Identification of the blast resistance gene Pit in rice cultivars using functional markers

DNA markers that allow for identification of resistance genes in rice germplasm have a great advantage in resistance breeding because they can assess the existence of the genes without laborious inoculation tests. Functional markers (FMs), which are designed from functional polymorphisms within the sequence of genes, are unaffected by nonfunctional allelic variation and make it possible to identify an individual gene. We previously showed that the resistance function of the rice blast resistance gene Pit in a resistant cultivar, K59, was mainly acquired by up-regulated promoter activity through the insertion of a long terminal repeat (LTR) retrotransposon upstream of Pit. Here, we developed PCR-based DNA markers derived from the LTR-retrotransposon sequence and used these markers to screen worldwide accessions of rice germplasm. We identified 5 cultivars with the LTR-retrotransposon insertion out of 68 rice accessions. The sequence and expression pattern of Pit in the five cultivars were the same as those in K59 and all showed Pit-mediated blast resistance. The results suggest that the functional Pit identified using the markers was derived from a common progenitor. Additionally, comparison of the Pit coding sequences between K59 and susceptible cultivars revealed that one nucleotide polymorphism, which caused an amino acid substitution, offered another target for a FM. These results indicate that our DNA markers should enhance prediction of Pit function and be applicable to a range of rice varieties/landraces cultivated in various regions worldwide and belonging to the temperate japonica, tropical japonica, and indica groups.     

Quantitative Trait Locus Mapping and Candidate Gene Analysis for Plant Architecture Traits Using Whole Genome Re-Sequencing in Rice

Plant breeders have focused on improving plant architecture as an effective means to increase crop yield. Here, we identify the main-effect quantitative trait loci (QTLs) for plant shape-related traits in rice (Oryza sativa) and find candidate genes by applying whole genome re-sequencing of two parental cultivars using next-generation sequencing. To identify QTLs influencing plant shape, we analyzed six traits: plant height, tiller number, panicle diameter, panicle length, flag leaf length, and flag leaf width. We performed QTL analysis with 178 F₇ recombinant in-bred lines (RILs) from a cross of japonica rice line ‘SNUSG1’ and indica rice line ‘Milyang23’. Using 131 molecular markers, including 28 insertion/deletion markers, we identified 11 main- and 16 minor-effect QTLs for the six traits with a threshold LOD value > 2.8. Our sequence analysis identified fifty-four candidate genes for the main-effect QTLs. By further comparison of coding sequences and meta-expression profiles between japonica and indica rice varieties, we finally chose 15 strong candidate genes for the 11 main-effect QTLs. Our study shows that the whole-genome sequence data substantially enhanced the efficiency of polymorphic marker development for QTL fine-mapping and the identification of possible candidate genes. This yields useful genetic resources for breeding high-yielding rice cultivars with improved plant architecture.     

Phenotypic screening and molecular analysis of blast resistance in fragrant rice for marker assisted selection

Experiments were conducted to identify blast-resistant fragrant genotypes for the development of a durable blast-resistant rice variety during years 2012–2013. The results indicate that out of 140 test materials including 114 fragrant germplasms, 25 differential varieties (DVs) harbouring 23 blast-resistant genes, only 16 fragrant rice germplasms showed comparatively better performance against a virulent isolate of blast disease. The reaction pattern of single-spore isolate of Magnaporthe oryzae to differential varieties showed that Pish, Pi9, Pita-2 and Pita are the effective blast-resistant genes against the tested blast isolates in Bangladesh. The DNA markers profiles of selected 16 rice germplasms indicated that genotype Chinigura contained Pish, Pi9 and Pita genes; on the other hand, both BRRI dhan50 and Bawaibhog contained Pish and Pita genes in their genetic background. Genotypes Jirakatari, BR5, and Gopalbhog possessed Pish gene, while Uknimodhu, Deshikatari, Radhunipagol, Kalijira (3), Chinikanai each contained the Pita gene only. There are some materials that did not contain any target gene(s) in their genetic background, but proved resistant in pathogenicity tests. This information provided valuable genetic information for breeders to develop durable blast-resistant fragrant or aromatic rice varieties in Bangladesh.     

Differential Gene Expression Reflects Morphological Characteristics and Physiological Processes in Rice Immunity against Blast Pathogen Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a serious pathogen that jeopardises the world’s most important food-security crop. Ten common Malaysian rice varieties were examined for their morphological, physiological and genomic responses to this rice blast pathogen. qPCR quantification was used to assess the growth of the pathogen population in resistant and susceptible rice varieties. The chlorophyll content and photosynthesis were also measured to further understand the disruptive effects that M. oryzae has on infected plants of these varieties. Real-time PCR was used to explore the differential expression of eight blast resistance genes among the ten local varieties. Blast disease has destructive effects on the growth of rice, and the findings of our study provide evidence that the Pikh, Pi9, Pi21, and Osw45 genes are involved in defence responses in the leaves of Malaysian rice at 31 h after inoculation with M. oryzae pathotype P7.2. Both the chlorophyll content and photosynthesis were reduced, but the levels of Pikh gene expression remained constant in susceptible varieties, with a developed pathogen population and mild or severe symptoms. The Pi9, Pi21, and Osw45 genes, however, were simultaneously upregulated in infected rice plants. Therefore, the presence of the Pikh, Pi9, Pi21, and Osw45 genes in the germplasm is useful for improving the resistance of rice varieties.     

Regions outside the leucine-rich repeat domain determine the distinct resistance specificities of the rice blast resistance genes Pik and Pik-m

Two alleles of the rice blast resistance (R) Pik locus, Pik-m and Pik, are each composed of a pair of nucleotide-binding site–leucine-rich repeat (NBS–LRR) genes, referred to as the first gene and the second gene. Pik-m and Pik are unique in that many of the amino acid substitutions between them are distributed in or near the N-terminal coiled-coil (CC) domain of the first gene, suggesting that the CC domain of the first gene plays an important role in determinating their R specificity. To examine this hypothesis, I investigated resistance phenotypes of transgenic plants carrying each of two kinds of domain-swapped Pik-m-based recombinant first genes. Replacement of the LRR domain of Pik-m with the equivalent region of Pik did not change the Pik-m-type specificity, indicating that regions outside the LRR domain are responsible for differentiating the R specificity of Pik-m from Pik. In contrast, replacement of both the NBS and LRR domains of Pik-m with the corresponding region of Pik resulted in loss of blast resistance, suggesting that co-adaptation of polymorphisms in the CC and NBS domains is necessary to maintain resistance.     

Whole-Genome Analysis Revealed the Positively Selected Genes during the Differentiation of indica and Temperate japonica Rice

To investigate the selective pressures acting on the protein-coding genes during the differentiation of indica and japonica, all of the possible orthologous genes between the Nipponbare and 93–11 genomes were identified and compared with each other. Among these genes, 8,530 pairs had identical sequences, and 27,384 pairs shared more than 90% sequence identity. Only 2,678 pairs of genes displaying a Ka/Ks ratio significantly greater than one were revealed, and most of these genes contained only nonsynonymous sites. The genes without synonymous site were further analyzed with the SNP data of 1529 O. sativa and O. rufipogon accessions, and 1068 genes were identified to be under positive selection during the differentiation of indica and temperate japonica. The positively selected genes (PSGs) are unevenly distributed on 12 chromosomes, and the proteins encoded by the PSGs are dominant with binding, transferase and hydrolase activities, and especially enriched in the plant responses to stimuli, biological regulations, and transport processes. Meanwhile, the most PSGs of the known function and/or expression were involved in the regulation of biotic/abiotic stresses. The evidence of pervasive positive selection suggested that many factors drove the differentiation of indica and japonica, which has already started in wild rice but is much lower than in cultivated rice. Lower differentiation and less PSGs revealed between the Or-It and Or-IIIt wild rice groups implied that artificial selection provides greater contribution on the differentiation than natural selection. In addition, the phylogenetic tree constructed with positively selected sites showed that the japonica varieties exhibited more diversity than indica on differentiation, and Or-III of O. rufipogon exhibited more than Or-I.     

Stacking of five favorable alleles for amylase content,fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker

Marker-assisted selection (MAS) for qualitative traits such as grain quality and resistance to certain diseases has proven to be highly effective. Multiple genes responsible for various quality components and disease resistances can be simultaneously stacked to boost the performance and to lengthen the commercial lifespan of high-yield varieties. Grain quality genes (fgr and Wx) and three disease resistance genes (Pita, Pik and Xa23) have been well characterized and used in MAS breeding. However, stacking all of them together into a single variety has not been reported. We reported here the stacking of the five genes into elite lines in rice. We achieved this through the development of functional markers from causal mutations at fgr, Wx and Pita, a gene-targeted marker at Pik and the use of a linked marker for Xa23. We employed and optimized the high-resolution melting (HRM) analysis method for use as the genotyping platform of fgr, Wx, Pik and Pita. By combining high-throughput DNA isolation, multiplex and nested-PCR methods, we showed that HRM could serve as cost-effective, highly automated, moderate-throughput and reliable non-gel genotyping platform for a small-scale MAS program.     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

The Genome Sequence of ‘Mycobacterium massiliense’ Strain CIP 108297 Suggests the Independent Taxonomic Status of the Mycobacterium abscessus Complex at the Subspecies Level

Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, ‘M. massiliense’, and ‘M. bolletii’. In 2011, ‘M. massiliense’ and ‘M. bolletii’ were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of ‘M. massiliense’ within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of ‘Mycobacterium massiliense’ (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of ‘M. bolletii’ and ‘M. massiliense’ at the subspecies level. The genome tree also clearly illustrated that ‘M. bolletii’ and ‘M. massiliense’ form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known ‘M. massiliense’ and ‘M. bolletii’ strains.     

Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers

Background and Aims The origin of limes and lemons has been a source of conflicting taxonomic opinions. Biochemical studies, numerical taxonomy and recent molecular studies suggested that cultivated Citrus species result from interspecific hybridization between four basic taxa (C. reticulata, C. maxima, C. medica and C. micrantha). However, the origin of most lemons and limes remains controversial or unknown. The aim of this study was to perform extended analyses of the diversity, genetic structure and origin of limes and lemons.Methods The study was based on 133 Citrus accessions. It combined maternal phylogeny studies based on mitochondrial and chloroplastic markers, and nuclear structure analysis based on the evaluation of ploidy level and the use of 123 markers, including 73 basic taxa diagnostic single nucleotide polymorphism (SNP) and indel markers.Key Results The lime and lemon horticultural group appears to be highly polymorphic, with diploid, triploid and tetraploid varieties, and to result from many independent reticulation events which defined the sub-groups. Maternal phylogeny involves four cytoplasmic types out of the six encountered in the Citrus genus. All lime and lemon accessions were highly heterozygous, with interspecific admixture of two, three and even the four ancestral taxa genomes. Molecular polymorphism between varieties of the same sub-group was very low.Conclusions Citrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia – ‘Mexican’ lime types of Tanaka’s taxa), C. reticulata (for C. limonia, C. karna and C. jambhiri varieties of Tanaka’s taxa, including popular citrus rootstocks such as ‘Rangpur’ lime, ‘Volkamer’ and ‘Rough’ lemons), C. aurantium (for C. limetta and C. limon – yellow lemon types – varieties of Tanaka’s taxa) or the C. maxima × C. reticulata hybrid (for C. limettioides – ‘Palestine sweet’ lime types – and C. meyeri). Among triploid limes, C. latifolia accessions (‘Tahiti’ and ‘Persian’ lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions (‘Tanepao’ lime types and ‘Madagascar’ lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations.     

Differential Responses of CO2 Assimilation,Carbohydrate Allocation and Gene Expression to NaCl Stress in Perennial Ryegrass with Different Salt Tolerance

Little is known about the effects of NaCl stress on perennial ryegrass (Lolium perenne L.) photosynthesis and carbohydrate flux. The objective of this study was to understand the carbohydrate metabolism and identify the gene expression affected by salinity stress. Seventy-four days old seedlings of two perennial ryegrass accessions (salt-sensitive ‘PI 538976’ and salt-tolerant ‘Overdrive’) were subjected to three levels of salinity stress for 5 days. Turf quality in all tissues (leaves, stems and roots) of both grass accessions negatively and significantly correlated with GFS (Glu+Fru+Suc) content, except for ‘Overdrive’ stems. Relative growth rate (RGR) in leaves negatively and significantly correlated with GFS content in ‘Overdrive’ (P<0.01) and ‘PI 538976’ (P<0.05) under salt stress. ‘Overdrive’ had higher CO₂ assimilation and F_v/F_m than ‘PI 538976’. Intercellular CO₂ concentration, however, was higher in ‘PI 538976’ treated with 400 mM NaCl relative to that with 200 mM NaCl. GFS content negatively and significantly correlated with RGR in ‘Overdrive’ and ‘PI 538976’ leaves and in ‘PI 538976’ stems and roots under salt stress. In leaves, carbohydrate allocation negatively and significantly correlated with RGR (r² = 0.83, P<0.01) and turf quality (r² = 0.88, P<0.01) in salt-tolerant ‘Overdrive’, however, the opposite trend for salt-sensitive ‘PI 538976’ (r² = 0.71, P<0.05 for RGR; r² = 0.62, P>0.05 for turf quality). A greater up-regulation in the expression of SPS, SS, SI, 6-SFT gene was observed in ‘Overdrive’ than ‘PI 538976’. A higher level of SPS and SS expression in leaves was found in ‘PI 538976’ relative to ‘Overdrive’. Accumulation of hexoses in roots, stems and leaves can induce a feedback repression to photosynthesis in salt-stressed perennial ryegrass and the salt tolerance may be changed with the carbohydrate allocation in leaves and stems.     

Characterization and fine mapping of the rice blast resistance gene <Emphasis Type="Italic">Pia</Emphasis>

Blast, caused by Magnaporthe oryzae, is one of the most widespread and destructive diseases of rice. Breeding durable resistant cultivars (cvs) can be achieved by pyramiding of various resistance (R) genes. Pia, carried by cv. Aichi Asahi, was evaluated against 612 isolates of M. oryzae collected from 10 Chinese provinces. The Pia gene expresses weak resistance in all the provinces except for Jiangsu. Genomic position-ready marker-based linkage analysis was carried out in a mapping population consisting of 800 F₂ plants derived from a cross of Aichi Asahi×Kasalath. The locus was defined in an interval of approximately 90 kb, flanked by markers A16 and A21. Four candidate genes (Pia-1, Pia-2, Pia-3, and Pia-4), all having the R gene conserved structure, were predicted in the interval using the cv. Nipponbare genomic sequence. Four candidate resistance gene (CRG) markers (A17, A25, A26, and A27), derived from the four candidates, were subjected to genotyping with the recombinants detected at the flanking markers. The first three markers completely co-segregated with the Pia locus, and the fourth was absent in the Aichi Asahi genome and disordered with the Pia locus and its flanking markers, indicating that the fourth candidate gene, Pia-4, could be excluded. Co-segregation marker-based genotyping of the three sets of differentials with known R gene genotypes revealed that the genotype of A26 (Pia-3) perfectly matched the R gene genotype of Pia, indicating that Pia-3 is the strongest candidate gene for Pia.     

Historical Introgression of the Downy Mildew Resistance Gene Rpv12 from the Asian Species Vitis amurensis into Grapevine Varieties

The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance–a hypersensitive response in leaves challenged with P. viticola–was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12⁺ haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12⁺ haplotype is shared by 15 varieties, the most ancestral of which are the century-old ‘Zarja severa’ and ‘Michurinets’. Before this knowledge, the chromosome segment around Rpv12⁺ became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12⁺ has an additive effect with Rpv3⁺ to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3⁺ plants.     

Genotyping and development of single-nucleotide polymorphism (SNP) markers associated with blast resistance genes in rice using GoldenGate assay

Development and large-scale genotyping of single-nucleotide polymorphism (SNP) is required to use identified sequence variation in the alleles of different genes to determine their functional relevance to the candidate gene(s). In the present study, Illumina GoldenGate assay was used to validate and genotype SNPs in a set of six major rice blast resistance genes, viz. Pi-ta, Piz(t), Pi54, Pi9, Pi5(1) and Pib, distributed over five chromosomes, to understand their functional relevance and study the population structure in rice. All the selected SNPs loci (96) of six blast (Magnaporthe oryzae) resistance genes were genotyped successfully in 92 rice lines with an overall genotype call rate of 92.0 % and minimum GenTrain cutoff score of ≥0.448. The highest genotyped SNPs were found in japonica type (97.1 %) rice lines, followed by indica (92.12 %), indica basmati (91.84 %) and minimum in case of wild species (82.0 %). Among the genotyped loci, the highest score (98.68 %) was observed in case of Piz(t), followed by Pi-ta, Pi5(1), Pib, Pi54 and Pi9. Polymorphism was obtained in 87.5 % SNPs loci producing 7,728 genotype calls. Minor allele frequency ranged from 0.01 to 0.49 and has good differentiating power for distinguishing different rice accessions. Population structure analysis revealed that a set of genotypes from four rice subpopulations had “admix” ancestry (>26 %) with more than one genetic background of indica, japonica and wild types. SNPs markers were validated in a set of 92 rice lines and converted into CAPS markers which can be used in blast resistance breeding programme.     

Genetic Features of the Spontaneous Self-Compatible Mutant, ‘Jin Zhui’ (Pyrus bretschneideri Rehd.)

‘Jin Zhui’ is a spontaneous self-compatible mutant of ‘Ya Li’ (Pyrus bretschneideri Rehd. S₂₁S₃₄), the latter displaying a typical S-RNase-based gametophytic self-incompatibility (GSI). The pollen-part mutation (PPM) of ‘Jin Zhui’ might be due to a natural mutation in the pollen-S gene (S₃₄ haplotype). However, the molecular mechanisms behind these phenotypic changes are still unclear. In this study, we identified five SLF (S-Locus F-box) genes in ‘Ya Li’, while no nucleotide differences were found in the SLF genes of ‘Jin Zhui’. Further genetic analysis by S-RNase PCR-typing of selfed progeny of ‘Jin Zhui’ and ‘Ya Li’ × ‘Jin Zhui’ progeny showed three progeny classes (S₂₁S₂₁, S₂₁S₃₄ and S₃₄S₃₄) as opposed to the two classes reported previously (S₂₁S₃₄ and S₃₄S₃₄), indicating that the pollen gametes of ‘Jin Zhui’, bearing either the S₂₁- or S₃₄-haplotype, were able to overcome self-incompatibility (SI) barriers. Moreover, no evidence of pollen-S duplication was found. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in ‘Jin Zhui’, rather than natural mutation in the pollen-S gene (S₃₄ haplotype). Furthermore, abnormal meiosis was observed in a number of pollen mother cells (PMCs) in ‘Jin Zhui’, but not in ‘Ya Li’. These and other interesting findings are discussed.