In the spring of 2006, we found a mutant with multi-tillers, dwarfism and semi-sterility (named as tdr) in the F₄ progeny line E20 from the cross between IR68888B (female parent) and Luxiang 90 (male parent). We also created several F1populations derived from the crosses between the tdr1 line (female parent) and other indica lines E20, 931, IR68888B and Luxiang 90 (male parents).

In order to character the mutant phenotype, the mutant tdr, its wild type, and 9311 were grown in paddy field in Mianyang, Sichuan. A total of 30 plants from each of the above three lines were used to investigate several traits including tiller number, plant height, inter-node length, kilo-grain weight and pollen fertility.

Because the phenotypes of multi-tiller, dwarfism and semi-sterility were generally associated with plant hormones, exogenous gibberellins (1 × 10⁻⁴ mol/L GA3) and sterile water as control were sprayed for the tdr line, its wild type and 9311 at elongation stage in the greenhouse. The length of panicle, 1^st, 2^nd, 3^rd, 4^th and 5^th upper internodes were measured for 10 plants each line at mature stage. Simultaneously, phytohormone levels were scored for young seedling using the ELISA kits (AndyGene) of four plant hormones including gibberellic acid (GA), cytokinin (CTK), brassinolide (BR) and strigolactone (SL) according to the corresponding protocols (http://www.andygene.com/index.php).

DNA was prepared using the modified hexadecyltrimethylammonium (CTAB) method. Ten normal plants and 10 dwarf plants from the F2 population derived from tdr and 9311 were used to create the dominant and recessive bulks, respectively. Fine mapping of TDR gene was conducted with SSR and InDel markers. InDel markers were designed according to the genomic sequences of indica line 9311 and japonica line Nipponbare.

The mutant tdr showed dwarfism, high tillering ability, small grain and low setting rate (Figure 1). Ten plants from each lines tdr, its wild type and 9311 were characterized. Compared with the wild type, the maximum number of tillers in tdr1 line is up to 81.4 and its plant height is not enough half (45.5 cm) of the plant height of its wild type (109.86 cm). Its kilo-grain weight is 15.36 gram, the setting rate is only 32.47% and lots of unstained pollens were observed.

To fully understand tillering dynamics of tdr1 line, we continuously investigated the number of tillers of 10 plants for tdr1 line, its wild type and 9311 every 5 days after transplantation. We found that the tdr1 line has rapidly increased the tiller number since June 13^th (Figure 2) and the number of tillers reached a plateau for the tdr1 line on July 8^rd. For the wild type and 9311, the number of tillers have become immobile since June 28^th, but new tillers occurred again on about August 7^th (the heading day of tdr1 line) for the tdr1 line, which indicates that the transition from vegetative to reproductive stages could inhibit the growth of axillary buds and the end of the transition phase could relieve the dormancy of axillary buds in the tdr1 line again.

In previous reports, GA plays an important role for plant height and tillering. In order to understand the effect of GA for the tdr1 line, we treated the tdr1 line, its wild type and 9311 with exogenous GA3 (1 × 10⁻⁴ mol/L). We found that exogenous GA can increase the plant height for the above three lines, but showed different effect for the three lines (Figure 3). For the tdr1 line, the 2^nd and 3^rd upper internodes were significantly elongated, but its plant height was not fully recovered. For its wild type, the length of the 1^st, 2^nd and 3^rd upper internodes were remarkably increased. For 9311, the panicle, 1^st, 2^nd, 3^rd and 4^th internodes were significantly elongated. These above results indicate that the phenotype of the tdr1 line is not caused by the signal transduction pathway of GA.

To fully understand the change of phytohormones in the tdr1 line, we measured the content of phytohormones such as gibberellins (GA), cytokinin (CTK), brassinolide (BR) and strigolactone (SL) in the tdr line and its wild type. Compared with in the wild type (GA 113.02 pmol/L, CTK 41.99 pmol/L, SL 37.08 pmol/L), the levels of GA (109.34 pmol/L), CTK (43.05 pmol/L) and SL (35.18 pmol/L) did not significantly change in the tdr1 line, but the level of BR (165.22

pmol/L) remarkably decreased in the tdr1 line (Figure 4), implying the biosynthesis of brassinolide might play roles for the phenotype of tdr1 line.

To determine the inheritance pattern of the phenotype of high-tillering, dwarfism and semi-sterility in the mutant tdr1 line, we used the tdr1 line and four normal semi-dwarf lines with different genetic background such as its wild type E20, 9311, IR68888B and Luxiang 90 to construct several F₂ populations and 1

backcross population (BC₁F₁). We scored plant height of the parent lines, their F₁, F₂ and BC₁F₁ progenies and investigated the segregation ratios of plant height in F₂ and BC₁F₁ populations (Table 1). All the F₁ plants from the four crosses showed the wild-type phenotype, and all of these F₂ progenies have a segregation ratio of 3:1 between wild-type and mutant plants (χ² < 3.84), indicating this phenotype is controlled by a recessive gene.

Genetic analysis showed that the phenotype of high-tillering, dwarfism and semi-sterility was controlled by a recessive gene. We selected the F₂ population

from the cross between 9311 and the tdr1 line to map the TDR gene. Thirty nine SSR markers showed the polymorphism between 9311 and tdr1 line in about 600 used SSR markers evenly distributed on 12 chromosomes in rice. We used these polymorphic SSR markers to perform the linkage analysis for 392 F₂ recessive plants and finally the underlying gene TDR1 was located in the 2.1 cM interval between RM252 and RM303 on Chr.4 (Figure 5, Table 2).

For fine mapping of the TDR1 gene, we further screened the polymorphic markers between RM252 and RM303 based on the Nipponbare reference genome and obtained 7 polymorphic markers in this interval. The 1620 recessive plants in the F₂ population were used to fine map the TDR1 gene. Finally, the TDR1 gene was mapped in the 105.4 kb interval between RM3288 and RM6590. Based on the Nipponbare reference genome (https://rapdb.dna.affrc.go.jp/), there are 20 annotated genes including D17/HTD1 (Os04g0550600) and OsSPL7 (Os04g0551500) (Table 3).