Genetic and Evolutionary Analysis of Purple Leaf Sheath in Rice

Abstract

Background: Anthocyanin accumulates in many plant tissues or organs, in rice for example leading to red, purple red and purple phenotypes for protection from damage by biotic and abiotic stresses and for reproduction. Purple leaf, leaf sheath, stigma, pericarp, and apiculus are common in wild rice and landraces and occasionally found in modern cultivars. No gene directly conferring anthocyanin deposited in a purple leaf sheath has yet been isolated by using natural variants. An F2 population derived from ssp. japonica cv. Tainung 72 (TNG72) with purple leaf sheath (PSH) crossed with ssp. indica cv. Taichung Sen 17 (TCS17) with green leaf sheath (GSH) was utilized to isolate a gene conferring leaf sheath color.

Results: By positional cloning, 10-and 3-bp deletions in the R2R3 Myb domain of OsC1 were uncovered in GSH varieties TCS17 and Nipponbare, respectively. Allelic diversity, rather than gene expression levels of OsC1, might be responsible for anthocyanin accumulation. Parsimony-based analysis of genetic diversity in 50 accessions, including cultivars, landraces, and A-genome wild rice, suggests that independent mutation occurred in Asian, African, South American, and Australian species, while O. meridionalis had a divergent sequence. OsC1 was thought of as a domestication related gene, with up to 90 % reduction of genetic diversity in GSH; however, no values from three tests showed significant differences from neutral expectations, implying that OsC1 had not been subjected to recent selection. Haplotype network analysis revealed that species from different continents formed unique haplotypes with no gene flow. Two major groups of haplotypes corresponding to 10-bp deletion and other sequences were formed in Asian rice, including O. rufipogon, O. nivara and O. sativa. Introgressions of OsC1 between subspecies through natural and artificial hybridization were not rare. Because artificial and natural selection imposed admixture on rice germplasm in Taiwan, the genealogy of OsC1 might not be congruent with the current distribution of alleles through lineage diversification.

Conclusion: OsC1 is responsible for purple leaf sheath, and much new information about OsC1 is provided e.g., new alleles, non-domestication syndrome, and incongruence of genealogy with geographic distribution.

Keywords: Genealogy; Genetic Diversity; OsC1; Purple Leaf Sheath.

Fig. 1

Phenotypes of Tainung 72 (TNG72) and Taichung Sen 17 (TCS17) for different tissues and growth stages. All of the figures are established that left is TNG72 and right is TCS17. Plants at (a) coleoptile, (b) 4-leaf stage, (c) active tillering stage, (d) caryopsis and (e) stigma at flowering time. Bar size is (a) 1 cm; (b), (c), and (d) 5 cm; (e) 1 mm. (f) Estimated anthocyanin contents in leaf sheath and blade at seedling and active tillering stages. * p < 0.05 using paired t-test

Fig. 2

Isolation and sequence analysis of the gene conferring responsible for purple leaf sheath. Coarse mapping of the purple leaf sheath gene between CH0639 and RM276 on chromosome 6. Fine mapping narrows down this region to 160.04 kb between CH0611 and RM253. The LOC_06g10350 locus was annotated as MYB family transcription factor by RGAP and corresponded to Os06g0205100 annotated as transcription factor MYB6 and O. sativa C1, and rice homolog of maize C1 by RAP. Sequence analysis of 3 cultivars, Tainung 72 (TNG72), Nipponbare and Taichung Sen 17 (TCS17). TNG72 has complete sequences translated into 272 amino acids. Nipponbare has 3-bp deletion in the second exon of OsC1, and is translated into 271 amino acids. TNG17 has 10-bp deletion in the third exon of OsC1, and is translated into 207 amino acids. The black lines are conserved domains of OsC1, R2R3 Myb domain. The triangles indicate sites of variation

Fig. 3

Anthocyanin accumulation and gene expression of OsC1 in the leaf sheath. (a) Phenotypes and anthocyanin contents of 1 green leaf sheath (GSH) accession and 14 purple leaf sheath (PSH) accessions with various degrees of purple. The 15 accessions are Tainung 67 (TNG67) (1), Chuan4 (2), G124 (3), Warisanmochi2 (4), Shui Pai Tiao (5), Tonsisai (6), IR13525-118-3-2-2-2 (7), 4233 8 (8), Jinya-149 (9), Tainung 72 (TNG72) (10), Ssall-Bye (11), Tainung Sen 20 (12), Shang Chi Tsao Tao (SCTT) (13), Asamurasaki (14), and Kun Shan Wu Siang Keng (KSWSK) (15). Leaf sheath phenotypes are characterized according to the RHS (Royal Horticultural Society, 5th edition) Color Charts. (b) Gene expression of OsC1 in the leaf sheath of 4 cultivars at tillering. Cultivars TNG67, TNG72, SCTT and KSWSK, exhibiting diverse degrees of purple color, were selected to evaluate gene expression by real-time PCR. Letters indicate differences at significance level p < 0.05 by using LSD analysis

Fig. 4

Sequence variation of OsC1 in 50 accessions. According to DNA sequence variation, 5 groups were classified. The accessions of Group I exhibit red purple, purple, tryan rose and green leaf sheath and have complete OsC1 sequences closely resembling that of Tainung 72. The accessions of Group II have red purple and tryan rose leaf sheath and share the sequence of Shang Chi Tsao Tao, which has one nonsynonymous mutation in position 1192. The accessions of Group III have blackish purple and green leaf sheath and share the sequence of Kun Shan Wu Siang Keng, which has one nonsynonymous mutation in position 918. The accessions of Group IV have green leaf sheath and share the sequence of Taichung Sen 17, which has a 10-bp deletion starting at position 796. The accessions of Group V include 6 wild A-genome species. Non-synonymous amino acid mutations were indicated to have similar function (yellow) or altered polarity (blue), charge (pink) and aromatic nature (green). d: deletion. i: insertion

Fig. 5

Haplotype network analysis for OsC1. A total of 17 haplotypes, H1-H17, were discerned, and each is represented by a rectangle. In Asian rice, two major groups, Group a and Group b, are classified. Group b can be subdivided into three clusters, B1, B2, and B3, indicated by ovals. Blue dots and triangles indicate indica and japonica accessions, respectively. Text color resembles the color of leaf sheath (green, purple). Box line color resembles the color of leaf sheath of majority of accessions, green, pansy purple, red purple, and blackish purple, respective, besides red resembles admixture of different colors. Black nodes represent inferred branch on which each single nucleotide polymorphism occurred