Breeding and adoption of biofortified crops and their nutritional impact on human health.

Micronutrient malnutrition has affected over two billion people worldwide and continues to be a health risk. A growing human population, poverty, and the prevalence of low dietary diversity are jointly responsible for malnutrition, particularly in developing nations. Inadequate bioavailability of key micronutrients, such as iron (Fe), zinc (Zn), and vitamin A, can be improved through agronomic and/or genetic interventions. The Consultative Group on International Agricultural Research prioritizes developing biofortified food crops that are rich in minerals and vitamins through the HarvestPlus initiative on biofortification. The objective of this review is to provide an overview of biofortified food crops along with evidence supporting their acceptability and adoption. Between 2004 and 2019, 242 biofortified varieties belonging to 11 major crops were released in 30 countries across Asia, Africa, and Latin America. These conventionally bred biofortified crops include Fe-enriched beans, pearl millet, and cowpea; Zn-enriched rice, wheat, and maize; both Fe- and Zn-enriched lentil and sorghum; and varieties with improved vitamin A in orange-fleshed sweet potato, maize, cassava, and banana/plantain. In addition to ongoing efforts, breeding innovations, such as speed breeding and CRISPR-based gene editing technologies, will be necessary for the next decade to reach two billion people with biofortified crops.

Genome-wide association study reveals significant genomic regions for improving yield, adaptability of rice under dry direct seeded cultivation condition.

BACKGROUND: Puddled transplanted system of rice cultivation despite having several benefits, is a highly labor, water and energy intensive system. In the face of changing climatic conditions, a successful transition from puddled to dry direct seeded rice (DDSR) cultivation system looks must in future. Genome-wide association study was performed for traits including, roots and nutrient uptake (14 traits), plant-morphological (5 traits), lodging-resistance (4 traits) and yield and yield attributing traits (7 traits) with the aim to identify significant marker-trait associations (MTAs) for traits enhancing rice adaptability to dry direct-seeded rice (DDSR) system. RESULTS: Study identified a total of 37 highly significant MTAs for 20 traits. The false discovery rate (FDR) ranged from 0.264 to 3.69 × 10- 4, 0.0330 to 1.25 × 10- 4, and 0.0534 to 4.60 × 10- 6 in 2015WS, 2016DS and combined analysis, respectively. The percent phenotypic variance (PV) explained by SNPs ranged from 9 to 92%. Among the identified significant MTAs, 15 MTAs associated with the traits including nodal root, root hair length, root length density, stem and culm diameter, plant height and grain yield were reported to be located in the proximity of earlier identified candidate gene. The significant positive correlation of grain-yield with seedling establishment traits, root morphological and nutrient-uptake related traits and grain yield attributing traits pointing towards combining target traits to increase rice yield and adaptability under DDSR. Seven promising progenies with better root morphology, nutrient-uptake and higher grain yield were identified that can further be used in genomics assisted breeding for DDSR varietal development. CONCLUSIONS: Once validated, the identified MTAs and the SNPs linked with trait of interest could be of direct use in genomic assisted breeding (GAB) to improve grain yield and adaptability of rice under DDSR.

Deciphering the genetic basis of root morphology, nutrient uptake, yield, and yield-related traits in rice under dry direct-seeded cultivation systems.

In the face of global water scarcity, a successful transition of rice cultivation from puddled to dry direct-seeded rice (DDSR) is a future need. A genome-wide association study was performed on a complex mapping population for 39 traits: 9 seedling-establishment traits, 14 root and nutrient-uptake traits, 5 plant morphological traits, 4 lodging resistance traits, and 7 yield and yield-contributing traits. A total of 10 significant marker-trait associations (MTAs) were found along with 25 QTLs associated with 25 traits. The percent phenotypic variance explained by SNPs ranged from 8% to 84%. Grain yield was found to be significantly and positively correlated with seedling-establishment traits, root morphological traits, nutrient uptake-related traits, and grain yield-contributing traits. The genomic colocation of different root morphological traits, nutrient uptake-related traits, and grain-yield-contributing traits further supports the role of root morphological traits in improving nutrient uptake and grain yield under DDSR. The QTLs/candidate genes underlying the significant MTAs were identified. The identified promising progenies carrying these QTLs may serve as potential donors to be exploited in genomics-assisted breeding programs for improving grain yield and adaptability under DDSR.

Microsatellite-based genetic diversity patterns in disjunct populations of a rare orchid.

We investigated the patterns of genetic diversity and structure in seven disjunct populations of a rare North American orchid, Cypripedium kentuckiense by including populations that represented the periphery and the center of the its range. Eight nuclear and two chloroplast microsatellites were used. Genetic diversity was low across the sampled populations of C. kentuckiense based on both nuclear (average An = 4.0, Ho = 0.436, He = 0.448) and cpDNA microsatellites (average An = 1.57, Nh = 1.57 and H = 0.133). The number of private alleles ranged from one to four per population with a total of 17 private alleles detected at five nuclear microsatellites. One private allele at one cpDNA microsatellite was also observed. Although the absolute values for nuclear microsatellite based population differentiation were low (Fst = 0.075; ϕPT = 0.24), they were statistically significant. Pairwise Fst values ranged from 0.038 to 0.123 and each comparison was significant. We also detected isolation by distance with nDNA microsatellites based on the Mantel test (r(2) = 0.209, P = 0.05). STRUCTURE analysis and the neighbor joining trees grouped the populations similarly whereby the geographically proximal populations were genetically similar. Our data indicate that the species is genetically depauperate but the diversity is distributed more or less equally across its range. Population differentiation and isolation by distance were detectable, which indicates that genetic isolation is beginning to manifest itself across the range in this rare species.

A narrowly endemic photosynthetic orchid is non-specific in its mycorrhizal associations.

Mycorrhizal association is a common characteristic in a majority of land plants, and the survival and distribution of a species can depend on the distribution of suitable fungi in its habitat. Orchidaceae is one of the most species-rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and some also for subsequent growth and survival. Given this obligate dependence, at least in the early growth stages, elucidating the patterns of orchid-mycorrhizal relationships is critical to orchid biology, ecology and conservation. To assess whether rarity of an orchid is determined by its specificity towards its fungal hosts, we studied the spatial and temporal variability in the host fungi associated with one of the rarest North American terrestrial orchids, Piperia yadonii. The fungal internal transcribed spacer region was amplified and sequenced by sampling roots from eight populations of P. yadonii distributed across two habitats, Pinus radiata forest and maritime chaparral, in California. Across populations and sampling years, 26 operational taxonomic units representing three fungal families, the Ceratobasidiaceae, Sebacinaceae and Tulasnellaceae, were identified. Fungi belonging to the Sebacinaceae were documented in orchid roots only at P. radiata forest sites, while those from the Ceratobasidiaceae and Tulasnellaceae occurred in both habitats. Our results indicate that orchid rarity can be unrelated to the breadth of mycorrhizal associations. Our data also show that the dominance of various fungal families in mycorrhizal plants can be influenced by habitat preferences of mycorrhizal partners.

Isolation and characterization of microsatellites in a medicinal plant, Phyllanthus emblica (Euphorbiaceae).

PREMISE OF THE STUDY: Microsatellite markers were isolated and characterized in a medicinal plant, Phyllanthus emblica, to study population genetics for designing an effective in situ and ex situ conservation of genetic resources of the species. • METHODS AND RESULTS: Six microsatellite markers were developed using an enrichment and magnetic separation protocol. They were characterized in two natural populations of P. emblica. Out of the six microsatellites, five showed polymorphism, with the number of alleles ranging from four to seven. Observed and expected heterozygosities ranged from 0.360 to 0.760 and 0.499 to 0.806, respectively. • CONCLUSIONS: The five polymorphic microsatellite markers will be useful for studying the genetic structure, reproductive biology, and for identification of clones and provenances of this important medicinal plant.

Higher fine-scale genetic structure in peripheral than in core populations of a long-lived and mixed-mating conifer--eastern white cedar (Thuja occidentalis L.).

BACKGROUND: Fine-scale or spatial genetic structure (SGS) is one of the key genetic characteristics of plant populations. Several evolutionary and ecological processes and population characteristics influence the level of SGS within plant populations. Higher fine-scale genetic structure may be expected in peripheral than core populations of long-lived forest trees, owing to the differences in the magnitude of operating evolutionary and ecological forces such as gene flow, genetic drift, effective population size and founder effects. We addressed this question using eastern white cedar (Thuja occidentalis) as a model species for declining to endangered long-lived tree species with mixed-mating system. RESULTS: We determined the SGS in two core and two peripheral populations of eastern white cedar from its Maritime Canadian eastern range using six nuclear microsatellite DNA markers. Significant SGS ranging from 15 m to 75 m distance classes was observed in the four studied populations. An analysis of combined four populations revealed significant positive SGS up to the 45 m distance class. The mean positive significant SGS observed in the peripheral populations was up to six times (up to 90 m) of that observed in the core populations (15 m). Spatial autocorrelation coefficients and correlograms of single and sub-sets of populations were statistically significant. The extent of within-population SGS was significantly negatively correlated with all genetic diversity parameters. Significant heterogeneity of within-population SGS was observed for 0-15 m and 61-90 m between core and peripheral populations. Average Sp, and gene flow distances were higher in peripheral (Sp = 0.023, σg = 135 m) than in core (Sp = 0.014, σg = 109 m) populations. However, the mean neighborhood size was higher in the core (Nb = 82) than in the peripheral (Nb = 48) populations. CONCLUSION: Eastern white cedar populations have significant fine-scale genetic structure at short distances. Peripheral populations have several-folds higher within-population fine-scale genetic structure than core populations. Anthropogenic disturbances and population fragmentation presumably have significant effects on fine-scale genetic structure in eastern white cedar. Core populations have higher neighborhood size than peripheral populations, whereas gene flow distances are higher in peripheral than in core populations. The results of our study contribute to the knowledge of poorly-understood spatial genetic structure of core versus peripheral populations in plants. As well, the information is of significance for conservation of genetic resources of eastern white cedar and perhaps of other long-lived forest trees with mixed-mating system.

Genetic diversity and differentiation of core vs. peripheral populations of eastern white cedar, Thuja occidentalis (Cupressaceae).

PREMISE OF THE STUDY: Geographically peripheral (marginal) populations are expected to have lower genetic diversity and higher genetic differentiation than geographically core (central) populations as a result of supposedly lower effective population size (N(e)) and higher genetic drift, founder effect, fragmentation, and isolation in peripheral than in core populations. Here we address this issue for a long-lived plant species, eastern white cedar (Thuja occidentalis). METHODS: Genetic diversity and population structure of 13 natural populations of eastern white cedar from its Canadian eastern peripheral and core natural ranges in New Brunswick, Nova Scotia, and Prince Edward Island were studied using six nuclear microsatellite DNA markers. KEY RESULTS: The core populations of eastern white cedar had significantly higher allelic diversity (mean A = 8.83, A(r) = 8.13, A(e) = 4.03) and N(e) (428) than the peripheral populations (A = 6.64, A(r) = 6.15, A(e) = 3.12, N(e) = 198). However, expected heterozygosity was similar in the core (H(e) = 0.64) and peripheral (H(e) = 0.60) populations. Genetic differentiation was significantly higher among the peripheral (F(ST) = 0.089) than among the core (F(ST) = 0.032) populations. No genetic differentiation (F(ST)/Φ(RT) = 0.000) was detected between core and peripheral regions. CONCLUSIONS: Peripheral populations have significantly lower N(e) and genetic diversity in terms of allelic diversity (richness) and significantly higher genetic differentiation than the core populations of eastern white cedar in its Canadian eastern range. However, core and peripheral populations have similar levels of expected heterozygosity. Implications for conservation of eastern white cedar genetic resources are discussed.

Genetic differentiation in natural populations of a keystone bunchgrass (Aristida stricta) across its native range.

Aristida stricta Michx. (Poaceae) is a perennial bunchgrass native to the Southeastern Coastal Plain of North America where it is a keystone species in the longleaf pine savannas and slash pine flatwoods from southeastern North Carolina to Florida, and westward to the coast of Mississippi. We examined genetic relationships within and among ten populations of A. stricta by using eight inter-simple sequence repeat (ISSR) markers to generate band frequency data for 32 individuals from each sampled population. An analysis of molecular variance showed that 38% of the variation resided among populations while 62% was attributable to variation within populations. Grouping the populations by habitat or by geographic location did not show significant differentiation between the groups. Overall, pair-wise geographic and genetic distances were not correlated. Data indicate that while individuals within each population are genetically diverse, there seemingly are barriers to gene flow across populations leading to their divergence. Each population contains several exclusive loci suggesting that limited gene flow and/or genetic drift are likely leading to this pattern of localization. Our results, coupled with those of the previous studies that presented evidence for local adaptation and phenotypic differences among populations, suggest that there is sufficient differentiation among populations of this species to warrant: (1) maintenance of the existing genetic diversity at individual sites, and (2) use of local seed and plant sources for conservation projects.

A simple method for estimating genetic diversity in large populations from finite sample sizes.

BACKGROUND: Sample size is one of the critical factors affecting the accuracy of the estimation of population genetic diversity parameters. Small sample sizes often lead to significant errors in determining the allelic richness, which is one of the most important and commonly used estimators of genetic diversity in populations. Correct estimation of allelic richness in natural populations is challenging since they often do not conform to model assumptions. Here, we introduce a simple and robust approach to estimate the genetic diversity in large natural populations based on the empirical data for finite sample sizes. RESULTS: We developed a non-linear regression model to infer genetic diversity estimates in large natural populations from finite sample sizes. The allelic richness values predicted by our model were in good agreement with those observed in the simulated data sets and the true allelic richness observed in the source populations. The model has been validated using simulated population genetic data sets with different evolutionary scenarios implied in the simulated populations, as well as large microsatellite and allozyme experimental data sets for four conifer species with contrasting patterns of inherent genetic diversity and mating systems. Our model was a better predictor for allelic richness in natural populations than the widely-used Ewens sampling formula, coalescent approach, and rarefaction algorithm. CONCLUSIONS: Our regression model was capable of accurately estimating allelic richness in natural populations regardless of the species and marker system. This regression modeling approach is free from assumptions and can be widely used for population genetic and conservation applications.

Successful cross-amplification of Shorea microsatellites reveals genetic variation in the tropical tree, Shorea robusta Gaertn.

Sal (Shorea robusta) is a tree species with very good quality timber found across tropical south Asia. Due to over-exploitation, its genetic resources are highly depleted. Here we report for the first time on molecular markers in sal. Cross-species amplification of 27 Shorea microsatellites resulted in 24 of them harboring amplicons of expected sizes. Five microsatellites were moderate to highly polymorphic (N(a)=6-19). Observed and expected heterozygosities ranged from 0.49 to 0.77 and 0.52 to 0.89, respectively. These microsatellites are found to be adaptively neutral and are useful for population genetic studies in this species.

Genetic relatedness and population differentiation of Himalayan hulless barley (Hordeum vulgare L.) landraces inferred with SSRs.

A set of 107 hulless barley (Hordeum vulgare L. subsp. vulgare) landraces originally collected from the highlands of Nepal along the Annapurna and Manaslu Himalaya range were studied for genetic relatedness and population differentiation using simple sequence repeats (SSRs). The 44 genome covering barley SSRs applied in this study revealed a high level of genetic diversity among the landraces (diversity index, DI = 0.536) tested. The genetic similarity (GS) based UPGMA clustering and Bayesian Model-based (MB) structure analysis revealed a complex genetic structure of the landraces. Eight genetically distinct populations were identified, of which seven were further studied for diversity and differentiation. The genetic diversity estimated for all and each population separately revealed a hot spot of genetic diversity at Pisang (DI = 0.559). The populations are fairly differentiated (theta = 0.433, R(ST) = 0.445) accounting for > 40% of the genetic variation among the populations. The pairwise population differentiation test confirmed that many of the geographic populations significantly differ from each other but that the differentiation is independent of the geographic distance (r = 0.224, P > 0.05). The high level of genetic diversity and complex population structure detected in Himalayan hulless barley landraces and the relevance of the findings are discussed.

Modeling population genetic data in autotetraploid species.

Allozyme and PCR-based molecular markers have been widely used to investigate genetic diversity and population genetic structure in autotetraploid species. However, an empirical but inaccurate approach was often used to infer marker genotype from the pattern and intensity of gel bands. Obviously, this introduces serious errors in prediction of the marker genotypes and severely biases the data analysis. This article developed a theoretical model to characterize genetic segregation of alleles at genetic marker loci in autotetraploid populations and a novel likelihood-based method to estimate the model parameters. The model properly accounts for segregation complexities due to multiple alleles and double reduction at autotetrasomic loci in natural populations, and the method takes appropriate account of incomplete marker phenotype information with respect to genotype due to multiple-dosage allele segregation at marker loci in tetraploids. The theoretical analyses were validated by making use of a computer simulation study and their utility is demonstrated by analyzing microsatellite marker data collected from two populations of sycamore maple (Acer pseudoplatanus L.), an economically important autotetraploid tree species. Numerical analyses based on simulation data indicate that the model parameters can be adequately estimated and double reduction is detected with good power using reasonable sample size.