Developing rice with high yield under phosphorus deficiency: Pup1 sequence to application

Abstract

The major quantitative trait locus (QTL) Phosphorus uptake1 (Pup1) confers tolerance of phosphorus deficiency in soil and is currently one of the most promising QTLs for the development of tolerant rice (Oryza sativa) varieties. To facilitate targeted introgression of Pup1 into intolerant varieties, the gene models predicted in the Pup1 region in the donor variety Kasalath were used to develop gene-based molecular markers that are evenly distributed over the fine-mapped 278-kb QTL region. To validate the gene models and optimize the markers, gene expression analyses and partial allelic sequencing were conducted. The markers were tested in more than 80 diverse rice accessions revealing three main groups with different Pup1 allele constitution. Accessions with tolerant (group I) and intolerant (group III) Pup1 alleles were distinguished from genotypes with Kasalath alleles at some of the analyzed loci (partial Pup1; group II). A germplasm survey additionally confirmed earlier data showing that Pup1 is largely absent from irrigated rice varieties but conserved in varieties and breeding lines adapted to drought-prone environments. A core set of Pup1 markers has been defined, and sequence polymorphisms suitable for single-nucleotide polymorphism marker development for high-throughput genotyping were identified. Following a marker-assisted backcrossing approach, Pup1 was introgressed into two irrigated rice varieties and three Indonesian upland varieties. First phenotypic evaluations of the introgression lines suggest that Pup1 is effective in different genetic backgrounds and environments and that it has the potential to significantly enhance grain yield under field conditions.

Figure 1.

Pup1 gene models and positions of markers. A, The genomic Pup1 region of the tolerant donor Kasalath is defined by the markers T5-4 and Ba76H14_7154 (Heuer et al., 2009; accession no. AB458444.1) and characterized by a large INDEL (gray line) with adjacent regions partially conserved in Nipponbare (black lines). The positions and names of the developed codominant and dominant markers are indicated. Markers K4 and K67 were not polymorphic. B to E, The targeted gene models and marker positions within the models. B, The putative gene OsPupK05-1 is located within an intron of gene OsPupK04-1, which is highly similar to the shown Nipponbare gene model Os12g26290. C, For the dirigent-like gene model OsPupK20-2, two markers were developed, and PCR products can be digested with Bsp1286I (white triangles) and MseI (gray triangle). D, The hypothetical protein gene OsPupK29-1 is partially similar to two genes in Nipponbare, and the markers target exon 1, exon 2, and an intron. E, For the protein kinase OsPupK46-2, the markers amplify a region flanking the protein kinase domain (K46-1) and the 3′ region (K46-2). Black boxes in the gene models indicate exons, and lines indicate introns and untranslated regions. Conserved protein domains are highlighted in gray.

Figure 2.

RT-PCR gene expression analysis. The Pup1 genes indicated were analyzed by RT-PCR using RNA samples derived from roots and shoots of plants grown in P-deficient soil with (+P) and without (−P) P fertilizer application. Two different tolerant Nipponbare-Pup1 NILs with the Kasalath Pup1 locus were used (NIL24-4 = 1+; NIL14-4 = 2+). Nipponbare (NB) and a NIL14-4 sister line without the tolerant Pup1 locus (NIL14-6 = −) were used as intolerant controls. cDNA samples were analyzed by PCR using the cycle number indicated on the right. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as a control. H₂O, Water control.

Figure 3.

Sequence comparisons of marker amplicons. A, PCR amplicons of five markers were sequenced in the rice varieties indicated to determine the specificity of the primers and validate sequence polymorphisms. For OsPupK20-2, a nonsynonymous SNP at +620 bp was identified that changed the amino acid sequence in exon 2 (T in Nipponbare, C in Kasalath; left) and created a Bsp1286I site in Kasalath-type alleles but not in non-Kasalath (N-type) alleles. In the adjacent intron, one SNP (+660 bp) and one INDEL (+665–671 bp) were identified. The SNP creates an MseI restriction site (right). B, For OsPupK29-1, a polymorphic region with one INDEL and four SNPs is amplified by the marker K29-1. C, Another INDEL in OsPupK29-1 is targeted by the marker K29-3. D, PCR products of the dominant marker K46-2 (stop codon is highlighted in gray) were sequenced in three varieties that possess the OsPupK46 gene. Only polymorphic regions of the sequenced DNA fragments are shown.

Figure 4.

PCR amplicons of Pup1 gene-specific markers. Seven representative rice varieties were genotyped with nine codominant (top panels) and nine dominant (bottom panels) Pup1-specific markers. The markers target a total of 12 Kasalath Pup1 gene models. PCR products K20-1 and K20-2 were digested with MseI (K20-1_Mse) and Bsp1286I (K20-2_Bsp), respectively, to enhance resolution. K, Kasalath; N, Nipponbare; B, Batur; D, Dodokan; SB, Situ Bagendit; H₂O, water control. The sizes of the DNA fragments in Kasalath and Nipponbare (K/N) and unspecific amplicons that sometimes occur are indicated on the right. The absence of PCR amplicons with dominant markers is indicated as “none.”

Figure 5.

Pup1 haplotype in diverse rice genotypes. Eighty-one diverse rice varieties and breeding lines were genotyped using the Pup1-specific markers indicated. Based on the Pup1 core marker set (highlighted with dashed lines; see text for details), an UPGMA cluster analysis was conducted using Powermarker version 3.25 (Sokal and Michener, 1958; Liu and Muse, 2005). Three main groups (I, II, III) with different allelic composition at the Pup1 locus were identified. The different allele calls (Kasalath, non-Kasalath, and heterozygous) are color coded as indicated at bottom left. The rice genotypes analyzed are adapted to different rice agroecosystems [upland (UL): aerobic, unfavorable, and drought prone; lowland/irrigated (LL): anaerobic, generally favorable conditions] as shown at right. For details on the genotypes analyzed, see text and Supplemental Table S1.

Figure 6.

Pup1 core markers. A GGT 2.0 analysis (Van Berloo 2008) was conducted with the marker data of the 81 genotypes analyzed with the indicated Pup1-specific markers. The r² values of the individual markers are represented as a heat plot (black indicates r² = 1). The approximate physical locations of the markers are indicated on the black line, which represents the Kasalath Pup1 locus. Two haploblocks can be identified that define the core marker set (thick lines). The marker K29-2 is not associated with the other two markers for the OsPupK29-1 gene (dashed lines; see text for details).

Figure 7.

Field evaluation and selection of IR64-Pup1 and IR74-Pup1 breeding lines. A, Sister lines with (+Pup1) and without (−Pup1) the tolerant Pup1 locus and control plants were grown under irrigated field conditions in P-deficient soil (P−) and in a P-fertilized parallel control plot (P+) in three replicates. Phenotypic data from the BC₂F₃ populations were collected in 2010. Data were analyzed by paired t test (95%). Significance levels are as follows: * 0.05 > P ≥ 0.01, ** 0.01 > P ≥ 0.001, *** 0.001 > P. No asterisk indicates not significant. B, For the final selection of IR64-Pup1 and IR74-Pup1 plants, contrasting BC₂F₄ sister lines were genotyped with selected Pup1 core markers (top). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as a control. K, Kasalath; N, Nipponbare. Representative plants are shown at bottom.

Figure 8.

Field evaluation of three Indonesian Pup1 breeding lines. Advanced generations (BC₂F₄) of Pup1 breeding populations of three Indonesian upland varieties were grown in a field in Sukabumi (West Java, Indonesia) under rain-fed conditions in 2010. The data show average grain yield per plant obtained with (P+; y axis) and without (P−; x axis) P fertilizer application. Grain yield of the Pup1 recipient parents is highlighted in the graphs as light gray marker. Horizontal and vertical lines indicate se. The gray bar at the top of each graph signifies the Pup1 locus of the recipient parent before introgression of the tolerant Pup1 locus. Kasalath-type alleles naturally present in the recipient parent detected with the indicated Pup1 markers are shown as black lines.