Map-based Cloning and Natural Variation Analysis of the PAL3 Gene Controlling Panicle Length in Rice

doi:10.11983/CBB21119

Abstract

Abstract: Panicle architecture is one of the key factors determining the rice (Oryza sativa) grain yield. In this study, four short panicle mutants were identified from an EMS (ethyl methane sulfonate) mutant library of a japonica vareity Shengdao 808 (SD808). The panicle length, primary branch number, secondary branch number and grain number per panicle of these mutants were decreased in various degrees. Map-based cloning showed that these mutants were controlled by the gene PAL3 (PANICLE LENGTH 3) which encodes a peptide transporter with 12 transmembrane domains. The point mutations of pal3-1 and pal3-2 resulted in non-synonymous mutations of amino acids in the conserved region; the point mutation of pal3-3 resulted in the mis-splicing of the first exon and intron; and the point mutation of pal3-4 resulted in the premature termination of translation and thus the deletion of the 12^th transmembrane domain. Through haplotype analysis, nine haplotypes of PAL3 were identified, including three major haplotypes (Hap1-Hap3). Hap1 is dominated by japonica accessions, Hap2 contains both indica and japonica accessions, while Hap3 is dominated by indica accessions. Hap1 originated from O. rufipogon, while Hap2 and Hap3 may originate from O. nivara. Statistical analysis showed that the panicle length of Hap3 was significantly higher than that of Hap1 and Hap2, indicating that Hap3 may have the potential to improve the panicle length. In conclusion, this study revealed the important role of the peptide transporter in regulating rice panicle architecture and thus provides a new theoretical basis for rice panicle architecture improvement.

Key words: rice, panicle architecture, map-based cloning, peptide transporter, natural variation

Jiangyuan Shang, Yan Chun, Xueyong Li. Map-based Cloning and Natural Variation Analysis of the PAL3 Gene Controlling Panicle Length in Rice[J]. Chinese Bulletin of Botany, 2021, 56(5): 520-532.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21119

https://www.chinbullbotany.com/EN/Y2021/V56/I5/520

Figures/Tables 11

Table 1 Primers used for mapping

Primer name	Forward primer (5′→3′)	Reverse primer (5′→3′)
R11-3	AGAGAGACATCCGGAGACAA	TAAGACGAAAGGTCAAACGT
R11-5	GTGCTAACGTTTCGTCTAAC	AATAGCCTTCGGTGGTCTCA
R11-10	GTTCGTAATGTGGGCGTCTT	TGGGCACTCTTCTCACACTG
R11-12	CAATCTTGCTCTACTAGCTAGTG	GTGGCAACTAACAGATTAGATG
M1	GCAGTATATATTCGGCGGCG	GCCGTCGCCATATAGCTG
M2	GAGCCTCTCCTACTGTGCTA	AGAGCCCTCAGTTCCTCAAT
M3	GCTGACTACAGTAAGATCATGC	AGACAAACGGTCAAACATGT
M4	AAGGATCCAAGCTAGCCTCC	CCTGACAGCAAGCGAGAGAT
M5	CTTCAGCAAGTGAACTACGA	CCTAAACTAGCACGGATCATAGC
M6	TGTGAGGTTTAGGTTCTCGGA	TGAATAGAGATGCGGTCCAAC
M7	GGATTCGGCCACTGGTTGTT	GAATGTACTCGGATAAACCC

Figure 1 Comparison of the rice phenotype between wild type and the pal3 mutants (A) Gross morphology of wild type and the pal3 mutants at mature stage (Bar=20 cm); (B) Closed panicle of wild type and the pal3 mutants (Bar=2 cm); (C) Spread panicles of wild type and the pal3 mutants (right) (Bar=2 cm); (D)-(G) Statistic analysis of panicle length (D), primary branch number (E), secondary branch number (F) and grain number per panicle (G) of wild type and the pal3 mutants. WT: Wild type. Data in figure are means±SD (n=20). ** indicate the significant differences at P<0.01 level by Students’t test.

Table 2 Genetic analysis of the pal3-1, pal3-2, pal3-3 and pal3-4 rice mutants

Hybrid combi- nations	Phenotype of F₁	F₂ population			χ²_3:1
Hybrid combi- nations	Phenotype of F₁	Wild-type plant number	Mutant-phenotypic plant number	Total number	χ²_3:1
pal3-1 × WT	WT	190	60	250	0.13
pal3-2 × WT	WT	196	68	264	0.08
pal3-3 × WT	WT	180	63	243	0.11
pal3-4 × WT	WT	201	69	270	0.04

Figure 2 Map-based cloning of the PAL3 gene (A) The PAL3 gene was mapped to a region between InDel markers R11-3 and R11-5 on chromosome 11; (B), (C) Fine-mapping of PAL3. The PAL3 gene was further delimited to the region between markers M3 and M5, which contains seven open reading frames (ORFs). The number of recombinants is indicated beneath the marker positions; (D) Schematic structure of the PAL3 gene and the mutation sites of pal3 mutants. WT: Wild type

Table 3 Annotations of candidate genes in the fine-mapping region

Gene ID	Annotation
LOC_Os11g12680	Expressed protein
LOC_Os11g12690	Retrotransposon protein, putative, unclassified, expressed
LOC_Os11g12700	Retrotransposon protein, putative, unclassified, expressed
LOC_Os11g12710	Retrotransposon protein, putative, unclassified, expressed
LOC_Os11g12720	Retrotransposon, putative, centromere-specific
LOC_Os11g12730	Transposon protein, putative, CACTA, En/Spm sub-class, expressed
LOC_Os11g12740	Peptide transporter PTR2, putative, expressed

Figure 3 Transmembrane regions and phylogenetic analysis of PAL3 (A) 12 transmembrane domains of PAL3 and the mutation sites of the pal3-1, pal3-2 and pal3-4 mutants; (B) Phylogenetic analysis of PAL3 and its homologs. The phylogenetic analysis was carried out by MEGA X with 1 000 bootstrap replicates and was constructed using the distance method with maximum likelihood.

Table 4 Locus ID and gene name of PAL3 and its homologs

Species	Locus ID	Gene name
Rice	LOC_Os11g12740	PAL3/SP1
	LOC_Os07g01070	OsPTR1
	LOC_Os12g44100	OsPTR2
	LOC_Os10g33210	OsPTR3
	LOC_Os07g41250	OsPTR4
	LOC_Os04g50940	OsPTR5
	LOC_Os04g50950	OsPTR6
	LOC_Os01g04950	OsPTR7
	LOC_Os03g51050	OsPTR8
	LOC_Os06g49250	OsPTR9
	LOC_Os03g13274	OsNTR1.1
	LOC_Os10g40600	OsNRT1.1B
	LOC_Os01g54515	OsNPF4.5
Arabidopsis	At1g12110	AtNRT1.1
	At5g62680	AtNRT1.10
	At1g52190	AtNRT1.11
	At1g33440	AtNRT1.13
	At1g59740	AtNRT1.14
	At1g72120	AtNRT1.15
	At1g72125	AtNRT1.16
	At1g69850	AtNRT1.2
	At3g21670	AtNRT1.3
	At2g26690	AtNRT1.4
	At1g32450	AtNRT1.5
	At1g27080	AtNRT1.6
	At1g69870	AtNRT1.7
	At4g21680	AtNRT1.8
	At1g18880	AtNRT1.9
	At3g54140	AtPTR1
	At2g02040	AtPTR2/AtNTR1
	At5g46050	AtPTR3
	At2g02020	AtPTR4
	At5g01180	AtPTR5
	At1g62200	AtPTR6
	At1g27040	AIT2
	At3g25260	AIT3
	At3g25280	AIT4
	At3g47960	GTR1
	At3g45650	NAXT1
	At1g68570	Nitr

Figure 4 Sequences alignment of PAL3 and its homologs The black background represent that all amino acid sequences are homologous. The gray part represent that some of the amino acid sequences are homologous. The white part represent that the amino acid sequences are completely different. * indicate the mutation sites of the pal3-1, pal3-2 and pal3-4 mutants in turn; △: The variation sites between Hap1 and Hap3.

Figure 5 Haplotype analysis of PAL3 (A) Structure of PAL3 gene and the location of haplotypes; (B) Haplotypes of PAL3 in the cultivated rice. Haplotype data were analyzed by Rice Molecular Breeding Knowledgebase (MBKbase) (Peng et al., 2020), including 2 187 cultivated rice varieties. Variations on intron, heterozygous variations and synonymous variations were removed during analysis (-: The deletion of base pair); (C) Sequence alignment around the variation sites between Hap1 and hap3 (Δ: The variation sites between Hap1 and Hap3).

Figure 6 Haplotype of PAL3 in the wild rice Haplotype data of wild rice were from Gramene and Rice Relatives-GD (Mao et al., 2019). *: D475 and G476 deletion; ●: No datum

Figure 7 Subspecies composition and panicle phenotype analysis of Hap1, Hap2 and Hap3 of the PAL3 gene (A) Subspecies composition of Hap1, Hap2 and Hap3; (B) Comparison of panicle length among Hap1, Hap2 and Hap3; (C) Statistic of panicle length of Hap1, Hap2 and Hap3. Different lowercase letters indicate significant differences (P<0.05).

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