[picture] | Phyton- International Journal of Experimental Botany | [picture]

DOI: 10.32604/phyton.2021.014880

ARTICLE

Fine Mapping and Candidate Gene Prediction of the Quantitative Trait Locus qPL8 for Panicle Length in Rice

Lin Zhang¹,²,*, Yiting Zou¹, Zhong Bian¹, Dong Xie¹, Hadi Yeilaghi¹, Xiaolei Fan¹,², Dongsheng Zhao¹,² and Qiaoquan Liu¹,²,*

1 Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou, 225009, China
2 Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou, 225009, China
Corresponding Authors: Qiaoquan Liu. Email: qqliu@yzu.edu.cn; Lin Zhang. Email: zhangl@yzu.edu.cn
Received: 04 November 2020; Accepted: 24 November 2020

Abstract: Rice panicle is the sink organ where assimilation product accumulates, and its morphology determines the rice yield. Panicle length has been suggested as a yield-related trait, but the genetic factor for its control is still limited. In this study, we carried out fine-mapping of qPL8, a QTL identified for panicle length in our previous work. Near isogenic line (NIL) with qPL8 exhibited elongated panicle without obvious effect on other panicle elements. With five key recombinants from NIL population, the locus was finally narrowed down to a 278-kb region, where 44 genes are annotated. By comparing the genomic sequence of two parents, 17 genes were identified with SNPs or InDels variations in the coding region. Expression analysis showed that eight genes were up-regulated in the NIL with qPL8. Considering both the coding variation and expression status, several candidate genes for the locus were identified, and OsMADS37 was raised as the most possible candidate. Interestingly, an expression QTL (eQTL) also resides in the locus, leading to a cluster of gene expression variation in the region. This study will facilitate the application of qPL8 locus in rice breeding for yield potential.

Keywords: Rice; QTL; panicle length; fine mapping; qPL8

1  Introduction

Rice (Oryza sativa L.) is a major food crop in the world, feeding over half of the global population. As sink organ, panicles are the key determinant for yield improvement. Several components make up the panicle, including panicle length, panicle branch number (primary and secondary) and panicle density, determining the final grain number per panicle [1,2]. In natural varieties, the panicle morphology is highly diversified and belongs to the quantitative traits, which is contributed by multiple loci/genes [3]. Breeding elite variety relies on those loci. However, excellent panicle traits are mainly selected empirically by the visual observation in traditional breeding, which is laborious and difficult to combine elite loci together. Therefore, it is valuable to unveil the genetic loci responsible for those panicle elements and use the information to facilitate marker-assistant selection (MAS) breeding.

The release of both japonica and indica genome information provided rich sequence polymorphisms for marker development [4,5], and in combination of robust analytical algorithm, the genetic dissection of quantitative trait loci (QTL) has become a common approach [6]. Up to now, many QTL loci have been identified to confer panicle morphology [7], however, most of them are located in large genomic region and the genetic effect is ambiguous in primary mapping populations, limiting the utilization of those loci in rice MAS. Therefore, it is necessary to confirm the effects of relative loci and then finely map and clone those genes. Developing near-isogenic lines has been a good strategy for QTL confirmation and for fine mapping with derived populations [8]. Even for the minor QTL, the NIL population could enlarge its genetic effect and the variance it explained, and map the QTL as single mendelian factor [9]. With this strategy, many QTLs have been finely mapped and constrained to a narrow genomic region, providing essential information for exploring the candidate genes [10].

By now, several important QTLs for panicle morphology have been cloned, such as Gn1a for grain numbers, Ghd7 and Ghd8/DTH8 for panicle size, dep1 for panicle density and erection and IPA1/WFP for panicle primary branch number, all of which showed the potential to increase rice yield [11–17]. Gn1a encodes a cytokinin oxidase that degrades the phytohormone cytokinin, and its reduced expression causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs [11]. Ghd7 encodes a CCT domain protein, which delays flowing time and increases panicle size by suppressing downstream Ehd1 expression under long-day conditions, so does Ghd8/DTH8, a gene encoding the OsHAP3 subunit of HAP complex [12–14]. dep1 is a gain-of-function mutation, causing truncation of a phosphatidylethanolamine-binding protein-like domain protein. The mutant can enhance meristematic activity, resulting in a reduced length of the inflorescence internode, an increased number of grains per panicle and a consequent increase in grain yield [15]. IPA1 encodes the OsSPL14, an SBP domain transcription factor, and higher expression of OsSPL14 in the reproductive stage promotes panicle branching and rice grain yield [16,17].

The mechanistic explanation of those genes facilitates our understanding of panicle development and the utilization of them in rice breeding [3]. However, identification of new locus should enrich the pathway and provide more choices for panicle shape adjustment. In our previous report, we identified two QTLs for panicle length from the BC3F3 populations, namely qPL6 and qPL8, and finely mapped the qPL6 locus [18]. Here, we report the validation and fine-mapping of qPL8 using the NIL population, and the candidate gene(s) was predicted according to the coding sequence variance and gene expression difference.

2  Materials and Methods

2.1 Plant Materials

As shown in our previous report, near-isogenic lines for qPL8 were developed from the BC3F6 generation derived from the cross of Nipponbare and WS3, a landrace bearing large panicle and strong culm [18]. The NILs with reciprocal qPL8 alleles were planted in the Shanghai experimental station for QTL confirmation and trait evaluation. For easy citation of the two lines, they are named NILqPL8+ and NILqPL8– hereafter, and plus and minus indicate the presence and absence of the superior qPL8 allele. To narrow down the QTL region, progenies of heterozygous NIL plants were planted for recombinant identification. Then, selected recombinants and their progenies were planted for phenotype confirmation. To evaluate the impact of qPL8 and qPL6 on expression of MADS-box genes, we also planted the NILs with four allele combinations of the two locus as previously reported [18].

2.2 Agronomic Trait Evaluation

To confirm the effect of qPL8 on agronomic traits, eight agronomic traits were evaluated by NIL comparison, including panicle length (PL), panicle primary branches number (PBN), panicle secondary branches number (SBN), spikelet number per main panicle (SPP), seed setting rate (SR), plant height (PH), tiller number (TN) and stem diameter of internode 3 (SD). For the progenies of key recombinants, PL from the main culm of each individual was obtained. The methods for these traits evaluation followed the previous report [18].

2.3 Marker Development for QTL Fine Mapping

Eight new InDel markers that evenly distributed in the qPL8 region were designed for QTL fine mapping with the software Primer premier 5.0 according to the InDel information from two parents, WS3 and Nipponbare. With these markers, five recombinants dividing the QTL region to different parts were identified. To clarify the borders of key recombinants, one additional InDel and three dCaps markers were developed. The progenies of those recombinants were genotyped with markers located in the heterozygous region, and homozygous plants with reciprocal genotypes were obtained for phenotype confirmation. The markers used in QTL fine mapping are listed in Tab. S1. To clarify the NILs genetic background, 276 polymorphic markers were used as previously reported [18].

2.4 DNA Extraction and PCR Condition

Genomic DNA of WS3 was extracted by the CTAB method with slight modification. DNA for NIL genotyping and recombinants selection was extracted by the previous method [19]. PCR for InDels and dCaps markers followed the same procedure described as before [18].

2.5 Identification of Sequence Variation

The WS3 genomic DNA was subjected to library construction for paired-end sequencing, and clean data was obtained after removing adapter and low quality sequence. Then the clean reads were aligned to the Nipponbare reference genome (version_7.0), and SNPs and InDels information were extracted by SOAPsnp [20] and SAMtools [21] separately. According to the genome annotation information, SNPs and InDels in coding sequence of mapping region were identified.

2.6 RNA Extraction, RT-PCR and qRT-PCR

To analyze gene expression in the mapping region, young panicles (1–2 cm long) from the NILs were sampled and total RNA was extracted by the TRIpure Regent (Aidelai, Beijing) following the manufacturer’s instruction, which was then reverse-transcribed by iscriptTM cDNA sythesis kit (Bio-Rad). Primers spanning the introns of candidate genes were designed for RT-PCR analysis at proper cycles, and Ubiquitin was amplified as internal control. The genomic DNA was also amplified to confirm the primer quality. The candidate genes LOC_Os08g41950 (OsMADS7) and LOC_Os08g41960 (OsMADS37) were subjected to qRT-PCR analysis, and OsActin was amplified as internal control. All the primers used for expression analysis are listed in Tab. S2.

3  Results

3.1 Effect Validation of qPL8

In our previous report, the qPL8 was identified as a QTL for panicle length, and it has no pleiotropic effect on other traits. To further validate the effect of qPL8, we planted the NILs with reciprocal alleles and performed a detailed trait analysis. As shown in Fig. 1, no visual morphological difference can be observed for the whole plant between the NIL lines (Fig. 1a), but difference in panicle length can be observed by close-up inspection of the panicle (Fig. 1b). The observation was confirmed by the statistical analysis, and in addition to panicle length, difference in plant height was also identified at significant level, but no difference was detected for other traits (Tab. 1). With 276 polymorphic markers evenly distributed on 12 chromosomes, genotypes of two NILs were clarified. The result showed that all the genetic backgrounds of the NILs were homozygous and only the region at qPL8 locus was different. The region covers about 2000-kb physical interval flanked by markers RM23466 and RM3120, laying the foundation for fine mapping (Fig. 2).

Figure 1: Phenotype of NILqPL8− and NILqPL8+. (a) The gross view of the whole plants at mature stage. (b) Close-up view of the panicles from main culms

Table 1: Agronomic traits comparison between NILqPL8− and NILqPL8+

Figure 2: Graphical genotype of NILqPL8− and NILqPL8+. All the chromosomes that include introgression from parent WS3 are shown. The white and black bars indicate the region of Nipponbare and WS3, respectively. Markers that confine the borders of introgression are labeled. Dotted boxes indicate the position of qPL8

3.2 Fine Mapping of qPL8

By genotyping the NIL derived population, five recombinants were identified to divide the qPL8 region to several parts (Fig. 3a). The progeny lines of these recombinants were subjected to trait measurement. If the locus resides in the heterozygous region, the progeny with different genotypes would exhibit difference in panicle length, otherwise, the locus should reside in the homozygous region. Accordingly, R1 denoted qPL8 to the left of the marker qPL8ID-7, and R2 and R3 confirmed the result and further denoted the locus to left of the marker qPL8ID-6. Finally, R4 and R5 confined the locus between markers qPL8ID-1 and qPL8ID-4, harboring a ~318-kb genomic region. Then, the breaking point of R4 and R5 were clarified by four new markers, and the qPL8 was finally mapped to a 287-kb interval flanked by markers qPL8dCaps1 and qPL8dCaps3 (Fig. 3b).

Figure 3: Fine mapping and candidate gene prediction of qPL8. (a) R1-R5 indicate the recombinants that divide the qPL8 region. Eight InDels markers were used to denote the breaking point, and the numbers between markers indicate physical interval in kb. Black and white bars indicate the homozygous region from parents WS3 and Nipponbare, and gray bar indicates the heterozyougs region. The progeny sibling lines of each recombinants with reciprocal genotypes from the heterozyous region were measured for panicle length (cm), and t-test were performed for difference comparison of two genotypes (n = 16). The resulting p-values were showed on the right with p < 0.01 as significance threshold. (b) Candidate genes in the 287-kb mapping region. All the 44 annotated genes are plotted along the mapping region according to the gene position and gene size, and genes with and without sequence variation are highlighted by the red and blue boxes respectively. Genes with expression difference were labeled with asterisks under corresponding boxes

3.3 Coding Variance of Candidate Genes

According to MSU rice genome annotation project database (http://rice.plantbiology.msu.edu), 44 genes are predicted in the mapping region (Fig. 3b). Mutations on coding regions usually have effect on the phenotype. We then compared the mapped genomic region between two parents to identify sequence variations that lead to amino acid change. The sequencing depth of parent WS3 is about 39×, which provides high fidelity for the sequence variation. In all, 23 SNPs and 5 Indels were identified on the protein coding region (Tabs. S3 and S4), which distributed on 17 genes of the mapping region (Fig. 3b). None of the SNPs produces mutation as premature termination. Among the five InDels, two of them are not in triplex form, which should change the predicted protein largely. A 2-bp deletion was identified in one of the two transcripts from LOC_Os08g41950, a gene encoding the MADS-box family protein (OsMADS7), and an 8-bp insertion was identified in the gene LOC_Os08g42060, which was annotated as expressed protein.

3.4 Expression Variation of the Candidate Genes

In addition to coding variation, expression difference would also contribute to the phenotype change, and we further compared the expression of candidate genes between the NILs. As panicle length was the major difference between the NILs, the gene expression analysis was performed with young panicles in the stage 5 of eight rice inflorescence stages defined by Ding Ying, the father of rice in China. Among the 44 genes, eight were annotated as transposon elements (TEs) and two were hypothetical proteins without expression evidence. The ten predicted genes were removed from the candidate list. For the rest genes, we succeeded in designing primers except for LOC_Os08g42240, which is rich in repeat element and could also be a putative TE. The RT-PCR results showed that eight genes were up-regulated in the NIL with superior qPL8 allele (Fig. 4), and five of them also bear coding sequence variation (Fig. 3b). Therefore, the candidate genes for qPL8 are suggested to be among the 19 genes with either coding variation or expression difference (Tab. 2). We also found that four of the 19 candidate genes including the gene with 8-bp insertion were not expressed in the young panicle (Fig. 4 and Tab. 2), reducing their possibility as the causative genes. Among the eight differentially expressed genes, expression difference of LOC_Os08g42310 and LOC_Os08g42320 was obvious between the NILs, and the expression could be hardly detected in NILqPL8− (Fig. 4). Interestingly, five of the six additional genes with expression difference are located tightly around the two genes (Fig. 3b), making it possible that this region affects gene expression and functions as a cis-expression quantitative trait locus (cis-eQTL).

Figure 4: RT-PCR analysis for 33 genes in the mapping region. The result is confirmed by four independent experiments and representative one is showed with gene IDs. cDNAs from young panicles of NILqPL8− and NILqPL8+ were amplified for different genes at suitable cycles as showed. UBI denotes the amplification of ubiquitin. Arrows designate the genes with expression difference

Table 2: The information of candidate genes with sequence variation or expression difference

3.5 MADS-Box Genes Might Function in Panicle Length Control

Although many candidate genes were identified, we suggested that the MADS-box genes might be the most likely candidate genes for phenotype variation, as such genes have been repeatedly reported to participate in inflorescence and floret development [22]. In the mapping region, two MADS-box genes, LOC_Os08g41950 (OsMADS7) and LOC_Os08g41960 (OsMADS37), were identified. Although there was a large deletion in the predicted transcript 1 of OsMADS7, we could not detect this transcript in both NILs. Amplification of transcript 2 of OsMADS7 was successful, but no difference was detected between the NILs (Fig. 4). These results suggested the deletion in OsMADS7 might have little effect on gene function. For OsMADS37, no coding variation was identified, but its expression was elevated significantly in NILqPL8+. Expression profile of two MADS-box genes were confirmed by qRT-PCR, and NILs with qPL6 background were also subjected to analysis (Fig. 5). Interestingly, expression of OsMADS7 was elevated by qPL6 background, suggesting a role of OsMADS7 in mediating the panicle length control by qPL6. In an analogous manner, the up-regulation of OsMADS37 might contribute to the qPL8-mediated panicle length control.

Figure 5: Quantitative RT-PCR for OsMADS7 (a) and OsMADS37. (b) Characters in parentheses indicate the NILs with superior qPL6 allele. Error bars represent S.D. of three replicates

4  Discussion

As a panicle component, panicle length is usually measured as the yield-related trait, and many QTL for this trait was identified throughout the 12 chromosomes in rice [23,24], but few of them have been finely mapped. In our previous study, we identified a PL QTL on chromosome 8, and named it qPL8 [18]. However, the QTL interval for qPL8 was too large, making it hard to identify the candidate gene and apply it to rice breeding. In this study, we clarified the background of two NILs, and made sure that only the QTL region is different, therefore the difference between the NILs should only be caused by the QTL region. In addition to the panicle length, we also found the region had slight effect on the plant height. The field test confirmed that the PL trait conferred by qPL8 is stable among different seasons, making fine mapping of the QTL feasible. During NIL development, the heterozygous plants were used to develop population for QTL fine mapping directly. Similar to the process of NIL development, each recombinant was selfed to produce sibling lines with reciprocal genotypes, which made phenotypes determination more accurate. Accordingly, we succeeded in narrowing down the QTL to 278-kb interval, demonstrating the availability of the strategy. However, we could not further narrow down the mapping interval due to low recombination rate in the region, similar to the case of Ghd7 [12]. Nevertheless, the present result still facilitates the prediction of the underlying gene for qPL8.

Recent advance in sequencing technology has enabled easy re-sequencing of multiple rice genomes, which facilitates identification of functional variations in elite varieties [25–27], and it has been used to predict candidate genes in genome wide association studies (GWAS) [28]. In this study, we provided high-quality coding variation in the mapping region to predict the candidate genes by re-sequencing the parent WS3, and found rich SNPs and deletions in this region. Although we could not exclude the possibility that small variant might lead to phenotype change as showed by some study [29], expression variation is likely the alternative mechanism under the qPL8 phenotype, given that obvious expression difference are usually associated with the tissues showing phenotypes [30,31]. Therefore, we further qualify the candidate gene list of qPL8 by expression analysis.

By surveying the published function, we suggested the two MADS-box genes as the most likely gene(s) for PL control. Although the expression of OsMADS7 was not affected by qPL8, it is under control of qPL6, reflecting the roles of MADS-box genes in panicle elongation. Similarly, the OsMADS37 was greatly up-regulated by the qPL8, reminiscent of the OsMADS7 up-regulation by qPL6. Several transgenic studies have been performed to elucidate the function of OsMADS7 [32,33]. In the OsMADS7-RNAi lines, no obvious phenotype was observed, but silencing of both OsMADS7 and its homolog OsMADS8 caused severe morphological alterations of floral organs. Interestingly, PL was greatly decreased by knocking down OsMADS1/5/7/8 simultaneously [32], and ectopic over-expression of OsMADS7 led to early flowering and dwarf phenotype [33]. OsMADS37 is one of five MADS-box genes that belong to the MIKC* subgroup, however, its biological function is still unknown [34]. It would be worthy to clarify the effect of two MADS-box genes on PL by over-expressing them with native promoter.

It is interesting that a cluster of genes were up-regulated in the fine mapping region of qPL8. We therefore postulate that the qPL8 region harbors an eQTL that affects gene expression in cis, which was conceptualized recently [35]. Genome-wide eQTL analysis has been performed in rice, linking the transcript variation to the genomic loci [36]. It has been reported that cis-control of gene expression has important roles in crop domestication [30,31,37,38], and understanding its molecular mechanism will facilitate the manipulation of relative traits. The NILs developed in our study could be ideal plant materials for further exploration of the cis-eQTL.

Fine-mapping of the qPL8 locus will facilitate its application in rice breeding for yield potential. Although the grain number per panicle was not significantly improved, the locus can be used to modify the panicle shape, which may improve final yield by changing canopy structure [39]. It is also notable that the rice yield could be improved by pyramiding minor yield-related genes even though each of them contributes little to the total yield [40]. In this scenario, qPL8 can be selected to pyramid with other loci to increase yield potential, and we did find the better effect of qPL8 in another background. Moreover, the markers developed in this study will simplify the application of qPL8 locus.

5  Conclusion

In this study, we confirmed the effect of qPL8 in shaping panicle length by detailed trait analysis of the NILs. Five recombinants were identified from the NIL populations using 12 newly-developed InDel/Caps markers, and the qPL8 locus was finally confined to a 278 kb interval covering 44 genes. Facilitated by whole genome sequencing and RT-PCR, 17 genes with sequence variation and eight genes with expression variation were identified among the candidate gene list. Especially, expression of OsMADS37 and OsMADS7 was responsive to the allele variation of qPL8 and qPL6, respectively, suggesting that MADS-box genes might mediate the control of panicle length. This work lays the foundation for gene cloning and breeding utilization of qPL8 in the future.

Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (31871217 and 32072037), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (20KJA210002) and the Key Research and Development Program of Jiangsu Province (BE2018357).

Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.

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Appendix

Table S1: Markers developed for qPL8 fine mapping

Table S2: Primers used for semi-quantitative and quantitative RT-PCR analysis

Table S3: SNPs information between two parents on protein coding region

Table S4: InDels information between two parents on protein coding region

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