed between the colonies (Figure 3).

Cross mating between the homozygous normal brown-eyed KBI13 (♀) and red-eyed mutant KLS13 (♂) produced F₁ offspring with wild-type brown eye color. The F₁ offspring from the reciprocal crosses also showed only normal brown eye color. Inbreeding between heterozygous F₁ progenies resulted in F₂ offspring with brown eye color and red eye color at the expected ratio of 3:1 for a single recessive gene inheritance. The sex ratio in the offspring produced from most crosses was equal based on a chi-square test analysis (Table 1). These results suggest that the inheritance of the red eye mutant phenotype is controlled by a single autosomal recessive gene same as the previous findings [2] [3] .

To detect the map position of the red locus, BSA was employed to identify SSR markers linked to the red eye mutant phenotype. Two bulks from F₂ offspring were generated, each consisting of 29 individual brown-eyed and red-eyed second and third in star nymphs. A total of 387 SSR markers distributed throughout 14 chromosomes were used to screen the pair of bulks. Only SSR markers on sex chromosomes were not selected for the analysis. The primer BM20 generated amplification products for heterozygous alleles in the B bulk and homozygous alleles in the R bulk. The brown-eyed F₂ offspring consisted of nymphs that were homozygous or heterozygous for the mutant allele, while the red-eyed group consisted of only nymphs that were homozygous at the locus. This result indicated that the red gene was linked to BM20 in the F₂ population.

We employed an additional 20 SSR markers surrounding the BM20 locus on chromosome 9 [10] to assay 95 F₂ offspring. To obtain accurate estimates of marker positions on the map, we only selected clearly polymorphic markers and carefully scored them to reduce errors. Unfortunately, many markers could not be scored, mostly due to banding patterns that were too difficult to interpret, including multiple bands and bands that did not show clear polymorphism on the 6% polyacrylamide gel. Of 20 SSR markers tested, only three markers showed clearly distinguishable polymorphisms and were easy to score. A genetic linkage map was constructed based on the segregation data. Finally, the red gene was mapped to a location between SSR markers BM20 and NLGS182 (Figure 4) on chromosome 9 according to Jing et al.’s map [10] or to the linkage group 2 according to our previous map [9] . This is the first study to successfully locate the red gene in the genome of this important insect pest of rice using marker- based genetic mapping. We have demonstrated that BSA coupled with SSR markers can

*F₂ offspring from this cross were used as a mapping population. All offspring were collected at the 2^nd-3^rd in star nymphal stages.

easily and rapidly identify DNA markers linked to the red gene. The results from genetic crosses and genetic inheritance from the present and previous studies [2] [3] revealed that the red eye color is inherited as a simple Mendelian autosomal recessive gene. The location of the red gene on BPH chromosome 9 confirms that the red eye mutation is located on an autosome.

The red-eyed mutant colony has the potential for utilization in BPH research as a visible genetic marker and can be applied in various studies of genetics and ecology at a molecular level (e.g., population dynamics and genetics, evolution of compound eyes, visual acuity, migration, mating behavior, transgenic and RNAi technologies [7] ). A xanthommatin-related gene, karmoisin (Nlka), reported to be involved in eye mutations in some insect species [18] , was expected to cause the red eye mutation phenotype in the BPH. Liu et al. [7] identified the karmoisin gene from the BPH and found that the red eye phenotype of the BPH has no close connection with this gene. To identify the gene related to the red eye mutation, map-based cloning is one of the most effective approaches for cloning the red gene. Our finding will facilitate fine-scale mapping experiments to isolate the genes responsible for the red-eye mutation in the BPH through genotyping-by-sequencing approaches.