Candidate genes affecting stomatal density in rice (Oryza sativa L.) identified by genome-wide association.

Stomata regulate photosynthesis and water loss. They have been an active subject of research for centuries, but our knowledge of the genetic components that regulate stomatal development in crops remains very limited in comparison to the model plant Arabidopsis thaliana. Leaf stomatal density was found to vary by over 2.5-fold across a panel of 235 rice accessions. Using GWAS, we successfully identified five different QTLs associated with stomatal density on chromosomes 2, 3, 9, and 12. Forty-two genes were identified within the haplotype blocks corresponding to these QTLs. Of these, nine genes contained haplotypes that were associated with different stomatal densities. These include a gene encoding a trehalose-6-phosphate synthase, an enzyme that has previously been associated with altered stomatal density in Arabidopsis, and genes encoding a B-BOX zinc finger family protein, a leucine-rich repeat family protein, and the 40 S ribosomal protein S3a, none of which have previously been linked to stomatal traits. We investigated further and show that a closely related B-BOX protein regulates stomatal development in Arabidopsis. The results of this study provide information on genetic associations with stomatal density in rice. The QTLs and candidate genes may be useful in future breeding programs for low or high stomatal density and, consequently, improved photosynthetic capacity, water use efficiency, or drought tolerance.

Functional Bph14 from Rathu Heenati promotes resistance to BPH at the early seedling stage of rice (Oryza sativa L.) as revealed by QTL-seq.

KEY MESSAGE: A QTL associated with BPH resistance at the early seedling stage was identified on chromosome 3. Functional Bph14 in Rathu Heenati was associated with BPH resistance at the early seedling stage. Brown planthopper (BPH; Nilaparvata lugens Stål) is considered the most important rice pest in many Asian countries. Several BPH resistance genes have previously been identified. However, there are few reports of genes specific for BPH resistance at the early seedling stage, a crucial stage for direct-seeding cultivation. In this study, we performed a QTL-seq analysis using two bulks (20 F2 lines in each bulk) of the F2 population (n = 300) derived from a cross of Rathu Heenati (RH) × HCS-1 to identify QTL/genes associated with BPH resistance at the early seedling stage. An important QTL was identified on chromosome 3 and Bph14 was identified as a potential candidate gene based on the differences in gene expression and sequence variation when compared with the two parents. All plants in the resistant bulks possessed the functional Bph14 from RH and all plants in the susceptible bulk and HCS-1 contained a large deletion (2703 bp) in Bph14. The functional Bph14 gene of RH appears to be important for BPH resistance at the early seedling stage of rice and could be used in conjunction with other BPH resistance genes in rice breeding programs that confer resistance to BPH at the early and later growth stages.

riceExplorer: Uncovering the Hidden Potential of a National Genomic Resource Against a Global Database.

Agricultural crop breeding programs, particularly at the national level, typically consist of a core panel of elite breeding cultivars alongside a number of local landrace varieties (or other endemic cultivars) that provide additional sources of phenotypic and genomic variation or contribute as experimental materials (e.g., in GWAS studies). Three issues commonly arise. First, focusing primarily on core development accessions may mean that the potential contributions of landraces or other secondary accessions may be overlooked. Second, elite cultivars may accumulate deleterious alleles away from nontarget loci due to the strong effects of artificial selection. Finally, a tendency to focus solely on SNP-based methods may cause incomplete or erroneous identification of functional variants. In practice, integration of local breeding programs with findings from global database projects may be challenging. First, local GWAS experiments may only indicate useful functional variants according to the diversity of the experimental panel, while other potentially useful loci-identifiable at a global level-may remain undiscovered. Second, large-scale experiments such as GWAS may prove prohibitively costly or logistically challenging for some agencies. Here, we present a fully automated bioinformatics pipeline (riceExplorer) that can easily integrate local breeding program sequence data with international database resources, without relying on any phenotypic experimental procedure. It identifies associated functional haplotypes that may prove more robust in determining the genotypic determinants of desirable crop phenotypes. In brief, riceExplorer evaluates a global crop database (IRRI 3000 Rice Genomes) to identify haplotypes that are associated with extreme phenotypic variation at the global level and recorded in the database. It then examines which potentially useful variants are present in the local crop panel, before distinguishing between those that are already incorporated into the elite breeding accessions and those only found among secondary varieties (e.g., landraces). Results highlight the effectiveness of our pipeline, identifying potentially useful functional haplotypes across the genome that are absent from elite cultivars and found among landraces and other secondary varieties in our breeding program. riceExplorer can automatically conduct a full genome analysis and produces annotated graphical output of chromosomal maps, potential global diversity sources, and summary tables.

Primary Root Excision Induces ERF071, Which Mediates the Development of Lateral Roots in Makapuno Coconut (Cocos nucifera).

Coconut (Cocos nucifera L.) is widely recognized as one of nature's most beneficial plants. Makapuno, a special type of coconut with a soft, jelly-like endosperm, is a high-value commercial coconut and an expensive delicacy with a high cost of planting material. The embryo rescue technique is a very useful tool to support mass propagation of makapuno coconut. Nevertheless, transplanting the seedlings is a challenge due to poor root development, which results in the inability of the plant to acclimatize. In this study, primary root excision was used in makapuno to observe the effects of primary root excision on lateral root development. The overall results showed that seedlings with roots excised had a significantly higher number of lateral roots, and shoot length also increased significantly. Using de novo transcriptome assembly and differential gene expression analysis, we identified 512 differentially expressed genes in the excised and intact root samples. ERF071, encoding an ethylene-responsive transcription factor, was identified as a highly expressed gene in excised roots compared to intact roots, and was considered a candidate gene associated with lateral root formation induced by root excision in makapuno coconut. This study provides insight into the mechanism and candidate genes involved in the development of lateral roots in coconut, which may be useful for the future breeding and mass propagation of makapuno coconut through tissue culture.

Identification and Validation of a QTL for Bacterial Leaf Streak Resistance in Rice (Oryza sativa L.) against Thai Xoc Strains.

Rice is one of the most important food crops in the world and is of vital importance to many countries. Various diseases caused by fungi, bacteria and viruses constantly threaten rice plants and cause yield losses. Bacterial leaf streak disease (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most devastating rice diseases. However, most modern rice varieties are susceptible to BLS. In this study, we applied the QTL-seq approach using an F2 population derived from the cross between IR62266 and Homcholasit (HSC) to rapidly identify the quantitative trait loci (QTL) that confers resistance to BLS caused by a Thai Xoc isolate, SP7-5. The results showed that a single genomic region at the beginning of chromosome 5 was highly associated with resistance to BLS. The gene xa5 was considered a potential candidate gene in this region since most associated single nucleotide polymorphisms (SNPs) were within this gene. A Kompetitive Allele-Specific PCR (KASP) marker was developed based on two consecutive functional SNPs in xa5 and validated in six F2 populations inoculated with another Thai Xoc isolate, 2NY2-2. The phenotypic variance explained by this marker (PVE) ranged from 59.04% to 70.84% in the six populations. These findings indicate that xa5 is a viable candidate gene for BLS resistance and may help in breeding programs for BLS resistance.

Identification of Gene Associated with Sweetness in Corn (Zea mays L.) by Genome-Wide Association Study (GWAS) and Development of a Functional SNP Marker for Predicting Sweet Corn.

Sweetness is an economically important eating quality trait for sweet-corn breeding. To investigate the genetic control of the sweetness trait, we conducted a genome-wide association study (GWAS) in an association panel consisting of 250 sweet corn and waxy corn inbred and recombinant inbred lines (RILs), together with the genotypes obtained from the high-density 600K maize genotyping single-nucleotide polymorphism (SNP) array. GWAS results identified 12 significantly associated SNPs on chromosomes 3, 4, 5, and 7. The most associated SNP, AX_91849634, was found on chromosome 3 with a highly significant p-value of ≤1.53 × 10-14. The candidate gene identified within the linkage disequilibrium (LD) of this marker was shrunken2 (Zm00001d044129; sh2), which encodes ADP-glucose pyrophosphorylase (AGPase), a 60 kDa subunit enzyme that affects starch metabolism in the maize endosperm. Several SNP markers specific to variants in sh2 were developed and validated. According to the validation in a set of 81 inbred, RIL, and popular corn varieties, marker Sh2_rs844805326, which was developed on the basis of the SNP at the position 154 of exon 1, was highly efficient in classifying sh2-based sweet corn from other types of corn. This functional marker is extremely useful for marker-assisted breeding in sh2-sweet corn improvement and marketable seed production.

Mining and validation of novel genotyping-by-sequencing (GBS)-based simple sequence repeats (SSRs) and their application for the estimation of the genetic diversity and population structure of coconuts (Cocos nucifera L.) in Thailand.

Coconut (Cocos nucifera L.) is an important economic crop in tropical countries. However, the lack of a complete reference genome and the limitations of usable DNA markers hinder genomic studies and the molecular breeding of coconut. Here, we present the results of simple sequence repeat (SSR) mining from a high-throughput genotyping-by-sequencing (GBS) study of a collection of 38 coconut accessions. A total of 22,748 SSRs with di-, tri-, tetra-, penta- and hexanucleotide repeats of five or more were identified, 2451 of which were defined as polymorphic loci based on locus clustering in 38 coconut accessions, and 315 loci were suitable for the development of SSR markers. One hundred loci were selected, and primer pairs for each SSR locus were designed and validated in 40 coconut accessions. The analysis of 74 polymorphic markers identified between 2 and 9 alleles per locus, with an average of 3.01 alleles. The assessment of the genetic diversity and genetic relationships among the 40 coconut varieties based on the analysis of population structure, principal coordinate analysis (PCoA), and phylogenetic tree analysis using the 74 polymorphic SSR markers revealed three main groups of coconuts in Thailand. The identified SSR loci and SSR markers developed in this study will be useful for the study of coconut diversity and molecular breeding. The SSR mining approach used in this study could be applied to other plant species with a complex genome regardless of the availability of reference genome.