Chromosome-level genome assembly of milk thistle (Silybum marianum (L.) Gaertn.).

Silybum marianum (L.) Gaertn., commonly known as milk thistle, is a medicinal plant belonging to the Asteraceae family. This plant has been recognized for its medicinal properties for over 2,000 years. However, the genome of this plant remains largely undiscovered, having no reference genome at a chromosomal level. Here, we assembled the chromosome-level genome of S. marianum, allowing for the annotation of 53,552 genes and the identification of transposable elements comprising 58% of the genome. The genome assembly from this study showed 99.1% completeness as determined by BUSCO assessment, while the previous assembly (ASM154182v1) showed 36.7%. Functional annotation of the predicted genes showed 50,329 genes (94% of total genes) with known protein functions in public databases. Comparative genome analysis among Asteraceae plants revealed a striking conservation of collinearity between S. marianum and C. cardunculus. The genomic information generated from this study will be a valuable resource for milk thistle breeding and for use by the larger research community.

A meta-QTL analysis highlights genomic hotspots associated with phosphorus use efficiency in rice (Oryza sativa L.).

Phosphorus use efficiency (PUE) is a complex trait, governed by many minor quantitative trait loci (QTLs) with small effects. Advances in molecular marker technology have led to the identification of QTLs underlying PUE. However, their practical use in breeding programs remains challenging due to the unstable effects in different genetic backgrounds and environments, interaction with soil status, and linkage drag. Here, we compiled PUE QTL information from 16 independent studies. A total of 192 QTLs were subjected to meta-QTL (MQTL) analysis and were projected into a high-density SNP consensus map. A total of 60 MQTLs, with significantly reduced number of initial QTLs and confidence intervals (CI), were identified across the rice genome. Candidate gene (CG) mining was carried out for the 38 MQTLs supported by multiple QTLs from at least two independent studies. Genes related to amino and organic acid transport and auxin response were found to be abundant in the MQTLs linked to PUE. CGs were cross validated using a root transcriptome database (RiceXPro) and haplotype analysis. This led to the identification of the eight CGs (OsARF8, OsSPX-MFS3, OsRING141, OsMIOX, HsfC2b, OsFER2, OsWRKY64, and OsYUCCA11) modulating PUE. Potential donors for superior PUE CG haplotypes were identified through haplotype analysis. The distribution of superior haplotypes varied among subspecies being mostly found in indica but were largely scarce in japonica. Our study offers an insight on the complex genetic networks that modulate PUE in rice. The MQTLs, CGs, and superior CG haplotypes identified in our study are useful in the combination of beneficial alleles for PUE in rice.

Inhibitory Effects of Polyphenol- and Flavonoid-Enriched Rice Seed Extract on Melanogenesis in Melan-a Cells via MAPK Signaling-Mediated MITF Downregulation.

Melanin production is an important process that prevents the host skin from harmful ultraviolet radiation; however, an overproduction of melanin results in skin diseases. In the present study, we determined the antioxidative and anti-melanogenic activities of polyphenol- and flavonoid-enriched rice seed extracts in melan-a cells. The polyphenol and flavonoid content of Hopum (HP) and Sebok (SB) rice seed extracts was measured. The antioxidant capacity was determined using the ABTS radical scavenging method. SB contained high amounts of polyphenols and flavonoids, which significantly increased antioxidative activity compared with HP. Various concentrations of these extracts were evaluated in a cytotoxicity using melan-a cells. At 100 µg/mL, there was no significant difference for all treatments compared with untreated cells. Therefore, 100 µg/mL was selected as a concentration for the further experiments. SB significantly suppressed the phosphorylation/activation of p-38 MAPK, increased the expression of phosphorylated ERK 1/2 and Akt, and downregulated the microphthalmia-associated transcription factor (MITF). This resulted in decreased levels of tyrosinase and tyrosinase-related protein-1 and -2. These results indicate the potential of polyphenol- and flavonoid-enriched rice seed as a treatment for hyperpigmentation.

A Genomic Evaluation of Six Selected Inbred Lines of the Naturalized Plants of Milk Thistle (Silybum marianum L. Gaertn.) in Korea.

Milk thistle (Silybum marianum) belongs to the Asteraceae family and is a medicinal plant native to the Mediterranean Basin. Silymarin in achene is a widely used herbal product for chronic liver disease. There is growing interest in natural medicine using milk thistle in Korea, but the raw material completely relies on imports. Despite its economic importance, phenotypic evaluations of native resources of milk thistle in Korea have not been carried out. In addition, genomic research and molecular marker development are very limited in milk thistle. In this study, we evaluated 220 milk thistle resources consisting of 172 accessions collected from the domestic market, and 48 accessions isolated from 6 accessions distributed by the National Agrobiodiversity Center in Korea. Six plant characteristics (height, seed weight, number of flowers, seed weight per flower, spine length, and color at harvest) were measured, and six samples (M01-M06) were selected to represent the genetic diversity of the population for genomic research. To develop PCR-based and co-dominant insertion/deletion (InDel) markers, we performed genome-wide InDel detection by comparing the whole-genome resequencing data of the six selected accessions with the reference genome sequence (GCA_001541825). As a result, 177 InDel markers with high distinguishability and reproducibility were selected from the 30,845 InDel variants. Unknowingly imported alien plant resources could easily be genetically mixed, and jeopardized seed purity can cause continuous difficulties in the development of high value-added agricultural platforms utilizing natural products. The selected plant materials and 177 validated InDel markers developed via whole-genome resequencing analysis could be valuable resources for breeding, conservation, and ecological studies of natives to Korea, along with acceleration of Silybum marianum industrialization.

Corrigendum: GWAS analysis to elucidate genetic composition underlying a photoperiod-insensitive rice population, North Korea.

[This corrects the article DOI: 10.3389/fgene.2022.1036747.].

GWAS analysis to elucidate genetic composition underlying a photoperiod-insensitive rice population, North Korea.

Heading date (Hd) is one of the main factors determining rice production and regional adaptation. To identify the genetic factors involved in the wide regional adaptability of rice, we conducted a genome-wide association study (GWAS) with 190 North Korean rice accessions selected for non-precocious flowering in the Philippines, a low-latitude region. Using both linear mixed models (LMM) and fixed and random model circulating probability unification (FarmCPU), we identified five significant loci for Hd in trials in 2018 and 2019. Among the five lead single nucleotide polymorphisms (SNPs), three were located adjacent to the known Hd genes, Heading date 3a (Hd3a), Heading date 5 (Hd5), and GF14-c. In contrast, three SNPs were located in novel loci with minor effects on heading. Further GWAS analysis for photoperiod insensitivity (PS) revealed no significant genes associated with PS, supporting that this North Korean (NK) population is largely photoperiod-insensitive. Haplotyping analysis showed that more than 80% of the NK varieties harbored nonfunctional alleles of major Hd genes investigated, of which a nonfunctional allele of Heading date 1 (Hd1) was observed in 66% of the varieties. Geographical distribution analysis of Hd allele combination types showed that nonfunctional alleles of floral repressor Hd genes enabled rice cultivation in high-latitude regions. In contrast, Hd1 alleles largely contributed to the wide regional adaptation of rice varieties. In conclusion, an allelic combination of Hd genes is critical for rice cultivation across wide areas.

Transcriptional repressor RST1 controls salt tolerance and grain yield in rice by regulating gene expression of asparagine synthetase.

Salt stress impairs nutrient metabolism in plant cells, leading to growth and yield penalties. However, the mechanism by which plants alter their nutrient metabolism processes in response to salt stress remains elusive. In this study, we identified and characterized the rice (Oryza sativa) rice salt tolerant 1 (rst1) mutant, which displayed improved salt tolerance and grain yield. Map-based cloning revealed that the gene RST1 encoded an auxin response factor (OsARF18). Molecular analyses showed that RST1 directly repressed the expression of the gene encoding asparagine synthetase 1 (OsAS1). Loss of RST1 function increased the expression of OsAS1 and improved nitrogen (N) utilization by promoting asparagine production and avoiding excess ammonium (NH4+) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1Hap III decreased its transcriptional repression activity and contributed to salt tolerance and grain weight. Together, our findings unravel a synergistic regulator of growth and salt tolerance associated with N metabolism and provide a new strategy for the development of tolerant cultivars.

Development of a Temperate Climate-Adapted indica Multi-stress Tolerant Rice Variety by Pyramiding Quantitative Trait Loci.

Successful cultivation of rice (Oryza sativa L.) in many Asian countries requires submergence stress tolerance at the germination and early establishment stages. Two quantitative trait loci, Sub1 (conferring submergence tolerance) and AG1 (conferring anaerobic germination), were recently pyramided into a single genetic background, without compromising any desirable agronomic traits, leading to the development of Ciherang-Sub1 + AG1 (CSA). However, little research has been conducted to enhance plant tolerance to abiotic stress (submergence) and biotic stress (rice blast), which occur in a damp climate following flooding. The BC2F5 breeding line was phenotypically characterized using the AvrPi9 isolate. The biotic and abiotic stress tolerance of selected lines was tested under submergence stress and anaerobic germination conditions, and lines tolerant to each stress condition were identified through phenotypic and gene expression analyses. The Ciherang-Sub1 + AG1 + Pi9 (CSA-Pi9) line showed similar agronomic performance to its recurrent parent, CSA, but had significantly reduced chalkiness in field trials conducted in temperate regions. Unexpectedly, the CSA-Pi9 line also showed salinity tolerance. Thus, the breeding line newly developed in this study, CSA-Pi9, functioned under stress conditions, in which Sub1, AG1, and Pi9 play a role and had superior grain quality traits compared to its recurrent parent in temperate regions. We speculate that CSA-Pi9 will enable the establishment of climate-resilient rice cropping systems, particularly in East Asia.

The Effect of Water Level in Rice Cropping System on Phosphorus Uptake Activity of Pup1 in a Pup1+Sub1 Breeding Line.

Pyramiding useful QTLs into an elite variety is a promising strategy to develop tolerant varieties against multiple abiotic stresses. However, some QTLs may not be functionally compatible when they are introgressed into the same variety. Here, we tested the functional compatibility of Pup1 and Sub1, major QTLs for tolerance to phosphorus (P)-deficiency and submergence conditions, respectively. Phenotypic analysis revealed that IR64-Pup1+Sub1 (IPS) plants harboring both Pup1 and Sub1 QTLs show significant tolerance to submerged conditions, similarly to IR64-Sub1, while IPS failed to tolerate P deficiency and mild drought conditions; only IR64-Pup1 showed P deficiency tolerance. In submerged conditions, Sub1A and OsPSTOL1, major genes for Sub1 and Pup1 QTLs, respectively, were expressed at the same levels as in IPS and IR64-Sub1 and in IPS and IR64-Pup1, respectively. On the other hand, in P-non-supplied condition, crown root number, root length, and OsPSTOL1 expression level were significantly lower in IPS compared to those of IR64-Pup1. However, there was no significant difference in P content between IPS and IR64-Pup1. These results imply that Pup1 does not compromise Sub1 function in submerged condition, while Sub1 suppresses Pup1 function in P-non-supplied condition, possibly by regulating the transcript level of Pup1. In conclusion, Pup1 and Sub1 are regarded as functionally compatible under submergence condition but not under P-non-supplied condition. Further study is needed to elucidate the functional incompatibility of Pup1 and Sub1 QTLs in IPS under P-non-supplied condition.

Genome-Wide Association Study of Root System Development at Seedling Stage in Rice.

Root network structure plays a crucial role in growth and development processes in rice. Longer, more branched root structures help plants to assimilate water and nutrition from soil, support robust plant growth, and improve resilience to stresses such as disease. Understanding the molecular basis of root development through screening of root-related traits in rice germplasms is critical to future rice breeding programs. This study used a small germplasm collection of 137 rice varieties chosen from the Korean rice core set (KRICE_CORE) to identify loci linked to root development. Two million high-quality single nucleotide polymorphisms (SNPs) were used as the genotype, with maximum root length (MRL) and total root weight (TRW) in seedlings used as the phenotype. Genome-wide association study (GWAS) combined with Principal Components Analysis (PCA) and Kinship matrix analysis identified four quantitative trait loci (QTLs) on chromosomes 3, 6, and 8. Two QTLs were linked to MRL and two were related to TRW. Analysis of Linkage Disequilibrium (LD) decay identified a 230 kb exploratory range for detection of candidate root-related genes. Candidates were filtered using RNA-seq data, gene annotations, and quantitative real-time PCR (qRT-PCR), and five previously characterized genes related to root development were identified, as well as four novel candidate genes. Promoter analysis of candidate genes showed that LOC_Os03g08880 and LOC_Os06g13060 contained SNPs with the potential to impact gene expression in root-related promoter motifs. Haplotype analysis of candidate genes revealed diverse haplotypes that were significantly associated with phenotypic variation. Taken together, these results indicate that LOC_Os03g08880 and LOC_Os06g13060 are strong candidate genes for root development functions. The significant haplotypes identified in this study will be beneficial in future breeding programs for root improvement.

Characterization of the Common Japonica-Originated Genomic Regions in the High-Yielding Varieties Developed from Inter-Subspecific Crosses in Temperate Rice (Oryza sativa L.).

The inter-subspecific crossing between indica and japonica subspecies in rice have been utilized to improve the yield potential of temperate rice. In this study, a comparative study of the genomic regions in the eight high-yielding varieties (HYVs) was conducted with those of the four non-HYVs. The Next-Generation Sequencing (NGS) mapping on the Nipponbare reference genome identified a total of 14 common genomic regions of japonica-originated alleles. Interestingly, the HYVs shared japonica-originated genomic regions on nine chromosomes, although they were developed through different breeding programs. A panel of 94 varieties was classified into four varietal groups with 38 single nucleotide polymorphism (SNP) markers from 38 genes residing in the japonica-originated genomic regions and 16 additional trait-specific SNPs. As expected, the japonica-originated genomic regions were only present in the japonica (JAP) and HYV groups, except for Chr4-1 and Chr4-2. The Wx gene, located within Chr6-1, was present in the HYV and JAP variety groups, while the yield-related genes were conserved as indica alleles in HYVs. The japonica-originated genomic regions and alleles shared by HYVs can be employed in molecular breeding programs to further develop the HYVs in temperate rice.

Mapping and Validation of QTLs for the Amino Acid and Total Protein Content in Brown Rice.

Highly nutritious rice production will be benefited with the improvement of amino acid content (AAC) and protein content (PC). The identification of quantitative trait loci (QTLs) associated with the PC and AAC of rice grains could provide a basis for improving the nutritional value of rice grains. Here, we conducted QTL analyses using recombinant inbred lines from the cross between indica (Milyang 23 or M23) and japonica (Tong 88-7 or T887) rice varieties, afterward employing genotyping-by-sequencing to obtain a high-density genetic map. A total of 17 and 3 QTLs were detected for AAC and PC, respectively. Among them, two QTLs associated with more than 10 AACs, qAAC6.1 and qAAC7.1, were identified for the first time in this study. Each favorable allele that increased the AAC of the two QTLs was derived from M23 and T887, respectively. Allelic combination of qAAC6.1 M23 and qAAC7.1T887 showed significantly higher content of associated amino acids (AAs) than other allelic combinations. Near-isogenic line (NIL) possessing qAAC7.1T887 with M23 genetic background had significantly higher AACs than both parents. These results indicate that the pyramiding of QTLs would be useful in developing brown rice with a high AA and protein content.

Evaluation of Whole-Genome Sequence, Genetic Diversity, and Agronomic Traits of Basmati Rice (Oryza sativa L.).

Basmati is considered a unique varietal group of rice (Oryza sativa L.) because of its aroma and superior grain quality. Previous genetic analyses of rice showed that most of the Basmati varieties are classified into the aromatic group. Despite various efforts, genomic relationship of Basmati rice with other varietal groups and genomic variation in Basmati rice are yet to be understood. In the present study, we resequenced the whole genome of three traditional Basmati varieties at a coverage of more than 25X using Illumina HiSeq2500 and mapped the obtained sequences to the reference genome sequences of Nipponbare (japonica rice), Kasalath (aus rice), and Zhenshan 97 (indica rice). Comparison of these sequences revealed common single nucleotide polymorphisms (SNPs) in the genic regions of three Basmati varieties. Analysis of these SNPs revealed that Basmati varieties showed fewer sequence variations compared with the aus group than with the japonica and indica groups. Gene ontology (GO) enrichment analysis indicated that SNPs were present in genes with various biological, molecular, and cellular functions. Additionally, functional annotation of the Basmati mutated gene cluster shared by Nipponbare, Kasalath, and Zhenshan 97 was found to be associated with the metabolic process involved in the cellular aromatic compound, suggesting that aroma is an important specific genomic feature of Basmati varieties. Furthermore, 30 traditional Basmati varieties were classified into three different groups, aromatic (22 varieties), aus (four varieties), and indica (four varieties), based on genome-wide SNPs. All 22 aromatic Basmati varieties harbored the fragrant-inducing Badh2 allele. We also performed comparative analysis of 13 key agronomic and grain quality traits of Basmati rice and other rice varieties. Three traits including length-to-width ratio of grain (L/W ratio), panicle length (PL), and amylose content (AC) showed significant (P < 0.05 and P < 0.01) differences between the aromatic and indica/aus groups. Comparative analysis of genome structure, based on genome sequence variation and GO analysis, revealed that the Basmati genome was derived mostly from the aus and japonica groups. Overall, whole-genome sequence data and genetic diversity information obtained in this study will serve as an important resource for molecular breeding and genetic analysis of Basmati varieties.

Population Structure and Genetic Diversity of Cucurbita moschata Based on Genome-Wide High-Quality SNPs.

Pumpkins (Cucurbita moschata) are one of the most important economic crops in genus Cucurbita worldwide. They are a popular food resource and an important rootstock resource for various Cucurbitaceae. Especially, C. moschata is widely used as a rootstock for the commercial production of bloomless cucumbers in East Asia. Since the genetic diversity of the commercial rootstock varieties is narrow, there has been an increasing demand for the trait development of abiotic and biotic stress tolerance breeding. In this study, 2071 high-quality SNPs that were distributed evenly across 20 chromosomes of pumpkins were discovered through the genotyping-by-sequencing (GBS) analysis of 610 accessions of C. moschata germplasm with a global origin. Using these SNPs, various analyses of the genetic diversity and the population structure were performed. Three subgroups were clustered from the germplasm collection, which included East Asia, Africa, and America, and these areas were included the most in each subgroup. Among those groups, accessions from Africa and South Asia showed the highest genetic diversity, which was followed by the Mexico accessions. This result reflected that large gene pools that consist of various native landraces have been conserved in those of countries. Based on the genetic diversity, we finally constructed the C. moschata core collection, which included 67 representative accessions from the 610 germplasms. Five morphological traits that are important in commercial grafting and rootstock seed production, which include the cotyledon length, the cotyledon width, the hypocotyl length, the internode length, and the number of female flowers, were investigated for three years and used to confirm the validity of the core collection selection. The results are expected to provide valuable information about the genetic structure of the worldwide C. moschata germplasm and help to create new gene pools to develop genetically diverse rootstock breeding materials.

Identification and Characterization of LARGE EMBRYO, a New Gene Controlling Embryo Size in Rice (Oryza sativa L.).

BACKGROUND: Although embryo accounts for only 2-3% of the total weight of a rice grain, it is a good source of various nutrients for human health. Because enlarged embryo size causes increase of the amount of nutrients and bioactive compounds stored within rice grain, giant embryo mutants of rice (Oryza sativa L.) are excellent genetic resources for improving the nutritional value of rice grains. RESULTS: Three giant embryo mutants, including large embryo (le), giant embryo (ge) and super-giant embryo (ges), with variable embryo size were used in this study. We investigated whether genes controlling embryo size in these mutants (le, ge and ges) were allelic to each other. Although ge and ges was allelic to GIANT EMBRY (GE), le was not allelic to ge and ges in allelism test. The GE gene carried a unique nucleotide substitution in each of the two mutants (ge and ges), resulting in non-synonymous mutations in exon 2 of GE in both mutants. However, the GE gene of the le mutant did not carry any mutation, suggesting that the enlarged embryo phenotype of le was governed by another gene. Using map-based cloning, we mapped the LE gene to the short arm of chromosome 3. The le mutant showed mild enlargement in embryo size, which resulted from an increase in the size of scutellar parenchyma cells. The LE encodes a C3HC4-type RING finger protein and was expressed to relatively high levels in seeds at a late developmental stage. Knockdown of LE expression using RNA interference increased the embryo size of rice grains, confirming the role of LE in determining the embryo size. CONCLUSION: Overall, we identified a new gene controlling embryo size in rice. Phenotypic and molecular characterization results suggest that the le mutant will serve as a valuable resource for developing new rice cultivars with large embryos and nutrient-dense grains.

Novel allelic variant of Lpa1 gene associated with a significant reduction in seed phytic acid content in rice (Oryza sativa L.).

In plants, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also known as phytic acid (PA), is a major component of organic phosphorus (P), and accounts for up to 85% of the total P in seeds. In rice (Oryza sativa L.), PA mainly accumulates in rice bran, and chelates mineral cations, resulting in mineral deficiencies among brown rice consumers. Therefore, considerable efforts have been focused on the development of low PA (LPA) rice cultivars. In this study, we performed genetic and molecular analyses of OsLpa1, a major PA biosynthesis gene, in Sanggol, a low PA mutant variety developed via chemical mutagenesis of Ilpum rice cultivar. Genetic segregation and sequencing analyses revealed that a recessive allele, lpa1-3, at the OsLpa1 locus (Os02g0819400) was responsible for a significant reduction in seed PA content in Sanggol. The lpa1-3 gene harboured a point mutation (C623T) in the fourth exon of the predicted coding region, resulting in threonine (Thr) to isoleucine (Ile) amino acidsubstitution at position 208 (Thr208Ile). Three-dimensional analysis of Lpa1 protein structure indicated that myo-inositol 3-monophosphate [Ins(3)P1] could bind to the active site of Lpa1, with ATP as a cofactor for catalysis. Furthermore, the presence of Thr208 in the loop adjacent to the entry site of the binding pocket suggests that Thr208Ile substitution is involved in regulating enzyme activity via phosphorylation. Therefore, we propose that Thr208Ile substitution in lpa1-3 reduces Lpa1 enzyme activity in Sanggol, resulting in reduced PA biosynthesis.

Genetic Dissection of Grain Nutritional Traits and Leaf Blight Resistance in Rice.

Colored rice is rich in nutrition and also a good source of valuable genes/quantitative trait loci (QTL) for nutrition, grain quality, and pest and disease resistance traits for use in rice breeding. Genome-wide association analysis using high-density single nucleotide polymorphism (SNP) is useful in precisely detecting QTLs and genes. We carried out genome-wide association analysis in 152 colored rice accessions, using 22,112 SNPs to map QTLs for nutritional, agronomic, and bacterial leaf blight (BLB) resistance traits. Wide variations and normal frequency distributions were observed for most of the traits except anthocyanin content and BLB resistance. The structural and principal component analysis revealed two subgroups. The linkage disequilibrium (LD) analysis showed 74.3% of the marker pairs in complete LD, with an average LD distance of 1000 kb and, interestingly, 36% of the LD pairs were less than 5 Kb, indicating high recombination in the panel. In total, 57 QTLs were identified for ten traits at p < 0.0001, and the phenotypic variance explained (PVE) by these QTLs varied from 9% to 18%. Interestingly, 30 (53%) QTLs were co-located with known or functionally-related genes. Some of the important candidate genes for grain Zinc (Zn) and BLB resistance were OsHMA9, OsMAPK6, OsNRAMP7, OsMADS13, and OsZFP252, and Xa1, Xa3, xa5, xa13 and xa26, respectively. Red rice genotype, Sayllebon, which is high in both Zn and anthocyanin content, could be a valuable material for a breeding program for nutritious rice. Overall, the QTLs identified in our study can be used for QTL pyramiding as well as genomic selection. Some of the novel QTLs can be further validated by fine mapping and functional characterization. The results show that pigmented rice is a valuable resource for mineral elements and antioxidant compounds; it can also provide novel alleles for disease resistance as well as for yield component traits. Therefore, large opportunities exist to further explore and exploit more colored rice accessions for use in breeding.

Identification of a novel SPLIT-HULL (SPH) gene associated with hull splitting in rice (Oryza sativa L.).

KEY MESSAGE: The split-hull phenotype caused by reduced lemma width and low lignin content is under control of SPH encoding a type-2 13-lipoxygenase and contributes to high dehulling efficiency. Rice hulls consist of two bract-like structures, the lemma and palea. The hull is an important organ that helps to protect seeds from environmental stress, determines seed shape, and ensures grain filling. Achieving optimal hull size and morphology is beneficial for seed development. We characterized the split-hull (sph) mutant in rice, which exhibits hull splitting in the interlocking part between lemma and palea and/or the folded part of the lemma during the grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull splitting. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive gene, sph (Os04g0447100), which encodes a type-2 13-lipoxygenase. SPH knockout and knockdown transgenic plants displayed the same split-hull phenotype as in the mutant. The sph mutant showed significantly higher linoleic and linolenic acid (substrates of lipoxygenase) contents in spikelets compared to the wild type. It is probably due to the genetic defect of SPH and subsequent decrease in lipoxygenase activity. In dehulling experiment, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine. Collectively, the results provide a basis for understanding of the functional role of lipoxygenase in structure and maintenance of hulls, and would facilitate breeding of easy-dehulling rice.

Association between sequence variants in panicle development genes and the number of spikelets per panicle in rice.

BACKGROUND: Balancing panicle-related traits such as panicle length and the numbers of primary and secondary branches per panicle, is key to improving the number of spikelets per panicle in rice. Identifying genetic information contributes to a broader understanding of the roles of gene and provides candidate alleles for use as DNA markers. Discovering relations between panicle-related traits and sequence variants allows opportunity for molecular application in rice breeding to improve the number of spikelets per panicle. RESULTS: In total, 142 polymorphic sites, which constructed 58 haplotypes, were detected in coding regions of ten panicle development gene and 35 sequence variants in six genes were significantly associated with panicle-related traits. Rice cultivars were clustered according to their sequence variant profiles. One of the four resultant clusters, which contained only indica and tong-il varieties, exhibited the largest average number of favorable alleles and highest average number of spikelets per panicle, suggesting that the favorable allele combination found in this cluster was beneficial in increasing the number of spikelets per panicle. CONCLUSIONS: Favorable alleles identified in this study can be used to develop functional markers for rice breeding programs. Furthermore, stacking several favorable alleles has the potential to substantially improve the number of spikelets per panicle in rice.

Large-scale deployment of a rice 6 K SNP array for genetics and breeding applications.

BACKGROUND: Fixed arrays of single nucleotide polymorphism (SNP) markers have advantages over reduced representation sequencing in their ease of data analysis, consistently higher call rates, and rapid turnaround times. A 6 K SNP array represents a cost-benefit "sweet spot" for routine genetics and breeding applications in rice. Selection of informative SNPs across species and subpopulations during chip design is essential to obtain useful polymorphism rates for target germplasm groups. This paper summarizes results from large-scale deployment of an Illumina 6 K SNP array for rice. RESULTS: Design of the Illumina Infinium 6 K SNP chip for rice, referred to as the Cornell_6K_Array_Infinium_Rice (C6AIR), includes 4429 SNPs from re-sequencing data and 1571 SNP markers from previous BeadXpress 384-SNP sets, selected based on polymorphism rate and allele frequency within and between target germplasm groups. Of the 6000 attempted bead types, 5274 passed Illumina's production quality control. The C6AIR was widely deployed at the International Rice Research Institute (IRRI) for genetic diversity analysis, QTL mapping, and tracking introgressions and was intensively used at Cornell University for QTL analysis and developing libraries of interspecific chromosome segment substitution lines (CSSLs) between O. sativa and diverse accessions of O. rufipogon or O. meridionalis. Collectively, the array was used to genotype over 40,000 rice samples. A set of 4606 SNP markers was used to provide high quality data for O. sativa germplasm, while a slightly expanded set of 4940 SNPs was used for O. sativa X O. rufipogon populations. Biparental polymorphism rates were generally between 1900 and 2500 well-distributed SNP markers for indica x japonica or interspecific populations and between 1300 and 1500 markers for crosses within indica, while polymorphism rates were lower for pairwise crosses within U.S. tropical japonica germplasm. Recently, a second-generation array containing ~7000 SNP markers, referred to as the C7AIR, was designed by removing poor-performing SNPs from the C6AIR and adding markers selected to increase the utility of the array for elite tropical japonica material. CONCLUSIONS: The C6AIR has been successfully used to generate rapid and high-quality genotype data for diverse genetics and breeding applications in rice, and provides the basis for an optimized design in the C7AIR.