Multi-trait association study identifies loci associated with tolerance of low phosphorus in Oryza sativa and its wild relatives

Abstract

We studied variation in adaptive traits and genetic association to understand the low P responses, including the symbiotic association of arbuscular mycorrhizal (AM) fungal colonization in Oryza species (O. sativa, O. nivara, and O. rufipogon). In the present experiment, we performed the phenotypic variability of the morphometric and geometric traits for P deficiency tolerance and conducted the association studies in GLM and MLM methods. A positive association between the geometric trait of the top-view area and root traits suggested the possibility of exploring a non-destructive approach in screening genotypes under low P. The AMOVA revealed a higher proportion of variation among the individuals as they belonged to different species of Oryza and the NM value was 2.0, indicating possible gene flow between populations. A sub-cluster with superior-performing accessions had a higher proportion of landraces (42.85%), and O. rufipogon (33.3%) was differentiated by four Pup1-specific markers. Association mapping identified seven notable markers (RM259, RM297, RM30, RM6966, RM242, RM184, and PAP1) and six potential genotypes (IC459373, Chakhao Aumbi, AC100219, AC100062, Sekri, and Kumbhi Phou), which will be helpful in the marker-assisted breeding to improve rice for P-deprived condition. In addition, total root surface area becomes a single major trait that helps in P uptake under deficit P up to 33% than mycorrhizal colonization. Further, the phenotypic analysis of the morphometric and geometric trait variations and their interactions provides excellent potential for selecting donors for improving P-use efficiency. The identified potential candidate genes and markers offered new insights into our understanding of the molecular and physiological mechanisms driving PUE and improving grain yield under low-P conditions.

Figure 1

Box plot showing the population distribution of traits measured under P deficiency for 155 rice genotypes. (a) Shoot length and leaf length; (b) leaf number (leaf no.) and maximum root length (max root lth); (c) leaf width (leaf wd) and root dry weight (RDW); (d) stem thickness and root P content (R_P); (e) shoot dry weight (SDW) and root-shoot dry weight ratio (R/S_DW); (f) tiller number (Tiller/pl) and root volume (RV); (g) total dry weight (TDW) and root-shoot length ratio (R/S_lth); (h) root tips and top-view area (TVA); (i) SPAD, total root projected area (TRPA), and mycorrhiza colonization (%) (M_col); (j) average root diameter (ARD) and shoot P content (S_P); and (k) total root length (T_root_lth) and (l) total root surface area (TRSA). The upper, median, and lower quartiles of the boxes represent the 75th, 50th, and 25th percentiles of the population, respectively. The square box inside the quartile box represents mean and the asterisk represents outliers.

Figure 2

Pearson correlation matrix among the 23 traits measured under P deficiency for 155 rice genotypes. The color denotes the correlation, where 1 represents a complete positive correlation (dark blue) and −1 represents a complete negative correlation (dark red) between two traits. A large circle denotes strong correlation and a small circle indicates a weaker correlation.

Figure 3

PCA biplot graph representing genotypes in two main principal components for traits measured under P-deprived conditions, and these two components explained 38.52% and 15.76% of the variance, respectively. The vector's direction and length indicate the traits' contribution to the first two components in the PCA. The transparency of the trait vectors represents the contribution to the variance in the dataset, ranging from 2% (lightest) to 6% (darkest). Genotypes were divided into five groups based on their level of tolerance. Groups 1 and 2 consisted of improved genotypes with high tissue P in root and more root growth, respectively. Group 3 consisted of the mixture of landraces, O. rufipogon, O.nivara, and positive checks. Group 4 consisted of a mixture of all species with more shoot P and Group 5 had O. nivara and O. rufipogon together with higher root-shoot dry weight ratio.

Figure 4

Principal coordinate analysis (PCoA) of the five sub-populations (Pop 1 (irrigated), Pop 2 (rainfed lowland), Pop 3 (upland), Pop 4 (O. nivara), and Pop 5 (O. rufipogon)) were plotted into three major clusters. (a) AMOVA showed maximum variation among the individuals, followed by within individuals and between populations. The genetic variability estimated by the fixation index revealed (Fst = 0.11) indicates the existence of moderate genetic differentiation within the population. (b) Nei genetic diversity among the assumed sub-population using principal coordinate analysis (PCoA). The assumed five sub-populations (Population 1 (irrigated), Population 2 (rainfed lowland), Population 3 (upland), Population 4 (O. nivara), and Population 5 (O. rufipogon)) were plotted into three major clusters.

Figure 5

Unrooted tree using unweighted neighbor-joining (UNJ) method depicting clustering pattern of a panel population of 120 accessions in response to all 78 primers collectively, with and without Pup1 markers. (a) 78 primers grouped genotypes into three major clusters. Cluster-I (blue) constitutes 45 genotypes that were divided into three major sub-clusters. Cluster-II (green) was divided into six sub-clusters with 65 genotypes, most of them being improved varieties, including CR Dhan 801 and Kasalath. Cluster-III (red) was grouped as a separate cluster with nine genotypes of northeastern states of India with two improved lines. (b) The cluster analysis with 65 low-P linked markers separated 120 genotypes into three groups. Cluster-I (green) represented only 65 improved lines Cluster-II (red) grouped 47 genotypes comprising wild species and a few O. sativa (landraces and improved lines) and Cluster-III (blue) had eight genotypes with seven improved varieties and one wild accession. (c) The cluster analysis with Pup1-specific markers grouped 120 genotypes into three major clusters. Cluster-I (red) consisted of 48 genotypes and was further divided into three sub-clusters. Cluster-II (green) separated 45 genotypes into three sub-clusters and Cluster-III (blue) represented 30 genotypes that were further divided into three sub-clusters with 14 (III-1), 13 (III-2), and 3 (III-3) genotypes. The positive checks Dular and Kasalath were grouped into sub-cluster III-2 with IC459373, multiple-stress-tolerant CR Dhan 801, Poongar, Sekri, Kouni, AC10062, AC100326, AC100284, AC 100281, AC 100135, and AC 100117.

Figure 6

Graph of ∆K-value and ad hoc statistics related to the rate of change in the log probability of data between successive K-values. (a) 78 markers identified the highest log-likelihood with the number of populations set at three (K = 3) with ΔK = 248.51, (b) 65 markers linked to low P identified the highest log-likelihood with the number of populations set at three (K = 3) with ΔK of 179.01, and (c) Pup1-specific markers identified the highest log-likelihood with the number of populations set at two (K = 2) with ΔK = 106.29.

Figure 7

Distribution pattern of 120 rice accessions based on low-P linked markers and Pup1-specific markers determined by a model-based simulation, STRUCTURE 2.3.4. Grouping of accessions is based on (a) 78 markers, (b) 65 markers linked to low P, and (c) Pup1-specific markers. The number indicates the order of genotypes as mentioned in Table 1S.

Figure 8

Relationships between shoot biomass, total root surface area (TRSA) and mycorrhizal colonization with P under deficient condition. (a) The negative association between shoot biomass and root P concentration suggests that an increase in biomass (shoot) in deprived P was associated with the dilution effect of P. (b) The line indicates the fitted results representing the relationship between total P of plant tissue and possible parameters (TRSA and mycorrhizal colonization) involved in P uptake under P deficient condition. The contribution of improved P uptake of TRSA was high compared to mycorrhizal colonization.

Figure 9

Root and AM colonization in different rice genotypes found in low-P soil as shown by trypan blue staining. (a,e) Dular, (b,f) Kasalath, (c,g) Sekri, and (d,h) AC100219.

Figure 10

Distribution of primers used for association mapping and detected QTLs on 11 chromosomes of rice. Distances on the map are in Mbp presented on the left-hand side of the chromosomes. Markers highlighted in red are found associated with adaptive traits under P-deprived conditions. [SL shoot length (cm), NT tillers plant⁻¹, NL leaf number plant⁻¹, LL leaf length (cm), LW leaf width (cm), SG stem thickness (mm), RL max. root length (cm), SPAD, SDW shoot dry weight (g), RDW root dry weight (g), TDW total dry weight (g), TRL total root length (cm), TRPA total root projected area (cm²), TSA total root surface area (cm²), ARD average root diameter (mm), RV root volume (cm³), RT root tips, TPA top-view area (mm²), MC mycorrhiza colonization (%), SP shoot P (mg g⁻¹), RP root P (mg g⁻¹)].