Identification one major thousand-grain weight QTL TGW12 based on recombinant inbred line population crossed by wild rice (Oryza minuta) introgression line K1561 and indica rice G1025 CURRENT STATUS: ACCEPTED

Abstract Background: Rice yield is needed to improve to meet the requirement of increasing population. Narrow genetic source in cultivated rice is limiting the yield improvement. Wild rice owned valuable genes that have been lost or lacked in cultivated rice. Identification of the quantitative trait locus (QTL) for yield related traits such as thousand-grain weight (TGW) from wild rice can be used to widen the genetic source of cultivated rice. Results: In the study, sixteen TGW QTL were identified based on a recombinant inbred line (RIL) population derived from the cross of one introgression line K1561 of Oryza minuta. and one cultivated rice G1025. One largest effective QTL TGW12 was mapped to the region of 241.47 kb between the Specific Locus Amplified Fragment (SLAF) Marker2768345 and Marker2853491. The origin of TGW12 was tested by means of three markers nearby or within the TGW12 region, but it is presently uncertain whether it was originated from Oryza minuta. or not. Preliminary prediction of TGW12 indicated thirty-two Open Reading Fragments (ORFs) in the region. RT-PCR analysis and sequence alignment showed that the coding domain sequence of one MADS-box gene were different between G1025 and K1561 due to alternative slicing, which caused premature transcription termination. The MADS-box gene was considered as one putative candidate of TGW12 Conclusion: We mapped a major QTL TGW12 by RILs population derived from the cross of one O. minuta introgression line and one cultivated rice, and preliminarily concluded its candidate genes. The region of TGW12 should be further narrowed and creation of transgenic lines will reveal its function. In addition, TGW12 can be directly applied for improvement of TGW in breeding program. CKX: Cytokinin oxidase/dehydrogenase; dCAPS: Derived cleaved amplified polymorphic sequences; GW2: Ggrain width and weight 2; GW6a: Grain weight of chromosome 6; IAA: Indole-3-acetic acid; ORFs: Open reading fragments; PCR: Polymerase chain reaction; QTL: Quantitative trait locus; RIL: Recombinant inbred line; RT-PCR: Reverse transcript (RT)-PCR; SLAF: Specific locus amplified fragment; SNP: Single Nucleotide Polymorphism; SSR: Simple sequence repeats; TGW: Thousand-grain weight; WTG1: Wide and thick grain 1.

To date, QTLs/genes associated with TGW were mostly cloned from the cultivated rice. However, it is well known that the genetics of cultivated rice was becoming narrower during the process of domestication from wild rice, which is limiting the yield improvement of the cultivated rice. The genetic resource of cultivated rice is needed to be expanded for high-yield breeding. Wild rice species contain many valuable genes that can be used for genetic improvements of cultivated rice [25], and it will be an effective way to widen the genetic basis of cultivated rice by sourcing and using favorable wild rice genes.
Oryza minuta (O. minuta) is a tetraploid wild rice that possesses a number of favorable yield related genes [26]. Rahman  minuta [26]. In previous work, we also detected 28 QTLs for yield related traits by using ILs derived from the backcross of IR24 (O. sativa L) and O. minuta, and found that 46.4% of notable QTLs were from O. minuta [27].
To further identify the favorable yield related genes from O. minuta, a recombinant inbred line has been developed by crossing of K1561 to G1025 [27]. K1561 is one out of 192 ILs derived from backcross progenies (BC 4 F 2 ) of IR24 and O. minuta, which shows excellent agronomic traits such as long panicles and high TGW. G1025 is an excellent restorer line that is widely used in Guangxi Province of China with dense grains but light TGW. In this study, QTL mapping was conducted on the advanced RILs population by SSR and SLAF markers. Thirteen TGW QTLs TGWs were detected under fiver environments, and one largest effective QTL TGW12 was narrowed to 241.47 kb region based on the high-density genome map constructed with SLAF. The candidate genes of TGW12 were preliminarily concluded, and one gene encoding MADS-box protein was considered as the putative candidate based on sequence alignment. The TGW12 allele for increasing TGW might originated from O. minuta and can be directly used to yield improvement.

Plant Materials and Field Trials
The parental lines G1025, K1561 along with 201 F 6 , F 7 RILs were planted in Nanning (NN) from February to July in 2013, 2014, respectively. The parental lines along with 201 F 8 RILs were planted in NN (February to July) in 2015, and parents and F 9 RILs were planted in NN (February to July) and Wuhan (WH) from May to October in 2016, respectively. The phenotypes of parents and RILs were collected to map TGW based on SSR or SLAFs. Grain weight was calculated based on 200 grains and converted to TGW after harvesting and sun-drying. The mean values of ten plants were used as input data to identify QTLs (Table S1).

SSR, Linkage, and QTL analysis
DNA was extracted from fresh leaves following the CTAB procedure [37]. SSR markers were used to analyze a polymorphism between the parents (Table S2). SSR were synthesized according to published sequences [28]. Polymerase chain reaction (PCR) was conducted in a 15 uL volume as follow: 50 ng of template DNA, 0.3 uL of 10 mM each dNTPs, 0.5 units of Taq DNA polymerase, 1.5 uL of 10×PCR buffer with Mg 2+ , and 0.5 uL of 10 umol/L forward and reverse primers. The reaction conditions was carried out as an initial denaturation at 94 ℃ for 5 min, followed by 35 cycles of 30 s at 94 ℃, 30 s at 56 ℃, and 30 s at 72 ℃, with a final extension at 72 ℃ for 10 min. PCR products were separated on 6% polyacrylamide denaturing gels, and the bands were revealed by the silverstaining protocol [39].
Linkage was constructed by Mapmaker/Exp 3.0 [39]. Genetic distance was calculated by the Kosambi function. QTLNetwork2.2 was used to analyze QTL at a threshold of LOD 3.0 [40].

Single Nucleotide Polymorphism (SNP) genotyping, Linkage Map Construction and QTL analysis
Genomic DNA was extracted from fresh leaves of the parents and RILs by CTAB [37]. Quantified DNA was used for SLAF sequencing by an Illumina HiseqTM 2500 [41]. SLAF markers, developed in previous work, were used for genotyping, linkage map construction and QTL analysis for TGW in this study as described procedure by Zhu et al [29].
Derived cleaved amplified polymorphic sequences (dCAPS) marker development dCAPs marker was development for SLAF Marker2758157. Primers were designed according to dCAPS

Candidate genes prediction and RT-PCR analysis
The predicted genes in the target region of QTL were analyzed according to the annotation of Nipponbare reference genome2. RT-PCR was conducted as described by Sha et al. [42]. In brief, total RNA was extracted from roots, leaves, stems, shoot apical meristem, young panicles (1-5

Phenotypic analysis
TGW of the two parents G1025 and K1561 showed highly significant differences under five environments with an average of 16.01 g and 32.07 g (Table 1) (Table S1).

QTL mapping of TGW by Simple Sequence Repeats (SSR)
TGW QTLs were preliminarily detected by 300 SSR markers with evenly distribution on the 12 chromosomes. The population were F 6 , F 7 RILs derived from the cross of G1025 and K1561 planted in NN in 2013, 2014. Four QTLs TGW3, TGW7, TGW9.2, and TGW12 were stably detected on the chromosomes 3, 7, 9 and 12 in the two environments (Table 2). TGW12 had the largest effect which located on the region of RM247 and RM7003 (Table 2), so it was selected for further analysis. There were other 166 SSR markers (Table S2) in the region based on the genome sequencing data of Nipponbare [28]. The polymorphism of the 166 SSRs were firstly detected between the parental lines G1025 and K1561. As a result, nine SSRs showed polymorphism but only five displayed clear bands.
The five SSRs were further used to detect F 6 , F 7 RILs population in 2013, 2014. Finally, TGW12 was mapped to the 5.1 cM region between RM27638 and RM27748 ( Figure 2).

QTL mapping of TGW by SLAF markers
We have developed 5, 521 SLAF markers by SLAF sequencing [29]. To further map TGW QTLs, Those SLAF markers were used to screen F 8 Figure  3). TGW7 and TGW12 were both detected in three environments, and TGW9.  (Table 2). TGW12 had larger effect than TGW7, which was consistent with the results analyzed by SSR mapping (mentioned above). Further analysis for TGW12 was conducted by comparing the linkage map constructed by SSR and SLAF markers.

Evaluation of TGW12 phenotype and identification of TGW12 segment genetic origin
In order to evaluate whether the phenotypes were determined by TGW12, 16 out of the 201 RILs containing the TGW12 region were identified by means of the markers nearby the region (Figure 4).
Then, the phenotypes and genotypes of the 16 RILs were compared. All the 16 RILs with one or two segments of K1561 showed TGW increase than the recurrent parent G1025, suggesting TGW12 control TGW (Figure 4). To clarify whether the increasing effect of TGW12 was originated from O. minuta, the genotypes of G1025, K1561, IR24, and O. minuta were detected using markers nearby or within TGW12. The genotype of K1561 was the same as that of IR24 but different from that of G1025 and O. minuta on the sites of RM27638 and RM27748, which are nearby TGW12 (Figure 2, Figure 5).
However, the genotype of K1561 was the same as those of IR24 and O. minuta, but it was different from that of G1025 at the site of Marker2758157, which is within TGW12. We cannot presently draw a conclusion whether TGW12 originated from IR24 or O. minuta based on the above results. It has been suggested that translocation through centric break-fusion occurred more frequently than recombination in the introgression lines with interspecific cross, which didn't always resulted in an O. minuta chromosome arm onto a complete or incomplete O. sativa chromosome [30]. Thus, we cannot simply identify the TGW12 origination by means of single marker. It is feasible to compare sequence of TGW12 candidate among O. minuta, IR24, and K1561 once it was fine mapped.

Preliminary prediction of candidate genes for TGW12
Analysis of annotated genes indicated that 32 ORFs located in the 241.47 kb region based on Nipponbare genome annotation (http://rice.plantbiology.msu.edu) ( Table 3)

Discussion
Rice is one of the most staple food widely consumed by one-half world's people and more product is needed in the future. However, further yield improvement of rice is constrained by the narrow genetic basis of cultivated varieties. Wild rice species are good candidates for widening the genetic basis of cultivated rice due to preserved many valuably original genes [25].
O. minuta possesses a number of outstanding genes associated with resistance and yield [26]. Lots of QTLs for yield related traits have been identified using the introgression lines (IL) consisting of O. minuta segments [26,27]. In this study, sixteen TGWs were detected using the advanced RILs population under five environments (Table 2). Among them, one largest effective QTL TGW12 was located on chromosome 12. Eight QTLs (AQAG040, AQCF014, AQAG053, AQGP079, AQDR045, AQDR047, AQCY020, CQAS153) have been mapped to Chromosome 12 (http://archive.gramene.org/). Location comparison indicated that TGW12 was partially overlapped with AQDR045. TGW12 and AQDR045 were located in 4037811-6150143 and 1589200-5829185 of Chromosome 12 on the physical map of Nipponbare genome [28], respectively, suggesting there might be a major QTL controlling grain weigh in this region. However, AQDR045 was mapped by using the popu1ation derived from two cultivated rice Lemont and Teqing [32], whereas TGW12 was mapped by using the population of one O. minuta introgression line and one cultivated lines. Although it was uncertain whether TGW12 allele originated from O. minuta or IR24, it showed a great increasing effect for TGW and could be directly applied in the breeding program.
There were 32 annotated ORFs between TGW12 region, and we concluded the four ORFs encoding TFs as candidate due to their regulatory roles in plant growth and development. Sequence analysis indicated the amino acid sequence encoded by ORF17 were different between K1561 and G1025 due to AS happened, whereas there were no sequence difference for other three TFs ORF19, ORF24, and ORF27 between K1561 and G1025. So we assumed that ORF17 as one candidate of TGW12. ORF17 encoded MADS-box protein, which have highly conserved DNA-binding MADS domain and are involved predominantly in developmental processes [33]. In Arabidopsis, there are 107 genes encoding MADSbox proteins [33], and almost all of them are involved in the process of flower and seed development [34]. In rice, 75 MADS-box genes were identified, and more than 20 were transcribed during the stages of panicle and seed development [35]. In addition, alternative splicing of one MADS-box transcription factor OsMADS1 encoded by OsLG3b (Os03g0215400) controls grain length and yield in japonica rice [36]. The results suggested that ORF17 was one potential candidate of TGW12, but other ORFs located in the region could not be excluded at present.

Conclusions
We mapped a major QTL TGW12 by RILs population derived from the cross of one O. minuta introgression line and one cultivated rice, and preliminarily concluded its candidate genes. The region of TGW12 should be further narrowed to determine the exact candidate gene by developing backcross population and screening recombinant individuals. Sequence analysis of candidates will be helpful for ascertaining the origin of TGW12, and creation of transgenic lines will reveal its function.

Authors' Contributions
XL and YW conducted SLAF analysis. FY performed SSR analysis. YC and JL conducted phenotype analysis. YHC predicted the candidate genes. AS and SG designed the experiments. XL, YW, and FY wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
Not applicable.

Figure 2
Mapping of TGW12 by SSR and SLAF Markers.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Table S1.docx Table S2.docx