Combining higher accumulation of amylopectin, lysine and tryptophan in maize hybrids through genomics-assisted stacking of waxy1 and opaque2 genes

Waxy maize rich in amylopectin has emerged as a preferred food. However, waxy maize is poor in lysine and tryptophan, deficiency of which cause severe health problems. So far, no waxy hybrid with high lysine and tryptophan has been developed and commercialized. Here, we combined recessive waxy1 (wx1) and opaque2 (o2) genes in the parental lines of four popular hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) using genomics-assisted breeding. The gene-based markers, wx-2507F/RG and phi057 specific for wx1 and o2, respectively were successfully used to genotype BC1F1, BC2F1 and BC2F2 populations. Background selection with > 100 SSRs resulted in recovering > 94% of the recurrent parent genome. The reconstituted hybrids showed 1.4-fold increase in amylopectin (mean: 98.84%) compared to the original hybrids (mean: 72.45%). The reconstituted hybrids also showed 14.3% and 14.6% increase in lysine (mean: 0.384%) and tryptophan (mean: 0.102%), respectively over the original hybrids (lysine: 0.336%, tryptophan: 0.089%). Reconstituted hybrids also possessed similar grain yield (mean: 6248 kg/ha) with their original versions (mean: 6111 kg/ha). The waxy hybrids with high lysine and tryptophan assume great significance in alleviating malnutrition through sustainable and cost-effective means. This is the first report of development of lysine and tryptophan rich waxy hybrids using genomics-assisted selection.

​​Identification of Groundnut Germplasm Lines for Foliar Disease Resistance and High Oleic Traits using SNP and Gene-based Markers and Their Morphological Characterization

Background: Resistance to foliar fungal diseases along with oleic acid trait, are important objectives of groundnut breeding. Among foliar fungal diseases, rust and Late Leaf Spot (LLS) cause significant economic loss and high oleic trait is preferred in industry that enhances economic values of crop. Methods: Morphological characterization of the 186-groundnut germplasm lines/genotypes for ten yield attributing traits and their significance of correlation was analyzed using SPSS ver. 19 software at 1% and 5% probability level of significance. Screening for LLS and rust diseases was done employing 10X SNP assay at ICRISAT, Hyderabad, India. Selected superior groundnut germplasm line(s) were screened for presence of FAD2B allele responsible for high oleic acid traits using allele specific marker. Result: Significant and positive correlation was found between dry weight and hundred pod weight (r=0.0.801) and harvest index (r=0.0.830). Molecular characterization along with morphological characterization identified highly diversified lines of groundnut. This study reports 78 foliar fungal disease resistant groundnut germplasm lines. Selected 11 groundnut germplasm lines represented resistance against LLS and rust diseases along with FAD2B allele for oleic acid trait.

Development of Maize Hybrids With Enhanced Vitamin-E, Vitamin-A, Lysine, and Tryptophan Through Molecular Breeding

Malnutrition is a widespread problem that affects human health, society, and the economy. Traditional maize that serves as an important source of human nutrition is deficient in vitamin-E, vitamin-A, lysine, and tryptophan. Here, favorable alleles of vte4 (α-tocopherol methyl transferase), crtRB1 (β-carotene hydroxylase), lcyE (lycopene ε-cyclase), and o2 (opaque2) genes were combined in parental lines of four popular hybrids using marker-assisted selection (MAS). BC1F1, BC2F1, and BC2F2 populations were genotyped using gene-based markers of vte4, crtRB1, lcyE, and o2. Background selection using 81–103 simple sequence repeats (SSRs) markers led to the recovery of recurrent parent genome (RPG) up to 95.45%. Alpha (α)-tocopherol was significantly enhanced among introgressed progenies (16.13 μg/g) as compared to original inbreds (7.90 μg/g). Provitamin-A (proA) (10.42 μg/g), lysine (0.352%), and tryptophan (0.086%) were also high in the introgressed progenies. The reconstituted hybrids showed a 2-fold enhancement in α-tocopherol (16.83 μg/g) over original hybrids (8.06 μg/g). Improved hybrids also possessed high proA (11.48 μg/g), lysine (0.367%), and tryptophan (0.084%) when compared with traditional hybrids. The reconstituted hybrids recorded the mean grain yield of 8,066 kg/ha, which was at par with original hybrids (mean: 7,846 kg/ha). The MAS-derived genotypes resembled their corresponding original hybrids for the majority of agronomic and yield-related traits, besides characteristics related to distinctness, uniformity, and stability (DUS). This is the first report for the development of maize with enhanced vitamin-E, vitamin-A, lysine, and tryptophan.

Identification of Candidate Gene-Based Markers for Girth Growth in Rubber Trees

Girth growth is an important factor in both latex and timber production of the rubber tree. In this study, we performed candidate gene association mapping for girth growth in rubber trees using intron length polymorphism markers (ILP) in identifying the candidate genes responsible for girth growth. The COBL064_1 marker developed from the candidate gene (COBL4) regulating cellulose deposition and oriented cell expansion in the plant cell wall showed the strongest association with girth growth across two seasons in the Amazonian population and was validated in the breeding lines. We then applied single molecule real-time (SMRT) circular consensus sequencing (CCS) to analyze a wider gene region of the COBL4 to pinpoint the single nucleotide polymorphism (SNP) that best explains the association with the traits. A SNP in the 3’ UTR showing linkage disequilibrium with the COBL064_1 most associated with girth growth. This study showed that the cost-effective method of ILP gene-based markers can assist in identification of SNPs in the candidate gene associated with girth growth. The SNP markers identified in this study added useful markers for the improvement of girth growth in rubber tree breeding programs.

Gene Based ­­­­Markers in Marker-Assisted Selection to Screen Tomato Genotypes Resistant to Fusarium Wilt, Late Blight, Verticillium Wilt, Leaf Mold, Bacterial Spot and Bacterial Speck

The authors have requested that this preprint be withdrawn due to author disagreement.

Genetic Diversity and Population Assessment of Musa L. (Musaceae) Employing CDDP Markers

Sixty-six accessions of Musa genus with different genomic groups that consisted of wild relatives and cultivated lines were obtained from the International Transit Center, Belgium, for DNA extraction using Cetyl trimethylammonium bromide method, followed by amplification with Conserved DNA-derived Polymorphism (CDDP) markers for genetic diversity and population assessment. A total of 421 alleles with major allele frequency of 2.051 were detected from the reproducible markers. High genetic diversity (GD, 11.093) and polymorphic information content (0.918) were revealed. The number of polymorphic loci and percentage of polymorphic loci ranged from 59 to 66 and 89.34 to 100, respectively. Using the potential genetic indicators including effective number of alleles, Nei’s genetic diversity, and Shannon’s information index, the AS genomic group was identified to have the highest GD, while the AAA accessions had the lowest GD indices. The GD parameters identified in the accessions were ranked as AS > AAB > AAAA > AA > ABB > wild diploidy > BB > AB > AAA from high to low based on polymorphic loci of the markers. Total intraspecific GD, interspecific GD, and estimate gene flow identified were 0.433, 0.404, and 7.113, respectively. The coefficient of gene differentiation of 0.066 was obtained, indicating 6.57% among the population and 93.43% within the population. Dendrogram analysis produced nine major groups with subgroups at similarity index of 0.814. These CDDP functional gene-based markers were informative and very efficient in resolving GD, and population indices among the banana and plantain accessions of different genomes. The identified CDDP markers might serve as potential tools for selecting suitable training populations for breeding and conservation of Musa species.

The use of effector gene based-markers to facilitate identification of Fusarium sp. infected shallot in Java, Indonesia

Herlina L, Istiaji B. 2020. The use of effector gene based-markers to facilitate identification of Fusarium sp. infected shallot in Java, Indonesia. Biodiversitas 21: 4677-4685. One of the most important diseases and become challenge in breeding resistant variety of shallots in Indonesia is Fusarium disease, caused by Fusarium oxysporum f.sp. cepae (FOC). To discriminate the F. oxysporum into forma speciales is uneasy, often managed through laborious and time-intensive disease assays., therefore molecular approaches become the most relevant choice. FOCs from Indonesia were examined by molecular criteria based on putative and effector genes-based markers, i.e.: SIX-genes, C5-gene, and CRX1-2-genes. The alignment analysis discovered some regions which sequences highly contained conserved bases, while amplification result bands vary in size, between 400 - 500 bp. The combined of 7 SIX-genes primers and 3 effector primers (C5 and CRX1-2) used in the clustering analysis of 15 FOC isolates in this study showed that they succeeded in separating 15 FOC isolates into 4 groups through NTSys. Clustering analysis showed that those markers succeeded in grouping 15 FOC isolates into different clades (by coefficient of similarity: 0.69). Phylogenetic analysis based on CRX genes sequence as putative effector genes confirmed that CRX1 and CRX2 genes were able to classify the FOC into their forma speciales. Those effectors genes are potential to serve as marker-templates to facilitate identification of FOC which infested shallots in Indonesia.

Molecular marker validation and identification of Fusarium wilt resistant chickpea genotypes

Fusarium wilt is one of the most destructive biotic stress reducing chickpea productivity worldwide. As a step towards understanding the basis of wilt resistance in chickpea, we investigated the morpho-physiological and biochemical traits of 29 desi and 15 kabuli chickpea genotypes and screened these genotypes for fusarium wilt disease using gene based molecular markers. The number of branches per plant were significant and positively correlated with number of pods per plant (r=0.635) and number of seed per plant (r=0.556) at 1% significance level. Biochemical parameters related to stresses were also analyzed for proline (1.19-3.92 µ mol/g), sugar (23.6-37.4 mg/g), malondialdehyde (MDA) (1.1 -3.67 nmol/g) and hydrogen peroxide (H2O2) (10.4-21.5 µ mol/g) in seeds of these genotypes grown under normal field conditions. Molecular screening was done by using15 gene-based markers. Polymorphic Information Content (PIC) value was in the range of 0.221 to 0.695, respectively. The genotypes JG-63 and Vijay identified during the investigation could be included in the hybridization programs during development of high yielding and wilt resistant varieties. The molecular markers TA194, TA-59, TA-96, TR-19, TR-29 and TR-31 can be used as marker assisted breeding tools for screening, validation and development of fusarium wilt resistant chickpea genotypes.