Physiological responses and variation in secondary metabolite content among Thai holy basil cultivars (Ocimum tenuiflorum L.) grown under controlled environmental conditions in a plant factory.

Holy basil (Ocimum Tenuiflorum L.) is a widely used herb containing several bioactive compounds of interest for the food and pharmaceutical industries. Plant factories using artificial lighting (PFAL) is a modern agricultural system that offers opportunity to improve crop production and stabilizes productivity in many herbal plants. However, little is known about the variation among holy basil varieties that can be cultivated and provide reasonable biomass and bioactive compounds in PFAL. We therefore evaluated 10 Thai accessions and two commercial cultivars in a PFAL (with hydroponic cultivation) to categorize cultivar characteristics by investigating physiological responses and secondary metabolite variation at plant flowering stage. Among Thai varieties, net photosynthetic rate (Pn) was significantly highest in varieties OC059 and OC081. The greatest growth and biomass measures were observed in OC064. Antioxidant capacity also varied, with the greatest accumulation of total phenolic compounds (TPC), flavonoids, and antioxidant activity by DPPH assay in OC064, and highest terpenoid content in OC194. The accumulation of major compounds confirmed by showing the highest levels of eugenol in OC057, OC063, OC194, and OC195 and methyl eugenol in OC072 and OC081. The highest α-humulene content was found in OC059. PCA based on physiological responses and secondary metabolites indicate that OC064 was clearly distinguished from other cultivars/accessions. These findings demonstrate variation across holy basil accessions for physiologic responses, antioxidant capacity, and secondary compounds in PFAL. These insights lead to identification of suitable varieties which is the most important step of developing an efficient method for producing high quality raw materials of Thai holy basil for supplying the foods and pharmaceutical industries.

Identification of Pathogenicity Loci in Magnaporthe oryzae Using GWAS with Neck Blast Phenotypic Data.

Magnaporthae oryzae (M. oryzae) is the most destructive disease of rice worldwide. In this study, one hundred and two isolates of M. oryzae were collected from rice (Oryzae sativa L.) from 2001 to 2017, and six rice varieties with resistance genes Pizt, Pish, Pik, Pib, and Pi2 were used in a genome-wide association study to identify pathogenicity loci in M. oryzae. Genome-wide association analysis was performed using 5338 single nucleotide polymorphism (SNPs) and phenotypic data of neck blast screening by TASSEL software together with haplotype block and SNP effect analysis. Twenty-seven significant SNPs were identified on chromosomes 1, 2, 3, 4, 5, 6, and 7. Many predicted genes (820 genes) were found in the target regions of six rice varieties. Most of these genes are described as putative uncharacterized proteins, however, some genes were reported related to virulence in M. oryzae. Moreover, this study revealed that R genes, Pik, Pish, and Pi2, were broad-spectrum resistant against neck blast disease caused by Thai blast isolate. Haplotype analysis revealed that the combination of the favorable alleles causing reduced virulence of isolates against IRBLz5-CA carrying Pi2 gene contributes 69% of the phenotypic variation in pathogenicity. The target regions and information are useful to develop marker-specific genes to classify blast fungal isolates and select appropriate resistance genes for rice cultivation and improvement.

Promotion of seed germination and early plant growth by KNO3 and light spectra in Ocimum tenuiflorum using a plant factory.

The plant factory with artificial light (PFAL) is a novel cultivation system of agriculture technology for crop production under controlled-environment conditions. However, there are a number of issues relating to low quality of seed germination and seedling vigor that lead to decreased crop yields. The present study investigates the optimal KNO3 concentration for seed germination, and the influence of different light spectra on early plant growth in holy basil (Ocimum tenuiflorum) under a PFAL system. Experiment 1 investigated the effects of KNO3 concentration (0, 0.2, 0.4 and 0.6%) on germination of seeds primed for 24 h under white Light emitting diodes (LED). Results show that sowing holy basil seeds in 0.4% KNO3 enhanced seed germination percentage (GP) and germination index (GI), while decreasing mean germination time (MGT). Experiment 2 investigated the effect of four light spectra on seed germination and early plant growth by sowing with 0 and 0.4% KNO3 and germinating for 15 days continuously under different monochromatic light settings: white, red, green and blue in PFAL. It was found that the green spectrum positively affected shoot and root length, and also decreased shortened MGT at 0 and 0.4% KNO3 when compared with other light treatments. Additionally, pre-cultivated seedlings under the green spectrum showed significant improvement in the early plant growth for all holy basil varieties at 15 days after transplanting by promoting stem length, stem diameter, plant width, fresh weights of shoot and root, and dry weights of shoot and root. These findings could be useful in developing seed priming and light treatments to enhance seed germination and seedling quality of holy basil resulting in increased crop production under PFAL.

Identification of broad-spectrum resistance QTLs against rice blast fungus and their application in different rice genetic backgrounds.

Rice blast disease is found worldwide leading to economic losses. Use of resistance gene is effective to improve rice resistance variety. Therefore, to deploy genomic regions harbouring resistance genes, a population of 587 F2:6 recombinant inbred lines (RILs) was developed from a cross between Jao Hom Nin, a Thai black rice variety with broad-spectrum resistance to blast disease, and Kao Dawk Mali 105, a susceptible Thai jasmine variety. The RILs were challenged with 17 blast isolates collected from Thailand and Laos PDR. Quantitative trait locus analysis identified genomic regions associated with broad-spectrum quantitative resistance (qBSRLs) and racespecific quantitative resistance (qRSRLs). Two qBSRLs were detected on chromosomes 1 and 11, and two qRSRLs were detected on chromosomes 8 and 12. The two qBSRLs were introgressed into two new genetic backgrounds through marker-assisted selection (MAS). Twelve breeding lines were tested for their spectra of resistance against 35 blast isolates. The results indicated that both qBSRLs were effective in new genetic backgrounds. The flanking markers and qBSRLs identified in the large mapping population showed high selection accuracy and effectiveness, suggesting the routine deployment of MAS technique in rice breeding programmes.

Identification of Bacterial Blight Resistance Loci in Rice (Oryza sativa L.) against Diverse Xoo Thai Strains by Genome-Wide Association Study.

Bacterial leaf blight (BLB) is a serious disease affecting global rice agriculture caused by Xanthomonas oryzae pv. oryzae (Xoo). Most resistant rice lines are dependent on single genes that are vulnerable to resistance breakdown caused by pathogen mutation. Here we describe a genome-wide association study of 222 predominantly Thai rice accessions assayed by phenotypic screening against 20 Xoo isolates. Loci corresponding to BLB resistance were detected using >142,000 SNPs. We identified 147 genes according to employed significance thresholds across chromosomes 1-6, 8, 9 and 11. Moreover, 127 of identified genes are located on chromosomal regions outside estimated Linkage Disequilibrium influences of known resistance genes, potentially indicating novel BLB resistance markers. However, significantly associated SNPs only occurred across a maximum of six Xoo isolates indicating that the development of broad-spectrum Xoo strain varieties may prove challenging. Analyses indicated a range of gene functions likely underpinning BLB resistance. In accordance with previous studies of accession panels focusing on indica varieties, our germplasm displays large numbers of SNPs associated with resistance. Despite encouraging data suggesting that many loci contribute to resistance, our findings corroborate previous inferences that multi-strain resistant varieties may not be easily realised in breeding programs without resorting to multi-locus strategies.

Genome-wide association mapping of virulence gene in rice blast fungus Magnaporthe oryzae using a genotyping by sequencing approach.

Magnaporthe oryzae is a fungal pathogen causing blast disease in many plant species. In this study, seventy three isolates of M. oryzae collected from rice (Oryza sativa) in 1996-2014 were genotyped using a genotyping-by-sequencing approach to detect genetic variation. An association study was performed to identify single nucleotide polymorphisms (SNPs) associated with virulence genes using 831 selected SNP and infection phenotypes on local and improved rice varieties. Population structure analysis revealed eight subpopulations. The division into eight groups was not related to the degree of virulence. Association mapping showed five SNPs associated with fungal virulence on chromosome 1, 2, 3, 4 and 7. The SNP on chromosome 1 was associated with virulence against RD6-Pi7 and IRBL7-M which might be linked to the previously reported AvrPi7.

Dissection of broad-spectrum resistance of the Thai rice variety Jao Hom Nin conferred by two resistance genes against rice blast.

BACKGROUND: Rice (Oryza sativa) is one of the most important food crops in the world. Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. To effectively cope with this problem, the use of rice blast resistance varieties through innovative breeding programs is the best strategy to date. The Thai rice variety Jao Hom Nin (JHN) showed broad-spectrum resistance against Thai rice blast isolates. Two QTLs for blast resistance in JHN were reported on chromosome 1 (QTL1) and 11 (QTL11). RESULTS: Monogenic lines of QTL1 (QTL1-C) and QTL11 (QTL11-C) in the CO39 genetic background were generated. Cluster analysis based on the disease reaction pattern of QTL1-C and QTL11-C, together with IRBLs, showed that those two monogenic lines were clustered with IRBLsh-S (Pish) and IRBL7-M (Pi7), respectively. Moreover, sequence analysis revealed that Pish and Pi7 were embedded within the QTL1 and QTL11 delimited genomic intervals, respectively. This study thus concluded that QTL1 and QTL11 could encode alleles of Pish and Pi7, designated as Pish-J and Pi7-J, respectively. To validate this hypothesis, the genomic regions of Pish-J and Pi7-J were cloned and sequenced. Protein sequence comparison revealed that Pish-J and Pi7-J were identical to Pish and Pi7, respectively. The holistic disease spectrum of JHN was found to be exactly attributed to the additive ones of both QTL1-C and QTL11-C. CONCLUSION: JHN showed broad spectrum resistance against Thai and Philippine rice blast isolates. As this study demonstrated, the combination of two resistance genes, Pish-J and Pi7-J, in JHN, with each controlling broad-spectrum resistance to rice blast disease, explains the high level of resistance. Thus, the combination of Pish and Pi7 can provide a practical scheme for breeding durable resistance in rice against rice blast disease.

Pseudo-backcrossing design for rapidly pyramiding multiple traits into a preferential rice variety.

BACKGROUND: Pyramiding multiple genes into a desirable genetic background can take years to accomplish. In this paper, a pseudo-backcrossing scheme was designed to shorten the backcrossing cycle needed. PinK3, an aromatic and potentially high-yielding rice variety-although one that is intolerant to flash flooding (Sub) and susceptible to bacterial leaf blight (BB), leaf-neck blast (BL) and the brown planthopper (BPH)-was used as a genetic basis for significant improvements through gene pyramiding. RESULTS: Four resistance donors with five target genes (Sub1A-C, xa5, Xa21, TPS and SSIIa) and three QTLs (qBph3, qBL1 and qBL11) were backcrossed individually using markers into the pseudo-recurrent parent 'PinK3' via one cycle of backcrossing followed by two cycles of pseudo-backcrossing and three selfings with rigorous foreground marker-assisted selection. In total, 29 pseudo-backcross inbred lines (BILs) were developed. Genome composition was surveyed using 61 simple sequence repeats (SSRs), 35 of which were located on six carrier chromosomes, with the remainder located on six non-carrier chromosomes. The recurrent genome content (%RGC) and donor genome content (%DGC), which were based on the physical positions of BC1F2, ranged from 69.99 to 88.98% and 11.02 to 30.01%, respectively. For the pseudo-BC3F3BILs, the %RGC and %DGC ranged from 74.50 to 81.30% and 18.70 to 25.50%, respectively. These results indicated that without direct background selection, no further increases in %RGC were obtained during pseudo-backcrossing, whereas rigorous foreground marker-assisted selection tended to reduce linkage drag during pseudo-backcrossing. The evaluation of new traits in selected pseudo-BC3F3BILs indicated significant improvements in resistance to BB, BL, BPH and Sub compared with PinK3, as well as significant improvements in grain yield (21-68%) over the donors, although yield was 7-26% lower than in 'PinK3'. All pyramided lines were aromatic and exhibited improved starch profiles, rendering them suitable for industrial food applications. CONCLUSIONS: Results show that our new pyramiding platform, which is based on marker-assisted pseudo-backcrossing, can fix five target genes and three QTLs into a high-yielding pseudo-recurrent background within seven breeding cycles in four years. This multiple pseudo-backcrossing platform decreases the time required to generate new rice varieties exhibiting complex, durable resistance to biotic and abiotic stresses in backgrounds with desirable qualities.