Detection of QTLs Regulating Six Agronomic Traits of Rice Based on Chromosome Segment Substitution Lines of Common Wild Rice (Oryza rufipogon Griff.) and Mapping of qPH1.1 and qLMC6.1

Abstract

Wild rice is a primary source of genes that can be utilized to generate rice cultivars with advantageous traits. Chromosome segment substitution lines (CSSLs) are consisting of a set of consecutive and overlapping donor chromosome segments in a recipient's genetic background. CSSLs are an ideal genetic population for mapping quantitative traits loci (QTLs). In this study, 59 CSSLs from the common wild rice (Oryza rufipogon Griff.) accession DP15 under the indica rice cultivar (O. sativa L. ssp. indica) variety 93-11 background were constructed through multiple backcrosses and marker-assisted selection (MAS). Through high-throughput whole genome re-sequencing (WGRS) of parental lines, 12,565 mapped InDels were identified and designed for polymorphic molecular markers. The 59 CSSLs library covered 91.72% of the genome of common wild rice accession DP15. The DP15-CSSLs displayed variation in six economic traits including grain length (GL), grain width (GW), thousand-grain weight (TGW), grain length-width ratio (GLWR), plant height (PH), and leaf margin color (LMC), which were finally attributed to 22 QTLs. A homozygous CSSL line and a purple leave margin CSSL line were selected to construct two secondary genetic populations for the QTLs mapping. Thus, the PH-controlling QTL qPH1.1 was mapped to a region of 4.31-Mb on chromosome 1, and the LMC-controlling QTL qLMC6.1 was mapped to a region of 370-kb on chromosome 6. Taken together, these identified novel QTLs/genes from common wild rice can potentially promote theoretical knowledge and genetic applications to rice breeders worldwide.

Keywords: QTL mapping; agronomic traits; chromosome segment substitution lines (CSSLs); common wild rice (Oryza rufipogon); molecular markers; qLMC6.1; qPH1.1.

Figure 1

Phenotypic morphology of DP15 and 93-11. (a), plant phenotype of DP15 and 93-11, bar = 20 cm; (b), panicle morphology of DP15 and 93-11, Bar = 5 cm; (c), phenotype of mature grain of DP15 and 93-11, bar = 1 cm; (d), length of brown rice of DP15 and 93-11, bar = 1 cm; (e), width of brown rice of DP15 and 93-11, bar = 1 cm.

Figure 2

Graphical genotypes of the 59 lines DP15-CSSLs. Note: Black bars indicate homozygous chromosome substituted segments derived from DP15; Yellow bars indicate heterozygous substituted segments derived from DP15; Grey bars indicate the genetic background of recipient parent 93-11.

Figure 3

Frequency distribution of 59 DP15-CSSLs and their two parents on four grain traits over two years. Note: (a), phenotypic distribution of TGW in DP15, 93-11, and the DP15-CSSLs; (b), phenotypic distribution of GL in DP15, 93-11, and the DP15-CSSLs; (c), phenotypic distributions of GW in DP15, 93-11, and the DP15-CSSLs; (d), phenotypic distribution of GLWR in DP15, 93-11, and the DP15-CSSLs; The marked solid arrow lines and dotted arrow lines indicate the phenotypic distribution of DP15 and 93-11 respectively.

Figure 4

Detection of QTLs related to four grain traits in the DP15-CSSLs. (a), detection of QTLs related to TGW in DP15-CSSLs; (b), detection of QTLs related to GL in DP15-CSSLs; (c), detection of QTLs related to GW in DP15-CSSLs; (d), detection of QTLs related to GLWR in DP15-CSSLs.

Figure 5

Phenotype of a PH-related DP15-CSSL line. (a), the plant architecture of ZN6 and 93-11 at heading stage, bar = 50 cm; (b), the internode of ZN6 and 93-11 at heading stage, bar = 5 cm; (c), the histogram of internode length of ZN6 and 93-11 at heading stage; the sign “*” in Figure 5c indicates a p ≤ 0.05 level; The sign “**” in Figure 5c indicates a p ≤ 0.01 level.

Figure 6

Tissue section, vessel scanning, and paraffin section figures of the uppermost internode at the grain filling stage. (a), the tissue section figure of the uppermost internode of ZN6, bar = 100 μm; (b), the tissue section figure of the uppermost internode of 93-11, bar = 100 μm; (c), the horizontal paraffin section figure of the uppermost internode of ZN6, bar = 100 μm; (d), the horizontal paraffin section figure of the uppermost internode of 93-11, bar = 100 μm; (e), the horizontal SEM section figure of the uppermost internode of ZN6, bar = 100 μm; (f), the horizontal SEM section figure of the uppermost internode of 93-11, bar = 100 μm; (g), the horizontal paraffin section figure of the uppermost internode of ZN6 and 93-11 at heading stage, bar = 200 μm; (h), the horizontal paraffin section figure of the uppermost internode of ZN6 and 93-11 at heading stage, bar = 200 μm; (i), the longitudinal paraffin section figure of the uppermost internode of ZN6, bar = 100 μm (j), the longitudinal paraffin section figure of the uppermost internode of 93-11, bar = 100 μm.

Figure 7

Genetic mapping of the qPH1.1 in rice. (a), the QTL detection figure of qPH1.1; (b), the BSA analysis for qPH1.1; (c), the recombinant identification and genetic mapping for qPH1.1.

Figure 8

Phenotype of ZN32, an LMC-related DP15-CSSL line. (a), the plant architecture of ZN32 and 93-11 at heading stage, bar = 20 cm; (b–d) in Figure 8 show the leaf margin morphology of ZN32 and 93-11 at heading stage, the red arrow in (b–d) indicates the leaf margin site, the bar in (b–d) are 1 cm, 5 mm, and 5 mm, respectively; (e), the ligule and auricle color of ZN32 and 93-11 at heading stage, the red arrow in (e) shows the auricle site, the scale bar = 5 mm; (f), the basal shoot of ZN32 and 93-11 at heading stage, the red arrow in (f) shows the basal shoot region, bar = 5 cm; (g), the leaf collar phenotype of ZN32 and 93-11 at heading stage, the red arrow in (g) shows the lamina joint site, bar = 5 mm; (h), the apiculus color of ZN32 and 93-11 at heading stage, the yellow arrow in (h) shows apiculus site; the red arrow shows stigma site, bar = 1 mm; (i), the stigma color of ZN32 and 93-11 at heading stage; The red arrow shows stigma site, bar = 1 mm; (j), the rice basal culm with leaf sheath surrounded of ZN32 and 93-11 at heading stage, the white arrow in (j) shows the zone of inner leaf sheath, bar = 5 mm; (k), the rice basal culm of ZN32 and 93-11 at heading stage, the white arrow in Figure 8k shows the borders of the culm, bar = 5 mm.

Figure 9

Phenotypic comparison of protoplasts extracted from ZN32 and 93-11 stigma under confocal microscopy. Note: (a–f), the protoplast of ZN32 showing fluorescence; (g–l), the protoplast of 93-11 showing no fluorescence; V indicates the position of vacuole; N indicates the position of nucleus in protoplast, bar = 5 μm.

Figure 10

Genetic mapping of the qLMC6.1 in rice. (a), the QTL detection figure of qLMC6.1; (b), the bulk segregation analysis for the qLMC6.1; (c), the recombinants identification and genetic mapping for the qLMC6.1; (I), simple model of Chromosome 6; (II), the distribution of primers used for primary mapping; (III), the distribution of primers used for fine mapping; (IV), the screening for recombinants for the mapping of qLMC6.1.