Quantitative Trait Loci Mapping of Flag-leaf Ligule Length in Rice and Alignment with ZmLG1 Gene

The family Gramineae contains 10 000–11 000 species, including many important crops, such as wheat, rice, maize and sorghum. The results from comparative genomics (Binelli et al. 1992; Berhan et al. 1993; Dunford et al. 1995), phylogenetic analysis using conserved DNA sequences (Hsiao et al. 1993), and hybrid characters from distantly related species (Laurie et al. 1990) all suggested that rice and maize evolved from one common ancestor (Moore et al. 1995a, 1995b). The divergence between rice (Oryza sativa) and maize (Zea mays) is believed to have diverged ∼50 Mooremya (Bennetzen and Freeling 1993) from a common ancestor.

Morphological features of leaf ligules are used as a key identification index of taxonomy in grasses. For example, cereal crops such as rice, maize and wheat, usually possess a ligule, whereas barnyard grasses (Echinochloa crusgalli) do not. The ligule length was also used in the infraspecific classification of Helictotrichon desertorum (Josef 1972). Although most rice varieties contain ligules, different varieties usually contain various ligule lengths or shapes. O. officinalis bears a short and triangular ligule. The ligule of O. meyeriana is nearly semicircular. African cultivated rice exhibits a shorter ligule with a rounded tip.

Because of the well descriptive characterization, Z. mays was as an excellent model for the study of leaf development (Sylvester et al. 1990). Some mutants involved in the elimination/development of ligule and auricle such as eta, kn1, lg1 and lg2, were identified or cloned in maize (Vollbrecht et al. 1991; Fowler and Freeling 1996; Moreno et al. 1997; Walsh et al. 1998; Osmont et al. 2003). lg1 removes the ligule and auricle in all but the uppermost leaves of the plant. The boundary between the sheath and the blade of lg1 is at a position similar to that in wild type but is less distinct (Becraft et al. 1990). LG1 encodes squamosa-promoter binding proteins and expresses in the developing ligular region (Moreno et al. 1997). lg2 was lacking in ligule and auricle only on the lowest leaves of the plant. Its ligule and auricle begin to appear at the leaf margins on higher leaves (Harper and Freeling 1996). It encodes a basic leucine zipper protein and is involved in the establishment of the leaf blade-sheath boundary (Walsh et al. 1998). In addition to LG1 and LG2, others are also involved in ligular region development in maize. The kn1 (knotted 1) mutant causes the formation of ectopic knots (Vollbrecht et al. 1991). The lg3 (Liguleless 3) mutant transforms blades to sheaths (Muehlbauer et al. 1997), and gnarley1 (gn1) produces altered ligules and sheaths (Foster et al. 1999). These mutants also affect ligular region development.

Liguless mutants were also found in rice and sorghum (Jones 1933; Zwick et al. 1998; Quan et al. 2003). Jones (1933) first found that lg is controlled by a single recessive gene in rice. Subsequently, Morinaga and Fukushima (1943) confirmed Jones' findings. Recently, lg1 was located on the long arm of rice chromosome 4 at a molecular level (Maekawa et al. 1991; Saito et al. 1991). To date, some researchers have found that the presence or absence of ligules in rice is controlled by a single gene, but the ligule length (LL) controlled by a quantitative trait has not been reported. In the present study, QTLs for the ligule length of flag leaf was analyzed, and the homologs related to ligule development were deduced and integrated into the existing simple sequence repeats (SSR) linkage map in rice. The functions of ligule are also discussed in the present paper. The results are helpful to understand the genetic basis of ligule development in rice.

Results

Ligule length of the DH population and their parents

Figure 1 shows the distribution for the ligule length of flag leaf in the parents and the DH population. There were significant differences in the ligule length between the two parental varieties, ‘CJ06’ and ‘TN1’. They were 9.36 mm and 15.64 mm, respectively. Thus the ligule length of ‘TN1’ was longer than that of ‘CJ06’. In the DH population, the ligule length ranged from 5.8 mm to 17.3 mm, mainly around 11 mm or so, showing continuous variation. A certain number of DH lines segregated transgressively over their parents, indicated ligule length as a typical quantitative trait.

QTL analysis

A molecular linkage map, which consisted of 178 markers evenly distributed over all 12 rice chromosomes, has been constructed by using this population (Sogawa et al. 2004, 2005; Zhang et al. 2006) and is suitable for QTL analysis. Interval QTL mapping for the ligule length identified five QTLs, qLL-2, qLL-4, qLL-6, qLL-10 and qLL-12, on chromosomes 2, 4, 6, 10 and 12, respectively (Figure 2 and Table 1). The additive effects of qLL-2, qLL-6 and qLL-1 were 2.06 3.07 and 1.95, respectively, and the QTL alleles from ‘TN1’ could increase the ligule length of flag leaf. qLL-4 and qLL-10 had additive effects of 2.25 and 2.74, respectively, indicating that ‘CJ06’ alleles at these two loci could increase the ligule length. The variances explained by these QTLs (qLL-2, qLL-4, qLL-6, qLL-10 and qLL-12) were 11.4%, 13.6%, 27.8%, 22.1%, and 11.0%, respectively. The total phenotypic variation explained by the five putative QTLs was 72.6% based on the multiple-QTL model in MAPMAKER/QTL.

Table 1. Quantitative trait loci (QTL) analysis for the ligule length of flag leaf in a double haploid (DH) population

Locus	Chromosome	Marker interval	LOD score	Variance explained (%)	Additive effect
qLL-2	2	RM240-RM250	2.53	11.4	2.06
qLL-4	4	RM255-RM280	2.84	13.6	−2.25
qLL-6	6	RM454-RM528	4.90	27.8	3.07
qLL-10	10	RM467-RM271	3.54	22.1	−2.74
qLL-12	12	RM270-RM17	3.07	11.0	1.95
			Total	72.6

• QTL nomenclature follows that of McCouch et al. (1997). LOD, logarithm of odds.

Chromosomal in silico mapping of maize LG locus in rice

The sequences of LG1, LG2, LG3 and LG4 in maize were obtained from the MaizeGDB web site (http://www.maizegdb.org/). The homologous genes in rice were identified via tblastn alignment against the maize sequences and integrated into the existing SSR linkage map by in silico mapping (Figure 2). The homologs of ZmLG1, ZmLG2, ZmLG3 and ZmLG4 were located on chromosome 4, 1, 1 and 5 in rice, respectively. Among them, a homologous gene of ZmLG1 named OsLG1, was located between RM255 and RM280, which is identical to the interval of qLL-4 on the long arm of chromosome 4. In addition to ZmLG1, the homologous genes of ZmLG2, ZmLG3 and ZmLG4 were not detected within the detected LL QTL intervals in rice (Figure 2).

According to the ZmLG1 sequence in maize, the homologous OsLG1 via BlastP search against GenBank database was gained, which shows 76% similarity to ZmLG1 (Figure 3). ZmLG1 encodes a squamosa-like protein with 399 amino acid residues and contains an extremely conservative squamosa promoter binding protein (SBP) domain in maize, which determines leaves with liglues or without. The predicted OsLG1 encodes a protein containing 416 amino acid residues, and also an extremely conservative SBP domain. The SBP domain consists of 81 amino acid residues, and is a type of specific transcription factor binding protein. The SBP domain is also highly homologous with part of the SBP1 and SBP2 functional domain in Antirrhinum majus.

Discussion

Rice is the staple food for more than half of the world's population. Humans have explored its evolution and differentiation for centuries. As early as the Han Dynasty, distinctions between indica and japonica rice were explored (Ding 1961). Later, Kato et al. (1928) formally identified two subspecies of cultivated Asian rice, O. sativa subsp. indica and O. sativa subsp. japonica based on both morphologic and physiologic differences. Chinese scholars, such as Ding (1949, 1983) proposed that indica and japonica are the xian subspecies and jing subspecies of China. Oka (1958) pointed out that no single trait could distinguish indica and japonica, but the integration of many traits, such as morphologic characteristics, could distinguish indica from japonica. A morphologic index based on leaf hairiness, hairiness of hull, color of hull when heading, length of the first and second panicle internodes and length/width of grain was used to discriminate indica and japonica (Cheng et al. 1984). Ligule shape and size are a significant trait for classification of Poaceae (Josef 1972; Borre and Watson 1994). QTL analysis for ligule length showed that the locus of qLL-6 overlaps that of qLH-6a and qLH-6b detected by Qian et al. (2000). Moreover, the ligule length of indica longer than that of japonica is confirmed in many native varieties (Hu et al., unpubl. data, 2007). It is speculated that the ligule length is possibly an important trace of indica-japonica differentiation.

Bioinformatics is a useful tool in the pre- and post-genomic eras and contribute to our understanding of gene function, expression profile and their evolution. Based on the cloning of wheat vernalization gene VRN1, Yan et al. (2003) obtained the orthologous gene in sorghum and rice. Akiko et al. (2005) transformed wheat TaISA1 into rice su mutant and resumed su to the normal phenotype. It confirmed that the rice su mutant was caused by the loss of ISA1. The Pi36 was cloned by the approach of in silico map-based cloning (Liu et al. 2007). Comparative mapping between rice and maize also indicates that the lg-1 linkage group is a highly conserved region of the genome (Ahn and Tanksley 1993). According to the QTL analysis of ligule length and comparative genetics studies, we located OsLG1 on chromosome 4 in rice. Based on the function of ZmLG1 in maize, we speculated that OsLG1 impacts the proper formation of ligules and auricles at the blade-sheath boundary in rice. The homologous genes of ZmLG2, ZmLG3 and ZmLG4 were not detected in the QTL intervals, which may be explained by evolutionary divergence between rice and maize.

Squamosa promoter binding protein-domain proteins are plant-specific putative transcription factors, which consists of 76 highly conserved amino acids and overlap with a nuclear localization signal (NLS). SBP proteins bind specifically to the GTAC core motif in the A. majus SQUA promoter and Arabidopsis thaliana AP1 promoter (Klein et al. 1996; Birkenbihl et al. 2005). In maize, lg1 mutant not only fails to induce ligule and auricle, but disrupts some form of intercellular communication that is necessary for the normally coordinated development of the ligular region. The lack of LG1 possibly obstructs the inductive signal, which originates near the midvein traverse to the leaf margin. LG1 may be involved in the reception and/or propagation of a ‘make ligule/auricle’ signal that originates on either side of the midrib (Becraft and Freeling 1991). The homology of LG1 in rice perhaps also functions in the development of the laminar joint.

The size and shape of ligules in rice/maize are not only a primary trait for taxonomy in grasses, but also a feature playing a role in physical isolation, notably the prevention of pest invasion. Many QTLs conferring different traits were located on the same or similar regions. For example, the qLL-6 on chromosome 6 was close to the QTL locus of leaf hairs detected by Qian et al. (2000). The qLL-12 was located on chromosome 12 and is adjacent to the qFLA-12 detected by Dong et al. (2003). The QTL allele from indica varieties ‘TN1’ and ‘Zhaiyeqing 8’ could increase ligule length and flag leaf angle, respectively. Furthermore, we also found that the qLL-2 is adjacent to the qSBR-2 for rice sheath blight resistance detected by Kunihiro et al. (2002). The overlapping or adjacent locus between ligule length and rice morphology index, flag leaf angle and sheath blight resistance suggested that rice ligule is the trace of evolvement, but carrying important biological functions in rice.

Materials and Methods