Identification of genomic regions governing moisture and heat stress tolerance employing association mapping in rice (Oryza sativa L.).

BACKGROUND: Rice crop is damaged extremely by abiotic stress world-wide. The best approach to enhance drought tolerance in rice varieties is to identify and introgress yield QTLs with major effects. The Association mapping approach helps in the identification of genomic regions governing physiological, yield and yield attributes under moisture and heat stress conditions in diverse collections of crop germplasm, based on historic recombination events and linkage disequilibrium across the genome. METHODS AND RESULTS: The association mapping panel of 110 rice germplasm lines exhibited significant variation for all the traits in both irrigated and moisture stress conditions. The extent of yield reduction ranged to 83% during rabi, 2018-19, 53% in rabi, 2019-20 and 68% in pooled analysis. The genotypes Badami, Badshabhog, Pankaj, Varalu, Vasundhara, Vivekdhan, Krishna and Minghui63 exhibited drought tolerance with least yield penalty under moisture stress conditions. The genotypes Konark, MTU3626, NLR33671, PR118 and Triguna exhibited minimal reduction in heat stress tolerance traits. Association mapping of germplasm using 37808 SNP markers detected a total of 10 major MTA (Marker-trait association) clusters distributed on chromosomes 1, 3, 4 and 11 through mixed linear model (MLM) governing multiple traits from individual data analysis which are consistent across the years and situations. The pooled data generated a total of five MTA clusters located on chromosome 6. In addition, several novel unique MTAs were also identified. Heat stress analysis generated a total of 23 MTAs distributed on chromosomes 1, 5, 6 and 11. Candidate gene analysis detected a total of 53 and 38 genes under individual and pooled data analysis for various yield and yield attributes under control and moisture stress conditions, respectively and a total of 11 candidate genes in heat stress Conditions. CONCLUSION: The major and novel MTAs identified in the present investigation for various drought and heat tolerant traits can be utilized for breeding climate-resilient rice varieties. The candidate genes predicted for key MTAs are of great value to deploy into the rice breeding after functional characterization.

Genome-Wide Association Mapping for Heat and Drought Adaptive Traits in Pea.

Heat and drought, individually or in combination, limit pea productivity. Fortunately, substantial genetic diversity exists in pea germplasm for traits related to abiotic stress resistance. Understanding the genetic basis of resistance could accelerate the development of stress-adaptive cultivars. We conducted a genome-wide association study (GWAS) in pea on six stress-adaptive traits with the aim to detect the genetic regions controlling these traits. One hundred and thirty-five genetically diverse pea accessions were phenotyped in field studies across three or five environments under stress and control conditions. To determine marker trait associations (MTAs), a total of 16,877 valuable single nucleotide polymorphisms (SNPs) were used in association analysis. Association mapping detected 15 MTAs that were significantly (p ≤ 0.0005) associated with the six stress-adaptive traits averaged across all environments and consistent in multiple individual environments. The identified MTAs were four for lamina wax, three for petiole wax, three for stem thickness, two for the flowering duration, one for the normalized difference vegetation index (NDVI), and two for the normalized pigment and chlorophyll index (NPCI). Sixteen candidate genes were identified within a 15 kb distance from either side of the markers. The detected MTAs and candidate genes have prospective use towards selecting stress-hardy pea cultivars in marker-assisted selection.

Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.

KEY MESSAGE: A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.

Stable SNP Allele Associations With High Grain Zinc Content in Polished Rice (Oryza sativa L.) Identified Based on ddRAD Sequencing.

Polished rice is widely consumed staple food across the globe, however, it contains limited nutrients especially iron (Fe) and zinc (Zn). To identify promising genotypes for grain Zn, a total of 40 genotypes consisting 20 rice landraces, and 20 released high yielding rice varieties were evaluated in three environments (wet seasons 2014, 2015 and 2016) for nine traits including days to 50% flowering (DFF), plant height (PH), panicle length (PL), total number of tillers (TNT), single plant yield (SPY), Fe and Zn in brown (IBR, ZBR) and polished rice (IPR, ZPR). Additive Main Effect and Multiplicative Interaction (AMMI), Genotype and Genotype × Environment Interaction (GGE) analyses identified genotypes G22 (Edavankudi Pokkali), G17 (Taraori Basmati), G27 (Chittimuthyalu) and G26 (Kalanamak) stable for ZPR and G8 (Savitri) stable for SPY across three environments. Significant negative correlation between yield and grain Zn was reaffirmed. Regression analysis indicated the contribution of traits toward ZPR and SPY and also desirable level of grain Zn in brown rice. A total of 39,137 polymorphic single nucleotide polymorphisms (SNPs) were obtained through double digest restriction site associated DNA (dd-RAD) sequencing of 40 genotypes. Association analyses with nine phenotypic traits revealed 188 stable SNPs with six traits across three environments. ZPR was associated with SNPs located in three putative candidate genes (LOC_Os03g47980, LOC_Os07g47950 and LOC_Os07g48050) on chromosomes 3 and 7. The genomic region of chromosome 7 co localized with reported genomic regions (rMQTL7.1) and OsNAS3 candidate gene. SPY was found to be associated with 12 stable SNPs located in 11 putative candidate genes on chromosome 1, 6, and 12. Characterization of rice landraces and varieties in terms of stability for their grain Zn and yield identified promising donors and recipients along with genomic regions in the present study to be deployed rice Zn biofortification breeding program.

Genome-Wide Association Mapping for Heat Stress Responsive Traits in Field Pea.

Environmental stress hampers pea productivity. To understand the genetic basis of heat resistance, a genome-wide association study (GWAS) was conducted on six stress responsive traits of physiological and agronomic importance in pea, with an objective to identify the genetic loci associated with these traits. One hundred and thirty-five genetically diverse pea accessions from major pea growing areas of the world were phenotyped in field trials across five environments, under generally ambient (control) and heat stress conditions. Statistical analysis of phenotype indicated significant effects of genotype (G), environment (E), and G × E interaction for all traits. A total of 16,877 known high-quality SNPs were used for association analysis to determine marker-trait associations (MTA). We identified 32 MTAs that were consistent in at least three environments for association with the traits of stress resistance: six for chlorophyll concentration measured by a soil plant analysis development meter; two each for photochemical reflectance index and canopy temperature; seven for reproductive stem length; six for internode length; and nine for pod number. Forty-eight candidate genes were identified within 15 kb distance of these markers. The identified markers and candidate genes have potential for marker-assisted selection towards the development of heat resistant pea cultivars.

Genome-Wide Association Mapping for Agronomic and Seed Quality Traits of Field Pea (Pisum sativum L.).

Genome-wide association study (GWAS) was conducted to identify loci associated with agronomic (days to flowering, days to maturity, plant height, seed yield and seed weight), seed morphology (shape and dimpling), and seed quality (protein, starch, and fiber concentrations) traits of field pea (Pisum sativum L.). A collection of 135 pea accessions from 23 different breeding programs in Africa (Ethiopia), Asia (India), Australia, Europe (Belarus, Czech Republic, Denmark, France, Lithuania, Netherlands, Russia, Sweden, Ukraine and United Kingdom), and North America (Canada and USA), was used for the GWAS. The accessions were genotyped using genotyping-by-sequencing (GBS). After filtering for a minimum read depth of five, and minor allele frequency of 0.05, 16,877 high quality SNPs were selected to determine marker-trait associations (MTA). The LD decay (LD1/2max,90) across the chromosomes varied from 20 to 80 kb. Population structure analysis grouped the accessions into nine subpopulations. The accessions were evaluated in multi-year, multi-location trials in Olomouc (Czech Republic), Fargo, North Dakota (USA), and Rosthern and Sutherland, Saskatchewan (Canada) from 2013 to 2017. Each trait was phenotyped in at least five location-years. MTAs that were consistent across multiple trials were identified. Chr5LG3_566189651 and Chr5LG3_572899434 for plant height, Chr2LG1_409403647 for lodging resistance, Chr1LG6_57305683 and Chr1LG6_366513463 for grain yield, Chr1LG6_176606388, Chr2LG1_457185, Chr3LG5_234519042 and Chr7LG7_8229439 for seed starch concentration, and Chr3LG5_194530376 for seed protein concentration were identified from different locations and years. This research identified SNP markers associated with important traits in pea that have potential for marker-assisted selection towards rapid cultivar improvement.

Whole Genome Sequencing and Comparative Genomic Analysis Reveal Allelic Variations Unique to a Purple Colored Rice Landrace (Oryza sativa ssp. indica cv. Purpleputtu).

Purpleputtu (Oryza sativa ssp. indica cv. Purpleputtu) is a unique rice landrace from southern India that exhibits predominantly purple color. This study reports the underlying genetic complexity of the trait, associated domestication and de-domestication processes during its coevolution with present day cultivars. Along-with genome level allelic variations in the entire gene repertoire associated with the purple, red coloration of grain and other plant parts. Comparative genomic analysis using 'a panel of 108 rice lines' revealed a total of 3,200,951 variants including 67,774 unique variations in Purpleputtu (PP) genome. Multiple sequence alignment uncovered a 14 bp deletion in Rc (Red colored, a transcription factor of bHLH class) locus of PP, a key regulatory gene of anthocyanin biosynthetic pathway. Interestingly, this deletion in Rc gene is a characteristic feature of the present-day white pericarped rice cultivars. Phylogenetic analysis of Rc locus revealed a distinct clade showing proximity to the progenitor species Oryza rufipogon and O. nivara. In addition, PP genome exhibits a well conserved 4.5 Mbp region on chromosome 5 that harbors several loci associated with domestication of rice. Further, PP showed 1,387 unique when SNPs compared to 3,023 lines of rice (SNP-Seek database). The results indicate that PP genome is rich in allelic diversity and can serve as an excellent resource for rice breeding for a variety of agronomically important traits such as disease resistance, enhanced nutritional values, stress tolerance, and protection from harmful UV-B rays.

Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen.

Sclerospora graminicola pathogen is the most important biotic production constraints of pearl millet in India, Africa and other parts of the world. We report a de novo whole genome assembly and analysis of pathotype 1, one of the most virulent pathotypes of S. graminicola from India. The draft genome assembly contained 299,901,251 bp with 65,404 genes. This study may help understand the evolutionary pattern of pathogen and aid elucidation of effector evolution for devising effective durable resistance breeding strategies in pearl millet.

Identification of stress-responsive genes in an indica rice (Oryza sativa L.) using ESTs generated from drought-stressed seedlings.

The impacts of drought on plant growth and development limit cereal crop production worldwide. Rice (Oryza sativa) productivity and production is severely affected due to recurrent droughts in almost all agroecological zones. With the advent of molecular and genomic technologies, emphasis is now placed on understanding the mechanisms of genetic control of the drought-stress response. In order to identify genes associated with water-stress response in rice, ESTs generated from a normalized cDNA library, constructed from drought-stressed leaf tissue of an indica cultivar, Nagina 22 were used. Analysis of 7794 cDNA sequences led to the identification of 5815 rice ESTs. Of these, 334 exhibited no significant sequence homology with any rice ESTs or full-length cDNAs in public databases, indicating that these transcripts are enriched during drought stress. Analysis of these 5815 ESTs led to the identification of 1677 unique sequences. To characterize this drought transcriptome further and to identify candidate genes associated with the drought-stress response, the rice data were compared with those for abiotic stress-induced sequences obtained from expression profiling studies in Arabidopsis, barley, maize, and rice. This comparative analysis identified 589 putative stress-responsive genes (SRGs) that are shared by these diverse plant species. Further, the identified leaf SRGs were compared to expression profiles for a drought-stressed rice panicle library to identify common sequences. Significantly, 125 genes were found to be expressed under drought stress in both tissues. The functional classification of these 125 genes showed that a majority of them are associated with cellular metabolism, signal transduction, and transcriptional regulation.