Evaluation and GWAS of radicle gravitropic response in a core rice germplasms population

Gravitropism is one of the primary determinants of root development, facilitating root penetration into soil and subsequent absorption of water and nutrients. In this study, the gravitropism of 226 Chinese rice micro-core accessions and drought-resistant core accessions were assessed. The average value of gravitropic response speed of radicle roots was 41.05°/h in the population ranging from 16.77°/h to 62.83°/h. We observed a highly signicant difference in gravity response speed between Indica (42.49°/h) and Japonica (39.71°/h) subspecies with p-value < 0.002. The correlation analysis showed that the gravitational response speed of radicle roots was signicantly positively correlated with the number of deep roots (correlation coecient = 0.16), the growth speed of radicle roots (correlation coecient = 0.21) and the drought resistance coecient (correlation coecient = 0.14). Using genome-wide association analysis (GWAS), 4 QTLs(quantitative traits) associating with gravitropic response speed were identied on chromosome 4,11 and 12. From within these intervals 5 candidate genes were screened for qPCR verication in 6 extreme rice varieties, demonstrating that gene LOC_Os12g29350 may regulate gravitropism negatively and conrming it’s candidacy for further study. providing found to be positively corelated with the deep roots and drought resistance. Five candidate genes have been chosen for further verication by qPCR in 6 extreme varieties, and LOC_Os12g29350 was higher expressed in the slow gravitropic response varieties. Some known QTLs of roots traits and drought resistance located nearby the associated QTLs identied in this study, which conrmed the close relationship between radicle gravitropism and the drought resistance.


Introduction
Scientists have been trying to understand gravitropism for more than 120 years (Darwin and Darwin 1880). Gravitropism is the orientation of growth in response to gravity, which is necessary for roots to grow into soil, to acquire water and nutrients and to anchor plants, providing stability and preventing lodging.
The starch-statolith hypothesis and the Cholodny-Went theory attempt to explain some aspects of gravitropism . The starch-statolith hypothesis proposes that the starch-lled amyloplasts of gravity-sensing cells act as statoliths, signalling the direction of gravity by their sedimentation. The Cholodny-Went theory indicated, gravi-bending is the result of differential accumulation of auxin on opposite sides of the elongation zone, resulting in differential growth and tip curvature. In addition, mechanosensitive ion channel hypothesis also could explain some parts of the gravitropism . Rice is a staple food for nearly half of the world's population. It has a typical brous root system. In rice, some mutants and genes related to gravitropism have been identi ed. LAZY1 gene controls rice shoot gravitropism through regulating polar auxin transport ), but the primary roots of lazy1 mutants show normal gravitropism and circumnutation ). Aem1 mutant causes defects in root development and gravity response ). Overexpression of OsRAA1 effects root development and root response to gravity ).
The mechanism of gravity sensing in plants is one of the most fascinating questions in molecular biology, and because of the new availability of high-throughput sequencing and phenotyping technology, we can expand our knowledge of this trait through association analysis. For example, in the common bean, Liao et al mapped the QTLs controlling basal root gravitropism ). Using a mapping population derived from a Bala × Azucena, two main QTLs for gravitropic response have been mapped to chromosome 6 and 11 ).
Measurement of gravitropism related traits with a throughput lending itself to the sample size required for association analysis is now more feasible with tools such as the ROTATO with an automated camera, that could help researchers to dissect the gravi-response .
Despite progress made in the past decades, processes involved in positive root gravitropic response in the root tip still remains largely unclear in rice. Because root gravitropism is widely believed to be regulated by a tipping-point mechanism , the gravitropic response speed could be represented by the bending angle of the seminal in agar-lled Perspex chambers after rotation as Uga and Price et al did ). Here, we measured gravitropic response speed in an association mapping population , and identi ed several QTLs related to gravitropic response that can be deployed into marker assisted selection programmes.

Results
Gravitropic response speed of radicle roots In this study, we detected the bending angle of radicle roots of 266 core rice accessions representing their gravitropic response speed. Among these accessions, the average value of gravitropic response speed of radicle roots was 41.05°/h. The fastest speed was 62.83°/h, meanwhile the slowest speed was 16.77°/h. The standard deviation and coe cient of variation were 6.42°/h and 15.63%, respectively. As shown in Fig. 1, the gravitropic response speed of radicle roots generally presented a normal distribution and was mostly distributed between 31°/h to 51°/h, accounting for 89.4% of the total accessions. This indicated that this set of data was suitable for association mapping analysis.
Comparison of gravitropic response speed between Indica and Japonica rice Notable differences of gravitropic response speed were found between Indica and Japonica rice ( Table 1). The gravitropic response speed of Indica rice was mostly distributed between 33.5°/h to 53.5°/h, accounting for 94.7% of the Indica accessions. As for the Japonica rice, it was mostly distributed at 33.5°/h to 48.5°/h, accounting for 90.3% of the Japonica accessions. The gravitropic response speed of Indica (42.49°/h) was faster than that of Japonica (39.71°/h). Likewise, the range of variation of Indica (46.06°/h) was larger than that of Japonica (39.91°/h). The standard deviation and coe cient of variation of Indica and Japonica accessions were 6.41°/h and 6.30°/h, 15.32% and 15.77%, respectively. Compared the gravitropic response speed of Indica and Japonica rice by student's (t) test resulted in a p value of inequality at 0.002. This result suggested that there was signi cant difference in the gravitropic response speed of Indica and Japonica subspecies, and the gravity response speed of Indica radicle roots was signi cantly faster than that of Japonica rice. Correlation among gravitropic response speed, deep rooting phenotype and drought resistant index Multiple measurements for root phenotypes of these 226 accessions have been measured in a previous study . The ratio of the yield in dry elds to the yield in paddy elds was calculated as the yield-based drought resistant index (DRI). The original data was obtained from previous studies in our laboratory ). And correlations were calculated between these measurements and the gravitropic response measurements to determine if these traits could be inherently linked. By comparing the correlation coe cient between gravitropic response speed and some agronomic traits (Table 2), we observed that the gravitropic response speed was signi cantly positively correlated with tiller number (TN), deep roots (DR), growth speed of radicle roots (GSR) and drought resistant index (DRI) with correlation coe cients of 0.13, 0.16, 0.22 and 0.14, respectively. The results indicated that the speed of the gravitropic response of radicle roots was highly signi cantly positively correlated with the speed of radicle roots growth. The faster the gravitropic response, the faster the growth speed. At α = 0.05, the speed of the gravitropic response of radicle roots was also positively correlated with TN, DR and DRI. The faster the response speed, the larger the deep roots number as well as the drought resistant index. There was no signi cant correlation between the speed of gravitropic response and plant height (PH), shallow roots (SR), ratio of deep roots (RDR) or roots per tiller (R/T). Drought resistant index (DRI) 0.14 * Note: "*"means signi cance at P < 0.05,"**"means signi cance at P < 0.01.
12 accessions with the fastest speed of gravitropic response were selected for further study, and the growth speed of radicle roots and DRI of this subset were measured (Supplementary Table 1). Among them, the DRI of Xiaohonggu, IAC1246 and Zaohandao was 1.06, 1.18 and 1.89, respectively. These 3 accessions could be chosen as donor parental lines for drought-resistant breeding in the future.

GWAS of the gravitropic response speed of radicle roots
The raw sequence data of this population have been uploaded to public databases: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA260762 and ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR123/SRR12 39601 . GWAS was carried out to associate gravitropic response speed to a responsible genomic location using the EMMA model. 4 signi cantly associated QTLs were detected at the threshold of p = 10 − 5 (Fig. 2, Table 3). 2 QTLs named qGRS4.1 and qGRS4.2 were mapped on the chromosome 4 (404424750 bp and 404774373 bp), and other 2 QTLs named qGRS11 and qGRS12 located on each of chromosomes 11 and 12 (1123451244 bp and 1217439378 bp).  Based on gravitropic response speed, the fastest and slowest 3 extreme accessions were selected respectively from the 226 accessions. The fastest 3 extreme accessions were Zaohandao(F1), C22(F2) and Xianggu(F3), and the slowest were BLCO.BRANCO(S1), IPEACO162(S2) and Gaoyangdiandao(S3). The head SNP (1217439378) of qGRS12 indicated different allele types between 2 groups of accessions, that all three slowest accessions with the SNP type of G, while all three fastest accessions with the SNP type of both A and G. The expression patterns of the ve genes in these 6 accessions were shown in Fig. 3. This demonstrate that the expression level of LOC_Os12g29350 in the three accessions with the fastest gravitropic response speed was much lower than that in the three accessions with the slowest gravitropic response speed, indicating that this gene may be involved in the negative regulation of gravitropic response. The expression level of ETR2, RPA49, OsGIF1 and RLCK341were low in the 6 accessions, and no signi cant difference was found between the two types of rice with opposite gravitropic response speed.

Discussion
This natural mapping population has been re-sequenced and soundly assessed on root morphological characteristics and drought related traits in our previous research ). Therefore, it is a good resource to study the relations between gravitropic response and other important agronomic traits, and to explore the genes controlling roots gravitropism.
The gravitropic response of radicle is primarily controlled by genetic factors but is also signi cantly in uenced by environmental conditions ). Therefore, producing uniform and homogeneous growth environment is the precondition to carry on such experiments. However, it remains challenging to maintain an even soil environment to observe the hidden half from the soil. Despite there will be obvious difference of the root phenotype of seeds growing in the agar and soil, this agar-based screening system still may re ect a biological process, and it has been extensively used to study gravitropic responses ). Compared with the method used in other labs(Norton and Price 2009; Uga et al. 2013), we modi ed the measurement. Before the sowing, all the seeds have to be screened and cold soaked to stratify the seeds resulting in normalized germination vigour. Then, the bending angle was recorded after a shorter period of 1 hour after 90° rotation, that is not only to save time but also to detect early variation in the trait more profoundly. Because the rst 1 hour is the most e cient time in gravi-bending, after then the gravitropic response reduces down rapidly. In order to gain an accurate representation of this trait, 50 seeds per accession were used in this study. After removal of non-germinating/infected seeds/ odd roots distribution a minimum of 20 valid samples per accession were assessed.
Root growth angle is an important trait that in uences the ability of rice to avoid drought stress (Uga et al. 2015a, b), because the deep roots help plants to absorb water from deep soil. The gravitropic response determines the shape of the root system, especially in the vertical dimension. As we found in this study, the gravitropic response speed signi cantly positively correlated with the number of deep roots and the drought resistant index. The varieties that have better gravitropic response would therefore infer better drought tolerance. This means that the gravi-bending angle could become an early indicator to predict plants' drought resistance in a cost effective high-throughput manner. Here we highlight three varieties that express the desirable gravitropism trait along with drought resistance that could be promising resources for drought resistant breeding and research (supplementary table1, bold).
There are 11 known QTLs, that function in roots morphology and drought resistance, close to the 4 associated SNPs' physical position in the genome (http://qtaro.abr.affrc.go.jp/, ±1 Mb) ( Table 5). One of them, QTL 11 − 1 controlling root thickness and number of roots past 100 cm near the associated SNP on chromosome 11, was found to be co-segregated with marker C189 . And C189 also co-segregated with a radicle root morphology QTL -SRM11 identi ed by Price et al in the same mapping population ). This interval is therefore very important in the development of the root morphology. The other 10 QTLs are all related to drought resistance, three of which are located on chromosome11, and 7 were on the chromosome 12 ). We can conclude that the gravitropic response speed is corelated with drought resistance indeed.

Conclusion
This study modi ed the assessment method of radicle gravitropic response to be more e cient and precise. Using a natural population that already has plenty of root and drought resistance data, 4 signi cant associated QTLs were identi ed by GWAS. The trait of radicle gravitropic response speed found to be positively corelated with the deep roots and drought resistance. Five candidate genes have been chosen for further veri cation by qPCR in 6 extreme varieties, and LOC_Os12g29350 was higher expressed in the slow gravitropic response varieties. Some known QTLs of roots traits and drought resistance located nearby the associated QTLs identi ed in this study, which con rmed the close relationship between radicle gravitropism and the drought resistance.

Plant material
The association population used in this study is composed of 131 rice accessions from the mini-core collection of Chinese rice germplasm along with 95 rice accessions from core drought-resistance core rice germplasm collection. Of these 133 accessions are Indica rice and 93 accessions are Japonica rice. All rice seeds were provided by Shanghai Agrobiological Gene Center and harvested in the same season.

Evaluation of root gravitropism
Based on the root gravitropic curvature experiments described by Uga and Price et al. , the gravitropic response speed of radicle roots was measured with some modi cation. The growth direction of the root tip was rst marked when the radicle root grew to 1-2 cm. The root tip was then rotated from the normal vertical axis to the horizontal axis by rotating the agarose plate by 90 degrees. Now under the effect of gravity, the growth direction of radicle root tip was observed, and its position marked again after a growth period of 1 hour. The angle between the two marked root tip growth directions was recorded as the gravitropic response speed. The ratio of radicle root length to the growth period, starting at the date of sowing, was recorded as the radicle root growth speed. The gravitropic response speed of each panel member was calculated after removing the outliers. At least 20 valid seeds for each accession were used to calculate for its average gravitropic response speed.
There were 7 steps to evaluation of root gravitropism, and the details were shown as tip was marked. The root length and bending angle of the root tip was measured according the two tangent lines, and gravitropic response speed and growth speed of radicle roots were then calculated as described above.

Genome-wide association study
To perform basic statistical analysis on the phenotypic traits, we calculated the average value, standard deviation, coe cient of variation and correlation coe cient as well as to make frequency distribution graph.
The GWAS analysis conducted via the e cient mixed-model association (EMMA) method which is available within the Genome Association According to the GWAS results, the annotation information of all genes within the range of 200 kb on the two anks of the lead associated SNP loci was analyzed and the genes whose function are known to may be relate to roots development were selected. Additionally, a further selection step was carried out using transcriptome data of rice root , where genes that were highly expressed in roots were preferentially selected for further analysis.

RNA extraction and expression veri cation
To determine if selected candidate genes were differentially expressed between lines in the population, qPCR was conducted. A total of 6 accessions were used for expression veri cation, with three extreme accessions with the fastest and three with the slowest gravitropic response speed selected from the association population of 226 rice accessions. The radicle roots were sampled when they grew to 1-2 cm and were ash frozen in liquid nitrogen, then stored at -80 °C for later use. The total RNA of 10 pooled radicle roots was extracted using the TRNzol reagent (TIANGEN), and cDNA was synthesized by EasyScript®One-step gDNA Removal and cDNA Synthesis SuperMix following the manufacturers protocol (TransGen Biotech). Primer Premier v5.0 was used to design primers using the genome sequence of Nipponbare as a sequence reference (Supplementary Table 2), the target fragment lengths were expected to be between 150 bp − 250 bp. Real time quantitative PCR was performed in 96-well plates with an Applied Biosystems CFX96 Real-Time PCR Detection System using TransStart Top Green qPCR SuperMix (TransGen Biotech). All assays were carried out in triplicate or greater and the expression levels were calculated using the relative quantitation method (ΔΔCT).

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The genetic data of this population can be downloaded from http://www.ncbi.nlm.nih.gov/bioproject/PRJNA260762 and ftp://ftptrace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR123/SRR12 39601.

Competing interests
The authors declare that they have no competing interests.  Figure 1 Frequency distribution of gravitropic response in the natural rice population. The X axis is the bending angle of radicle for the rst hour after rotation 90.

Figure 2
Manhattan plots of GWAS of gravitropic response speed. The threshold is p=10-5.  The detailed process of the experiment measuring gravitropic response speed.