QTL Mapping for Hull Thickness and Related Traits in Hybrid Rice Xieyou 9308

Rice Science, 2014, 21(1): 29−38 Copyright © 2014, China National Rice Research Institute Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(13)60156-0

QTL Mapping for Hull Thickness and Related Traits in Hybrid Rice Xieyou 9308 LUO Li-li1, 2, #, ZHANG Ying-xin1, #, CHEN Dai-bo1, ZHAN Xiao-deng1, SHEN Xi-hong1, CHENG Shi-hua1, CAO Li-yong1 (1National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310036, China; 2College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China; #These authors contributed equally to this paper)

Abstract: We conducted a quantitative trait locus (QTL) analysis of 165 rice recombinant inbred lines derived from a cross between Zhonghui 9308 (Z9308) and Xieqingzao B (XB) in Hainan and Hangzhou, China. Grain thickness (GT), brown rice thickness (BRT), hull thickness (HT) and milling quality were used for QTL mapping. HT was significantly and positively correlated with GT and BRT. Twenty-nine QTLs were detected with phenotypic effects ranging from 2.80% to 21.27%. Six QTLs, qGT3, qBRT3, qBRT4, qHT6.1, qHT8 and qHT11, were detected repeatedly across two environments. Inherited from XB, qHT6.1, qHT8 and qHT11 showed stable expression, explaining 9.92%, 21.27% and 10.83% of the phenotypic variances in Hainan and 9.61%, 6.40% and 6.71% in Hangzhou, respectively. Additionally, the QTL cluster between RM5944 and RM5626 on chromosome 3 was probably responsible for GT and milling quality. The cluster between RM6992 and RM6473 on chromosome 4 played an important role in grain filling. Three near isogenic lines (NILs), X345, X338 and X389, were selected because they contained homozygous fragments from Zhonghui 9308, corresponding to qHT6.1, qHT8 and qHT11, respectively. The hull of XB was thicker than those of X345, X338 and X389. In all the lines, qHT6.1, qHT8 and qHT11 that regulated rice HT were stably inherited with obvious genetic effects. Key words: rice; hull thickness; milling quality; QTL mapping

Rice is not only a model organism for monocotyledons, but also an important food crop. Rice grain consists of a hull reciprocally hooked by the lemma and palea, inner and outer bran layers, and brown rice (Yang, 2005). And the grain weight is determined by the volume and shape of the hull that acts in a sink capacity (Venkateswarlu and Visperas, 1987). Rice hull plays important roles in preventing damage and maintaining humidity during the development of brown rice (Zhou et al, 2003; Abebe et al, 2004). Additionally, it has been reported that the percent of hull is negatively correlated with the milling quality (Jongkaewwattana and Geng, 2001). Thus, the shape, capacity and thickness of the hull are important aspects affecting yield and milling quality. However, there has been little regard for rice hull thickness (HT) in previous studies. The developing rice hull consists of an outer epidermis, skin tissues, soft tissues and endepidermis,

Received: 10 March 2013; Accepted: 28 June 2013 Corresponding author: CAO Li-yong ([email protected])

and its thickness is mainly determined by the soft tissues (Yang, 2005). No genetic assays for genes or QTL controlling HT have been conducted previously. There are two types of cloned genes for grain size and hull mutants that might relate to rice HT. Several genes regulating grain size have been cloned, such as DTH8 (Wei et al, 2010), GS3 (Fan et al, 2006), SRS5 (Segami et al, 2012), GIF1 (Wang et al, 2008), gw5 (Weng et al, 2008), GW2 (Song et al, 2007), GS5 (Li et al, 2011), qGW8 (Wang et al, 2012) and qSW5 (Wan et al, 2005). Among these, most of the genes act in regulating cell division or extension, resulting in the alteration of grain size. For example, DTH8/GHD8 encodes a putative HAP3 subunit of the CCAAT-boxbinding transcription factor that regulates yield, plant height and flowering time (Zhang et al, 2006; Wei et al, 2010). GS3 was reported to encode 232 amino acids with a putative PEBP-like domain, a transmembrane region, a putative TNFR/NGFR family cysteine-rich domain and a VWFC module. It is involved in regulating seed length, stigma length and stigma exsertion (Fan et al, 2006; Noriko et al, 2011). These two genes might

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also function in regulating rice hull development. Genetic studies of rice hulls always focused on rice hull mutants affecting flower development. Specifically, a palea formation controlling gene, depressed palea 1 (dp1), which regulates palea formation and floral organ number, has been cloned on chromosome 6 (Jin et al, 2011). The DP1 gene encodes a nuclear-localized AT-hook DNA binding protein, resulting in a primary defect in the main structure of the palea. The stunted lemma palea 1 (slp1) rice mutant displays severely degenerated lemmas/paleae, and SLP1 is localized at a 46.4-kb genomic region containing three putative genes, OsSPL16, OsMADS45 and OsMADS37 (Wang et al, 2011). Two other genes, G1 and OsMADS1, were also reported to regulate rice hull development (Jeon et al, 2000; Yoshida et al, 2009). Additionally, three genes controlling hull color, black hull 4 (Bh4), gh1 and gh2 were reported (Tobias and Chow, 2005; Zhu et al, 2011; Hong et al, 2012). Until now, little study associated with rice HT has been conducted. The objective of this study was to establish a measuring method for HT and conduct a genetic assay on rice HT, grain thickness (GT) and brown rice thickness (BRT) using QTL analysis. Additionally, the relationships of HT, GT, BRT with milling quality, brown rice rate (BR), milled rice rate (MR) and head rice rate (HR) were also used for association analysis and QTL mapping.

MATERIALS AND METHODS Rice materials A total of 165 recombinant inbred lines (RILs) derived from Xieyou 9308, the cross between Zhonghui 9308 (Z9308, a typical indica restorer) and Xieqingzao B (XB, an indica maintainer with 25% genes from japonica, http://www.ricedata.cn/variety/), were used. The trials were conducted in the experimental fields of the China National Rice Research Institute, Hangzhou (30.3° N, 20.2° E), Zhejiang Province, and Lingshui (18.2° N, 108.9° E), Hainan Province, China, in 2011, respectively. RILs and the two parental lines were sown in December 2011 and grown under natural conditions in Hainan. Field trials were conducted in randomized complete blocks with two replicates. The F1 hybrid of Z9308 × XB was backcrossed with Z9308 three times to produce the BC4F1 population. The BC4F2 lines were selected from the BC4F1 selfing population by marker-assisted selection (MAS). Using

gained near isogenic lines (NILs) based on the background of XB, rice hull QTLs were validated in the BC4F2 generation. Phenotypic evaluation Forty days after heading, four plants from the individual lines were harvested. Two panicles from each plant were selected for the evaluation of GT, BRT and HT. Grains in the middle of the panicle were threshed and dropped into water, and finally the submerged grains were gathered. Ten grains were selected randomly for evaluation. The remaining harvested panicles from each line were mixed and threshed for milling quality analysis. The water content of the sample was approximately 12% as measured by moisture analyzer (MB35, Switzerland). Milling quality, containing BR, MR and HR was measured against a national reference standard. GT and BRT were measured using a Vernier Caliper (TESA CAL IP67, SWISS) and the HT was calculated as follows: HT = ∑ (GT – BRT) / (2 × 10). Scanning electron microscope analysis For the two parental lines, spikelets were sampled from the middle of the panicle every 5 d after the heading day until 25 days after heading (DAH). A scanning electron microscope (SEM) examination was conducted following the protocol reported by Li et al (2010) with some modifications. In brief, fresh rice spikelets from the two parents were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0). The fixed samples were rinsed three times for 15 min each time with phosphate buffer, and they were fixed overnight with 1% OsO4 in phosphate buffer at 4 °C, and then washed three times for 15 min each time in the phosphate buffer and dehydrated through an ethanol series of 50%, 70%, 80%, 90%, 95% and 100% for 15 min with each step. Subsequently, they were incubated in 1:1 ethanol-isoamyl acetate mixture for 30 min and transferred to pure isoamyl acetate for 1 h. Finally, the samples were dried to a critical point with liquid CO2 and then coated with gold-palladium before mounted for observation. They were photographed under an SEM (Hitachi TM-1000 Tabletop Microscope, Japan). The thickness of the palea and lemma in the photos was determined using Adobe Photoshop CS6. The dynamic graph was drawn with Microsoft Office Excel 2007. QTL analysis QTL analysis was conducted using the composite

LUO Li-li, et al. QTL Mapping of Rice Hull Thickness

interval mapping (CIM) method with the software Windows QTL Cartographer V2.5 (http://statgen.ncsu. edu/qtlcart/WQTLCart.htm). The CIM analysis was run using Model 6 with a forward and backward stepwise regression, a window size of 10 cM, and a step size of 1 cM. Experiment-wide significance (P < 0.05) thresholds for QTL detection were determined with 500 permutations. A LOD score of 2.5 and significance level of 0.01 were used as thresholds to declare the presence of putative QTL. The location of a QTL was described according to its LOD peak location and the surrounding region with a 95% confidence interval. QTL validation Three NILs were constructed under background of XB using an MAS strategy. Genomic DNAs from the BC4F2 population and two parents were extracted from fresh leaves following the modified CTAB method. The PCR amplification reaction for verifying QTL was as follows: denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 5 min. The PCR products were separated by 8% polyacrylamide gels. After electrophoresis, the amplified DNA bands were visualized using silver staining. A total of 29 new InDel markers were developed in this experiment (Supplemental Table 1).

RESULTS HT development of Z9308 and XB To investigate the HT of Z9308 and XB in micro-

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structures clearly, rice hulls from the initial heading to maturity were observed under an SEM (Fig. 1). The dynamic development process of HT is shown in Fig. 2. Both the lemma and palea of Z9308 were thicker than that of XB on the heading day. Both the lemma and palea thicknesses of XB increased quickly after heading, inducing significantly larger values than those of Z9308 at 5 DAH. From 5 to 25 DAH, the thicknesses of XB remained stable and larger than those of Z9308. From 5 to 25 DAH, the thicknesses exhibited a slight decline, probably due to loss of water and accumulation of silicon. The thicknesses of the lemmas in Z9308 and XB were 67.9 and 72.8 µm on 25 DAF, respectively, showing a significant difference at the 0.05 level. However, the thickness of the paleae in Z9308 and XB were 67.3 and 70.1 µm on 25 DAF, respectively, which was not significantly different. Phenotypic variation For six traits across two environments, the phenotypic values of the RILs and the two parents are shown in Table 1 and Fig. 3. For GT, BRT and HT, XB showed significantly larger values than Z9308, but in verse for BR, MR and HR (Fig. 3). There may be a negative correlation between grain size and milling quality. XB (111.8 µm in Hainan and 97.7 µm in Hangzhou) showed significantly thicker hull than Z9308 (96.3 µm in Hainan and 79.1 µm in Hangzhou), which corresponds to the results in Fig. 1. The values for all six traits showed a continuous and normal distribution across two environments, although there was some transgressive segregation. Thus, the correlation analysis and QTL mapping were necessary to investigate genes

Fig. 1. Scanning electron microscope images of dynamic development process of lemmas and paleae in Zhonghui 308 (Z9308) and Xieqingzao B (XB). DAH, Days after heading; el, Exocuticle layer; ct, Cortex tissue; tt, Tender tissue; ea, Endocuticle layer.

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Fig. 2. Change trends of rice hull thickness. Error bars represent the standard errors.

of quantitative inheritance. Correlation analysis of six traits The correlation analysis was conducted among six traits across the two environments (Table 2). HT, GT and BRT were positively correlated with each other (P < 0.01) across the two environments, indicating the thicknesses of hull, grain and the brown rice rate were probably inherited from the same genetic factors. For milling quality, BR, MR and HR also showed significantly positive correlations with each other (P < 0.01). Unfortunately, there was no significant correlation between HT and milling quality as expected across the two environments (P < 0.05). The correlation between GT and MR was unstable across the two environments,

and the same was true for BRT and BR. It is necessary to continue studying the correlation between milling quality and GT/BRT. In conclusion, there was a stable correlation among HT, GT and BRT, as well as BR, MR and HR. QTL analysis Twenty-nine QTLs, located on chromosomes 2 to 12, were detected under the two environments. The LOD values of all QTLs ranged from 2.80 to 15.53 (Table 3). Chromosomal locations of detected QTLs were present in Fig. 4. The percentage of variance explained (PVE) by each QTL varied from 3.94% to 21.27% (Table 3). Details were as follows: I) Among seven QTLs detected for GT, the qGT3 was detected in both

Table 1. Phenotypic values of recombinant inbred lines (RILs) in two environments. Hangzhou Hainan Mean ± SD Range Mean ± SD Range GT (µm) 1 903.6 ± 79.5 1 685.0–2 177.0 1 973.6 ± 72.2 1 818.0–2 281.0 BRT (µm) 1 740.0 ± 72.9 1 540.0–1 992.0 1 785.3 ± 67.4 1 635.0–2 095.0 HT (µm) 81.8 ± 6.3 66.5–98.5 94.1 ± 6.6 76.0–110.5 BR (%) 78.3 ± 2.9 51.2–81.8 76.9 ± 2.7 59.2–81.3 MR (%) 68.5 ± 3.7 31.6–73.3 69.4 ± 3.2 53.2–77.3 HR (%) 58.7 ± 8.9 15.9–71.2 45.4 ± 11.9 12.4–67.8 GT, Grain thickness; BRT, Brown rice thickness; HT, Hull thickness; BR, Brown rice rate; MR, Milled rice rate; HR, Head rice rate. Trait

Table 2. Correlation among six traits in two environments. Trait

Hangzhou

Hainan

GT BRT HT BR MR HR GT BRT HT BR MR GT 1.000 1.000 BRT 0.989** 1.000 0.985** 1.000 HT 0.563** 0.434** 1.000 0.462** 0.303** 1.000 BR 0.134 0.141 0.026 1.000 0.189* 0.219** -0.082 1.000 MR -0.148* -0.163* 0.011 0.843** 1.000 0.062 0.081 -0.073 0.795** 1.000 HR -0.119 -0.128 -0.014 0.549** 0.669** 1.000 -0.104 -0.093 -0.096 0.264** 0.493** GT, Grain thickness; BRT, Brown rice thickness; HT, Hull thickness; BR, Brown rice rate; MR, Milled rice rate; HR, Head rice rate. * and **, Significant at the 0.05 and 0.01 levels, respectively.

HR

1.000

LUO Li-li, et al. QTL Mapping of Rice Hull Thickness

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75.4

Fig. 3. Frequency distribution of six traits in recombinant inbred lines (RILs) in two enviroments (Hainan and Hangzhou). Z 9308, Zhonghui 9308; XB, Xieqingzao B.

Hainan and Hangzhou, with PVEs of 10.29% to 12.77% and additive effects of 2.57 to 2.73. II) For BHT, seven QTLs, qBRT2, qBRT3, qBRT4, qBRT6, qBRT7, qBRT11 and qBRT12, were detected. Among these, the mediumeffect qBRT3 was detected under both environments,

with PVEs of 10.02% to 11.52% and additive effects of 2.31 to 2.50. The minor effect of qBRT4 was measured under both environments, with PVEs of 5.56% to 6.27% and additive effects of -1.64 to -1.77. III) For HT, five QTLs were detected. Among these, three

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QTLs, qHT6.1, qHT8 and qHT11, were observed under both environments. qHT6.1 showed PVEs ranging from 9.61% to 9.92% and had additive effects of 2.05 to 2.26. qHT8 showed the PVEs ranging from 6.40% to 21.27% and additive effects of 1.66 to 3.17. qHT11 showed the PVEs ranging from 6.71% to 10.83% and additive effects of 1.72 to 2.30. IV) For BR, three QTLs were measured. qBR2 was detected in Hangzhou, with a PVE of 8.84% and an addictive effect of -0.53. V) For MR, two QTLs, qMR3 and qMR7, were detected in Hangzhou, explaining 9.37% and 5.28% of phenotypic variances, with additive effects of -0.87 and 0.62, respectively. VI) For HR, five QTLs, qHR3.1, qHR3.2, qHR7.1, qHR7.2 and qHR10, were detected. qHR3.1, qHR3.2 and qHR7.2 were detected in Hangzhou, with PVEs of 5.81%, 6.00% and 7.90%, and addictive effects of -2.02, 1.96 and 2.22, respectively.

QTL validation To evaluate QTLs for HT, the BC4F2 population was subjected to MAS based on mapping results. Three individual lines (X345, X338 and X389) were obtained from the BC4F2 population, and their HT was significantly less than that of XB (Fig. 5). X345, X338 and X389 possessed the critical homozygous region that overlaps qHT6.1, qHT8 and qHT11 (Fig. 6). In all, qHT6.1, qHT8 and qHT11 were stably inherited with an obvious genetic effect that regulated rice hull thickness.

DISCUSSION HT is an important trait Rice hull limits the grain size, which is a main factor affecting yield (Yang, 2005). In this study, HT showed

Table 3. QTLs for hull thickness and related traits detected in recombinant inbred lines (RILs) in two environments. Trait GT

QTL qGT2 qGT3

Marker

Chr

RM106 RM6283

2 3

LOD

PVE (%)

A

Environment

4.18 5.96 -1.92 Hangzhou 5.85 10.29 2.57 Hangzhou 4.28 12.77 2.73 Hangzhou qGT4 RM3319 4 3.79 5.78 -1.85 Hangzhou qGT6.1 RM6734 6 3.30 6.20 1.81 Hainan qGT6.2 RM3827 6 4.71 7.27 2.08 Hangzhou qGT7 RM2 7 3.54 5.05 1.74 Hangzhou qGT11 RM1812 11 4.88 8.96 2.18 Hainan BRT qBRT2 RM106 2 4.02 5.86 -1.72 Hangzhou qBRT3 RM6283 3 7.27 11.52 2.50 Hangzhou 5.27 10.02 2.31 Hangzhou qBRT4 RM3319 4 3.59 6.27 -1.77 Hangzhou 3.00 5.56 -1.64 Hangzhou qBRT6 RM3827 6 4.08 6.21 1.77 Hangzhou qBRT7 RM2 7 2.83 3.99 1.42 Hangzhou qBRT11 RM1812 11 4.43 8.00 1.94 Hainan qBRT12 RM5479 12 3.20 9.55 -2.09 Hainan HT qHT6.1 RM587 6 7.30 9.92 2.26 Hangzhou 4.71 9.61 2.05 Hainan qHT6.2 RM6302 6 3.34 3.94 1.43 Hangzhou qHT7 RM3235 7 3.08 6.34 -1.66 Hainan qHT8 RM8243 8 15.53 21.27 3.17 Hangzhou 3.11 6.40 1.66 Hainan qHT11 RM1812 11 8.61 10.83 2.30 Hangzhou 2.97 6.71 1.72 Hainan BR qBR2 RM324 2 4.78 8.84 -0.53 Hangzhou qBR5 RM6972 5 2.80 5.95 0.68 Hainan qBR9 RM6491 9 3.26 8.24 0.80 Hainan MR qMR3 RM282 3 4.93 9.37 -0.87 Hangzhou qMR7 RM234 7 2.97 5.28 0.62 Hangzhou HR qHR3.1 RM282 3 3.16 5.81 -2.02 Hangzhou qHR3.2 RM168 3 3.30 6.00 1.96 Hangzhou qHR7.1 RM7454 7 3.88 8.11 3.46 Hainan qHR7.2 RM70 7 3.59 7.90 2.22 Hangzhou qHR10 RM271 10 3.47 9.34 3.87 Hainan GT, Grain thickness; BRT, Brown rice thickness; HT, Hull thickness; BR, Brown rice rate; MR, Milled rice rate; HR, Head rice rate; Chr, Chromosome; LOD, Logarithm of odds; PVE, Percentage of total phenotypic variance explained by the QTL; A, Additive effect. Bold font represents QTL detected across two environments. The symbol “-” before the additive effect value represents contribution from Zhonghui 9308.

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Fig. 4. Chromosomal locations of detected QTLs.

QTLs denoted with blank mark were detected in Hangzhou (HZ) and black in Hainan (HN). GT, Grain thickness; BRT, Brown rice thickness; HT, Hull thickness; BR, Brown rice rate; MR, Milled rice rate; HR, Head rice rate; Chr, Chromosome.

significant correlations with GT and BRT, corroborating the results of the previous study. Milling quality is a complex quantitative trait. In breeding practices, indica varieties with good milling quality always have thin hulls. However, in the RILs derived from Xieyou 9308, no significant correlation was found between HT and milling quality. Thus, the negative correlation between HT and milling quality in XB and Z9308 was probably not real due to the complex genetic composition of milled quality. Meanwhile, rice hull plays a very important role in rice flower development (Kellogg, 2001; Preston et al,

2009). In previous studies, several hull developmentrelated genes were cloned from various mutants, such as slp1, dp1 and G1 (Yoshida et al, 2009; Wang et al, 2011; Jin et al, 2011). However, hull mutants with inferior phenotypes could not be used for breeding. Hence, it is necessary to investigate the QTLs controlling rice hull development. Since no attempt has been made to determine QTL for HT, a QTL analysis was performed to map quantitative genes in this study. QTLs for rice hull thickness For HT, five QTLs were detected. Among these,

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** **

**

Fig. 5. Rice hull thickness of Xieqingzao B (XB) and three near isogenic lines (X345, X338 and X389). ** means significantly difference between XB and the corresponding line (P < 0.01).

Fig. 6. Graphical genotypes of Xieqingzao B (XB) and three near isogenic lines (NILs) with target QTL from Zhonghui 9308 (Z9308). Twelve chromosomes are represented by 12 pairs of bars. The horizontal lines on each chromosome indicate positions of marker loci. White squares show the chromosomal segment of XB, black ones of Z9308 and gray of heterozygous genotype. 1, InDel 85; 2, RM190; 3, RM5556; 4, InDel 113; 5, RM286; 6, InDel 144.

qHT6.1, qHT8 and qHT11 were determined across the two environments, indicating stable expression in Hainan and Hangzhou. qHT6.1 inherites from the thicker XB explained approximately 10% of phenotypic variance. It is tightly linked with the famous glutinous

endosperm (Wx) and physically corresponded to depressed palea1 (dp1) (Sano, 1984; Jin et al, 2011; Su et al, 2011; http://www.gramene.org/). If the qHT6.1 is a variant allele of dp1, it will be useful for theoretical research and breeding programs. Thus, the relationship of qHT6.1 and dp1 should be further investigated. The second QTL, qHT8 was located between RM126 and RM515 on chromosome 8, representing 21.27% and 6.40% of PVE in Hangzhou and Hainan, respectively. On chromosome 8, two other QTLs, qGW8 and DTH8, were identified using a map-based cloning strategy (Zhang et al, 2006; Wang et al, 2012). qGW8 physically located at the interval harboring qHT8 (http://www.gramene.org/) is a positive regulator of cell proliferation in grain that might also be responsible for hull development. Owing to the major effect of qHT8 in Hangzhou, it should be backcrossed to NILs for further study. To determine the allelic relationship of qHT8 and qGW8, gene sequencing and expression analysis will be conducted in XB and Z9308. The QTL qHT11 was mapped to the interval from RM286 to RM167 on the short arm of chromosome 11 in both Hangzhou and Hainan, with PVEs of 6.71% to 10.83%. In the same interval of qHT11, a small and round seed 5 (SRS5) was identified with the function of altering the lemma cell length (Segami et al, 2012; http://www.gramene.org/). It will be interesting to determine if SRS5 is the putative gene of qHT11 regulating the development of hulls in XB and Z9308. Three near isogenic lines (X345, X338 and X389) of the BC4F2 population were selected because they possessed homozygous fragments from the genotype of Z9308 that overlapped with the QTLs qHT6.1, qHT8 and qHT11. Moreover, the HT of XB was greater than those of X345, X338 and X389. In all, qHT6.1, qHT8 and qHT11 were stably inherited with obvious genetic effects that regulated rice HT. QTL clusters for the other five traits Although the 29 QTLs detected were distributed across 11 chromosomes, some QTLs tended to cluster on the short arm of chromosome 3 and the long arm of chromosome 4 across the two environments. On the short arm of chromosome 3, qGT3, qBRT3, qMR3 and qHR3.1 clustered between RM5944 and RM5626. Among these, qGT3 and qBRT3 were inherited from XB and detected across the two environments. However, qMR3 and qHR3.1 were detected in Hangzhou with an additive effect from Z9308. We can conclude that there was a stably expressed locus

LUO Li-li, et al. QTL Mapping of Rice Hull Thickness

responsible for thinner grains and better milling quality from Z9308. Residing between RM5499 and RM5626 (physically locating at 12 to 24 Mb on chromosome 3), the GRAIN SIZE 3 (GS3, 16 Mb on chromosome 3) was reported to regulate seed length, stigma length and to participate in stigma exsertion (Fan et al, 2006; Noriko et al, 2011). Thus, GS3 is probably the putative gene of this clustered QTL. Further investigation is necessary to validate the GS3 allele in Z9308 and XB. It will also be interesting to study the effect of GS3 on milling quality. On the long arm of chromosome 4, qBRT4 was detected under the two environments, while qGT4 was only detected in Hangzhou. Residing at the same interval as qBRT4 and qGT4, GIF1 plays an important role in the grain-filling process (Wang et al, 2008). Thus, this locus that controls GT and BR was probably allelic with GIF1, which validates the accuracy of mapping results of this study. Additionally, two other cluster loci were located on the long arm of chromosome 6 and the short arm of chromosome 7, respectively. No known genes reside in these two loci. However, they were only detected in Hangzhou. Thus, it is necessary to validate the existence of these two environmentally dependent QTLs. The correlation analysis showed that the development of HT was coordinated with the GT and BR, but not with milling quality. Thus, it is possible that HT is not an important element responsible for the milling quality in Xieyou 9308.

CONCLUSIONS In conclusion, 29 QTLs were determined using RILs for rice hull thickness related traits and milling quality. Among these, three stably expressed QTLs associated with the thickness of rice hull were detected on chromosomes 6, 8 and 11. qHT8 and qHT11 physically corresponded to DTH8 and SRS5, which both regulate grain size. The QTL cluster between RM5944 and RM5626 on chromosome 3 probably harbors GS3, which might affect grain thickness and milling quality. In addition, GIF1 might be the putative gene of qBRT4 and qGT4 with a function of grain filling. However, the alleles of DTH8, SRS5, GS3 and GIF1 in XB and Z9308 require validation by gene expression analysis and sequencing. X345, X338 and X389 possess the critical homozygous region that overlaps qHT6.1, qHT8 and qHT11. In further study, it will be necessary to fine map qHT6.1, qHT8 and qHT11.

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ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 31071398 and 31101203), and the National Program on Super Rice Breeding, the Ministry of Agriculture, China.

SUPPLEMENTAL DATA The following materials are available in the online version of this article at http://www.sciencedirect.com/ science/journal/16726308; http://www.ricescience.org. Supplemental Table 1. InDel markers used in marker assisted selection.

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