Fine Mapping and Cloning of the Novel Gene Qph-IAA30, which Simultaneously Affects the Plant Height, Panicle Length, Spikelet Number and Yield in Rice (Oryza Sativa L.)

Background: The plant height is one of the most important agronomic traits in rice (Oryza sativa L.), and the introduction of semidwarf rice led to record yield increases throughout Asia in the 1960s. Near-isogenic lines (NILs) are the most powerful tools for the detection and precise mapping of quantitative trait loci (QTLs). Results: In this study, 176 NILs were produced from the crossing and back-crossing of two rice cultivars. Specically, Jiafuzhan, an indica rice cultivar, served as the recipient, and Hui1586, a restorer japonica cultivar, served as the donor. Using the 176 NILs, we identied a novel QTL for plant height in NIL36. First, we mapped the QTL to a 31-kb region between the markers Indel12-29 and Indel12-31. The rice genome annotation indicated the presence of three candidate genes in this region. Through gene prediction and cDNA sequencing, we conrmed that the target gene in NIL36 was Osiaa30, hereafter referred to as qPH-iaa30. Further analysis showed that qPH-iaa30 was produced by a 1-bp deletion in the rst exon that resulted in the premature termination of OsIAA30. Knockout experiments showed that qPH-IAA30 was responsible for the plant height phenotype. Although qPH-IAA30 from Jiafuzhan showed a higher plant height, the plant also exhibited a longer panicle length, more spikelets and a higher yield. Taken together, our results demonstrate that qPH-IAA30 has good specic application prospects in future rice breeding. Conclusions: 176 NILs are produced from two rice cultivars, using the 176 NILs, a novel qPH-iaa30 for plant height is identied, and the qPH-IAA30 gene is responsible for the plant height phenotype.

annotation indicated the presence of three candidate genes in this region. Through gene prediction and cDNA sequencing, we con rmed that the target gene in NIL36 was Osiaa30, hereafter referred to as qPH-iaa30. Further analysis showed that qPH-iaa30 was produced by a 1-bp deletion in the rst exon that resulted in the premature termination of OsIAA30. Knockout experiments showed that qPH-IAA30 was responsible for the plant height phenotype. Although qPH-IAA30 from Jiafuzhan showed a higher plant height, the plant also exhibited a longer panicle length, more spikelets and a higher yield. Taken together, our results demonstrate that qPH-IAA30 has good speci c application prospects in future rice breeding.
Conclusions: 176 NILs are produced from two rice cultivars, using the 176 NILs, a novel qPH-iaa30 for plant height is identi ed, and the qPH-IAA30 gene is responsible for the plant height phenotype.

Background
Plant height is an important factor determining the architecture and grain yield of cereal plants [1][2]. The semidwarf genes, which result in a shortened culm, improved lodging resistance and an increased harvest index, contributed to the "Green Revolution" in wheat and rice [3][4]. However, the wide application of dwarf germplasm resources and their narrow genetic range coupled with the excessive use of pesticides and fertilizers have led to serious environmental problems [5][6], and these problems have encouraged the study of genetic and molecular mechanisms for establishing an "ideal" plant structure through height regulation. Most of the identi ed genes related to plant height, such as semidwarf1 (sd1) [7], GA-insensitive dwarf1 (gid1)[8], GA-insensitive dwarf2 (gid2) [9], BR-de cient dwarf1 (brd1) [10], BRinsensitive mutant (d61) [11], and BR-de cient mutant (osdwarf4-1) [12], are related to the metabolism or signaling of the phytohormones gibberellin (GA) and brassinosteroid (BR) [6].
Although the GA-and BR-related genes associated with plant height have been extensively studied, an increasing number of novel plant height-related genes that rely on pathways other than the GA and BR pathways are being discovered. For instance, the carotenoid-derived phytohormone strigolactone has become a focus of research on plant architecture patterning [13]. Recent studies have shown that OsCKX9, which encodes a cytokinin oxidase that catalyzes the degradation of cytokinin, functions as a primary strigolactone-responsive gene that regulates rice tillering, plant height, and panicle size, likely via the secondary response gene OsRR5, which encodes a cytokinin-inducible rice type-A response regulator. This pathway demonstrates that strigolactone regulates the rice shoot architecture by enhancing cytokinin catabolism through modulation of the expression of OsCKX9 [14].
Auxin exerts pleiotropic effects on plant cell elongation, cell division and differentiation, root initiation, apical dominance, and tropic responses by regulating the expression of the early auxin-responsive auxin/indoleacetic acid (Aux/IAA) genes [15]. By screening the available databases, Jain et al. (2006) identi ed 31 Aux/IAA genes in rice and found that these genes have different functions [15]. OsIAA1 and OsIAA3 play important roles in the crosstalk between the auxin and brassinosteroid signaling pathways and plant morphogenesis [16,17]. The gain-of-function mutation in OsIAA11 inhibits lateral root development in rice [18]. OsIAA13-mediated auxin signaling is involved in lateral root initiation in rice [19]. OsIAA6 is involved in drought tolerance and tiller outgrowth [20], and the OsIAA10 protein directly targets the rice dwarf virus P2 protein and enhances viral infection and disease development [21]. Near-isogenic lines (NILs) carry one or more donor chromosome segments, which provides distinct advantages for QTL identi cation [22]. Moreover, NILs can block background genetic noise, undoubtedly enhance our understanding of complex traits and promote plant genomic studies [22,23].
In this study, we described the development of NILs of rice through the crossing and back-crossing of two rice cultivars. Here, the japonica cultivar Hui1586 served as the donor, and the indica cultivar Jiafuzhan served as the recipient. Using 176 NILs, we identi ed the plant height gene, which was designated qPH-iaa30 based on subsequent analyses. qPH-iaa30 exhibits a 1-bp deletion in the rst exon that results in premature termination of qPH-IAA30, which encodes an auxin-responsive protein. Using CRISPR/Cas9 genome editing, we knocked out qPH-IAA30 in Jiafuzhan and observed that the knockout mutants showed the NIL36 plant height phenotype, which demonstrated that qPH-IAA30 is a functional auxinresponsive protein that regulates the height of rice plants. Moreover, the ndings showed that qPH-IAA30 simultaneously affects the plant height, panicle length, spikelets and yield and will have speci c application prospects in future rice breeding.

Plant materials
The indica rice cultivar Jiafuzhan and the japonica rice cultivar Hui1586 were preserved at the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. Suxiu867 (Food Crops Research Institute, Jiangsu Academy of Agricultural Sciences), a japonica cultivar, was used as the recipient, and Minghui86 (Rice Research Institute, Fujian Academy of Agricultural Sciences), a restorer indica cultivar, was used as the donor. The F 1 plants were generated from Suxiu867 as the female and Minghui86 as the male. The F 1 plants were backcrossed with Suxiu867 to produce the BC 1 F 1 generation. These BC 1 F 1 plants were backcrossed with Suxiu867 to produce BC 2 F 1 plants, and these plants were self-pollinated to produce BC 2 F 2 lines. The resulting lines were self-interbred to obtain Six generations, and a stable line QTLs were identi ed based on signi cant differences between parents. Plant height, panicle length, effective panicle number, spikelets per panicle, seed setting rate and 1000-grain weight were measured at maturity stage.
All plants were planted in accordance with standard commercial practices, with row spacing ranging from 13.3 cm to 26.4 cm, and eld management generally followed normal rice eld management practices.
Construction of a genetically mapped population NIL36 hybridized with Jiafuzhan to form a mapping population. The F 2 location population was constructed by self-crossing of F 1 population. A primary linkage of the QTL for plant height was obtained using 45 recessive plants from the F 2 population, and 1264 recessive plants in the F 2 population were selected for ne localization.

PCR ampli cation and marker detection analysis
The CTAB method [24] with minor modi cations was used for the extraction of plant DNA from frozen leaves of the rice plants. For PCR ampli cation, each 20-µL reaction mixture contained 30 ng DNA, 0.4 µM primers and 2× Es Tag MasterMix (Dye). The ampli cation program includes the following procedures: 2 min at 94°C; 33 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; and a nal extension at 72°C for 2 min. The ampli ed PCR product underwent 3% agarose gel electrophoresis and was stained with ethidium bromide [25].

Molecular mapping of QTLs for plant height
We used phenotypic data and SSR markers for the detection of QTLs. Genetic distance was estimated using MapDraw V2.1 [26]. The genetic linkage map obtained in this study is basically consistent with that reported by Rahman et al [27].

Physical map construction and bioinformatics analysis of QTLs for plant height
The physical map of QTLs for plant height was constructed through a bioinformatics analysis using the published sequences of BAC and P1-derived arti cial chromosome (PAC) clones of cv. Nipponbare released by the International Rice Genome Sequencing Project (IRGSP, http://rgp.dna.affrc.go.jp/IRGSP/index.html). Target gene linkage markers were used to clone and sequence alignment was performed using the matching Basic Local Alignment Search Tool. According to the existing sequence annotation database (http://rice.plantbiology.msu.edu/; http://www.tigr.org/). Candidate genes of the prediction were based on the existing sequence annotation database analysis (http://rice.plantbiology.msu.edu/; http://www.tigr.org/). Targeted knockout of OsIAA30 in rice using the CRISPR/Cas9 system The rst exon of the OsIAA30 gene in Jiafuzhan was targeted with one gRNA spacer. Highly speci c gRNA spacer sequences were designed using CRISPR plant database and website (Supplementary Table 1) [28]. Genome editing mutations of target genes in regenerated plants were analyzed. The deletion and insertion of genes were detected by PCR. PCR products were selected from transgenic CRISPR-edited strains for sequencing to identify speci c mutations. The degradation sequence decoding method was used to analyze the double peaks [29]. The primers used in CRISPR/Cas9 studies were shown in Table  S1.

NILs development
For development of the NILs, Jiafuzhan, an indica cultivar, was used as the recipient, and the restorer japonica cultivar Hui1586 was used as the donor. The F 1 plants were generated from Jiafuzhan as the female and Hui1586 as the male, and the F 1 plants were back-crossed with Jiafuzhan to produce the BC 1 F 1 generation. These BC 1 F 1 plants were then backcrossed with Jiafuzhan to produce BC 2 F 1 plants.
Using the same approach, 118 BC 3 F 1 individuals were obtained, and these plants were self-pollinated to produce the BC 3 F 2 lines. Based on their characteristics, we selected one or two individual plants from each line. As a result, 176 NILs were obtained (Fig. 1).

Identi cation and analysis of QTLs for plant height in the NILs
To evaluate the potential advantages of the NILs for QTL detection, the phenotypic variations in plant height were observed in 176 NILs, and NIL36 exhibited a lower plant height than Jiafuzhan. Further investigations and analyses showed that the plant height of Jiafuzhan was 116.22 cm, whereas that of Jiafuzhan NIL36 was 79.52 cm. The difference in plant height between these lines reached a very signi cant level (Fig. 2).
The phenotypic comparisons between NIL36 and Jiafuzhan are presented in Table 1. The results showed some signi cant differences in major agronomic traits, including the plant height, panicle length, spikelets per panicle and yield per plant, between NIL36 and Jiafuzhan. However, no signi cant difference in the number of effective panicles, seed setting rate, 1,000-grain weight, grain length or grain width was found (Table 1).

Linkage analysis of the QTL for plant height in NIL36
To identify the gene responsible for the NIL36 phenotype, we located the QTL for plant height in NIL36, and a total of 506 SSR markers from the rice molecular map were selected for polymorphism surveys between Hui1586 and Jiafuzhan [30]. Of these, 296 pairs exhibited polymorphism. Based on these 296 primer pairs, 45 recessive plants from the F 2 population (NIL36/Jiafuzhan) were used for a linkage analysis between markers and the QTL. One of these SSR markers, RM3326 on chromosome 12, was found to be linked to the trait in the 45 F 2 individuals.

Initial localization of the QTL for plant height
Published markers around RM3326 were used to initially locate the QTL. A genetic linkage analysis revealed that the QTL was located between the molecular markers RM2854 and RM235, which are located at a distance of 7.7 cM (Fig. 3-a).
To determine the location of the QTL within a smaller region, we identi ed 1264 recessive plants from the F 2 population from Jiafuzhan/NIL36, and six polymorphic indel markers were screened from 18 newly developmental indels (Table 3). Indel markers from the open rice genome sequences were designed and tested to predict the likelihood of polymorphism between NIL36 and Jiafuzhan by comparing sequences from Nipponbare (http://rgp.dna.affrc.go.jp/) and the indica cultivar 93 − 11 (http://rice.genomics.org.cn/). The genotyping of all recombinant genes was performed using six polymorphic markers. The results showed that the QTL was located in the 295-kb region between the molecular markers Indel12-7 and Indel12-9 on chromosome 12 ( Fig. 3-b and Table 3). For the ne mapping of the QTL, eight polymorphic indel markers were screened from 26 newly developed indels (Table 3). Recombinant screening with eight markers located in a more internal position within the target locus detected 13, 11, eight, six, two, one, two, and ve recombinant plants, respectively (Fig. 3-c). Thus, the QTL was precisely located within the 31-kb region between the molecular markers Indel12-29 and Indel12-31.

Candidate genes in the 31-kb region
According to the available sequence annotation databases (http://rice.plantbiology.msu.edu/; http://www.tigr.org/), three annotated genes were located in the 31-kb region (Fig. 3-d), and all had a corresponding full-length cDNA. Among these genes, LOC_Os12g40860 encodes the leucine-rich repeat family protein, LOC_Os12g40880 encodes the uridine kinase family protein, and LOC_Os12g40890 is the auxin-responsive Aux/IAA gene family member OsIAA30.

Sequence analyses of the QTL for plant height
To identify the gene responsible for the observed phenotype, we then sequenced three genes of Jiafuzhan and NIL36. A deletion of only 1 bp (120:C) was found in LOC_Os12g40890 (Fig. 4), and no further difference was observed in the remaining two gene sequences. Thus, we hypothesized that LOC_Os12g40890 and OsIAA30 corresponded to the QTL for plant height in NIL36, and this gene was tentatively designated qPH-iaa30.
The analysis of the open reading frame (ORF) region showed that qPH-IAA30 had ve exons. qPH-iaa30 exhibited a 1-bp deletion in the 120th base of the rst exon, which resulted in the premature termination of OsIAA30 (Fig. 4).
qPH-iaa30 is responsible for the plant height phenotype of NIL36 To con rm that qPH-IAA30 confers a plant height phenotype, we examined whether the knockout of qPH-IAA30 in Jiafuzhan would lead to the NIL36 phenotype. One sequence-speci c guide RNA (sgRNA) was designed to knock out qPH-IAA30 using the CRISPR/Cas9 gene editing system. A total of three plants from three independent events (OsIAA30KO-line1, OsIAA30KO-line2 and OsIAA30KO-line3) were obtained, and sequencing con rmed that these plants carry mostly insertions or deletions in the targeted sites ( Supplementary Fig. 1).
We then investigated the plant height phenotype of these three homozygous lines at maturity and found that all three lines showed the NIL36 phenotype ( Fig. 5 and Table 1). Therefore, the targeted mutation of qPH-IAA30 led to the NIL36 plant height phenotype, which indicated that the loss of function of OsIAA30 was responsible for this phenotype. Most importantly, OsIAA30KO-line1, OsIAA30KO-line2 and OsIAA30KO-line3 showed shorter panicle lengths, fewer spikelets per panicle and lower yields than Jiafuzhan ( Fig. 5 and Table 1). Therefore, we hypothesized that the qPH-IAA30 gene not only affected the plant height but also regulated the panicle length, spikelets per panicle and yield in rice.

Comparative analysis of the hormone content between Jiafuzhan and OsIAA30KO-lines
To analyze whether qPH-IAA30 affects changes in the Aux/IAA levels, we measured the Aux/IAA content in Jiafuzhan (CK) and OsIAA30KO-line1. The results showed that the Aux/IAA content of OsIAA30KO-line1, OsIAA30KO-line2, OsIAA30KO-line3 and NIL36 was signi cantly lower than that of Jiafuzhan (CK) (Fig. 6).

Discussion
NILs have been developed and used for genetic studies and the ne mapping of QTLs for genome-wide target traits [22]. For example, each NIL carries one or more donor chromosome segments, which provides distinct advantages for QTL identi cation, and a QTL can be visualized as a single Mendelian factor by blocking background genetic noise. Several QTLs, such as qDTY 2.2 [23], Cn1a [31], GS3 [32], GW2 [33], Ghd7 [34], DEP1 [35], cold tolerance QTLs[36], 99 putative QTLs [37] and qHD19[38], have been identi ed or cloned based on NILs. Moreover, NILs block background genetic noise, undoubtedly enhance the understanding of complex traits and promote plant genomic studies [22,23]. In the present study, we developed 176 NILs with the genetic background of Jiafuzhan rice. Using these lines, we mapped the plant height gene as the qPH-iaa30 gene. Map-based cloning and knockout experiments con rmed that the plant height phenotype of NIL36 was caused by loss of function of the OsIAA30 gene.
Previous studies have indicated that Aux/IAA genes play important roles in plant growth and development by regulating the expression of early auxin-responsive genes [15]. For example, OsIAA1 and OsIAA3 affect plant morphogenesis [16,17], OsIAA11 and OsIAA13 affect root development in rice [18,19], and OsIAA6 is involved in tiller outgrowth [20]. The present study demonstrated that the qPH-iaa30 gene signi cantly reduces plant height in rice, and knockout experiments con rmed that qPH-iaa30 was responsible for the plant height phenotype. Three homozygous knockout lines also exhibited the NIL36 phenotype, which includes a lower plant height, a shorter panicle length, fewer spikelets per panicle and a lower yield per plant compared with Jiafuzhan (Table 1). Further analysis of the hormone content showed that the Aux/IAA contents of NIL36, OsIAA30KO-line1, OsIAA30KO-line2 and OsIAA30KO-line3 were signi cantly lower than that of Jiafuzhan (CK) (Fig. 6). Therefore, we hypothesized that qPH-IAA30 could simultaneously regulate the plant height, panicle length, spikelets per panicle and yield per plant by affecting the auxin levels.
Although qPH-IAA30 affects certain traits, such as the plant height, panicle length, spikelets per panicle and yield per plant, qPH-IAA30 has speci c application prospects in the improvement of rice breeding. First, to further increase the rice yield, breeders can transform qPH-IAA30 into excellent material via molecular marker-assisted selection. Second, the qPH-iaa30 gene is controlled by a single recessive gene. Therefore, to breed a new hybrid rice variety with an ideal plant height, breeders can transfer this gene into both restorer and sterile lines through molecular marker-assisted selection.

Conclusions
In this study, 176 NILs were produced from the crossing and back-crossing of two rice cultivars. Using the 176 NILs, we identi ed a novel qPH-iaa30 for plant height in NIL36. Further analysis show that qPH-iaa30 is produced by a 1-bp deletion in the rst exon that results in the premature termination of OsIAA30 and the qPH-IAA30 gene is responsible for the plant height phenotype.
Declarations Figure 1 Flowchart of the development of NILs in the present study. Jiafuzhan was used as the recipient, and Hui1586 was used as the donor. The F1 plants were continuously backcrossed with Jiafuzhan to produce BC1F1, BC2F1 and BC3F1 plants.  Genetic and physical maps of qPH-iaa30. a: Primary mapping of qPH-iaa30. The gene was mapped to the region between the markers RM2854 and RM235. b: Further mapping of qPH-iaa30. The gene was mapped to the region between markers Indel12-7 and Indel12-9. c: Fine mapping of qPH-iaa30. qPH-iaa30 was localized to a 31-kb region between the markers Indel12-29 and Indel12-31, and the recombinant number between the markers and target genes is indicated under the linkage map. d: Candidate genes in the 31-kb target region.