Einkorn genomics sheds light on history of the oldest domesticated wheat

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago$^{1,2}$. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat

An online database for einkorn wheat to aid in gene discovery and functional genomics studies

Diploid A-genome wheat (einkorn wheat) presents a nutrition-rich option as an ancient grain crop and a resource for the improvement of bread wheat against abiotic and biotic stresses. Realizing the importance of this wheat species, reference-level assemblies of two einkorn wheat accessions were generated (wild and domesticated). This work reports an einkorn genome database that provides an interface to the cereals research community to perform comparative genomics, applied genetics and breeding research. It features queries for annotated genes, the use of a recent genome browser release, and the ability to search for sequence alignments using a modern BLAST interface. Other features include a comparison of reference einkorn assemblies with other wheat cultivars through genomic synteny visualization and an alignment visualization tool for BLAST results. Altogether, this resource will help wheat research and breeding. Database URL  https://wheat.pw.usda.gov/GG3/pangenome

Cloning and Characterization of Limonoid Glucosyltransferase from Kinnow Mandarin (Citrus reticulata Blanco)

Mandarina sorte Kinnow (Citrus reticulata Blanco) popularna je biljka iz roda citrusa u sjeverozapadnoj Indiji, koja se najviše uzgaja u državi Punjab. Međutim, limonoid aglikoni poput limonina uzrokuju gorak okus, što predstavlja najveći problem u industrijskoj proizvodnji soka od agruma. Iz tkiva mandarine sorte Kinnow izoliran je gen limonoid glukoziltransferaza (LGT) što kodira enzim za odgorčavanje, te je ispitan metabolizam razgradnje limonoida u agrumima. Nakon što je gen identificiran i karakteriziran, njegova cjelokupna sekvencija (1533 bp) deponirana je u bazu Nacionalnog centra za biotehnološke informacije (NCBI). Glukozil transferaza iz vrste Citrus reticulata (CrLGT) nalazi se na zasebnoj grani najveće podgrupe i filogenetski se razlikuje od onih u ostalih sorata mandarina, kao što je C. unshiu, te je jedinstvena po mnogim svojstvima. Ekspresija gena CrLGT na razini transkripcije ispitana je pomoću semikvantitativne metode u različitim tkivima biljke, kao što su mladi list, flavedo, albedo, opna i sjeme, tijekom rane (90 dana nakon cvatnje), srednje rane (150-210 dana nakon cvatnje) i kasne (240 dana nakon cvatnje) faze razvoja ploda, te je potvrđeno da je gen imao najveću ekspresiju u flavedu. Zaključeno je da izolirani gen LGT utječe na metaboličku razgradnju limonoida u agrumima. Pojačana ekspresija ovog gena može ublažiti gorčinu soka od agruma i povećati akumulaciju specifičnih glukozida koji imaju antikancerogena svojstva.Kinnow mandarin (Citrus reticulata Blanco) is a popular citrus crop of northwestern India and it occupies maximum fruit area in Punjab. However, citrus juice processing industry is still suffering from delayed bitterness problem caused mainly by limonoid aglycones such as limonin. In order to study citrus limonoid metabolism, limonoid glucosyltransferase (LGT) gene, which encodes a natural debittering enzyme, was isolated from the fruit tissues of Kinnow mandarin. After confirmation and characterization, its full-length gene sequence (1533 bp) was submitted to National Centre for Biotechnology Information. Citrus reticulata limonoid glucosyltransferase (CrLGT) occupies a position on an independent branch in the largest subgroup and is phylogenetically different from those in other mandarin species like C. unshiu, showing its uniqueness in several features. The transcript expression of CrLGT, evaluated in different tissues such as young leaf, flavedo, albedo, sac covering and seed of Kinnow mandarin during early (90 days after flowering (DAF)), mid (150-210 DAF) and late (240 DAF) fruit developmental stages using semi-quantitative method, showed the highest expression in flavedo. Thus, it was concluded that the isolated LGT gene has an effect on limonoid metabolic engineering in citrus. Overexpression of this gene can reduce the delayed bitterness problem in citrus juice and enhance the accumulation of specific glucosides that have anticancer effects

Novel Alleles of Phosphorus-Starvation Tolerance 1 Gene (PSTOL1) from Oryza rufipogon Confers High Phosphorus Uptake Efficiency

Limited phosphorus availability in the soil is one of the major constraints to the growth and productivity of rice across Asian, African and South American countries, where 50% of the rice is grown under rain-fed systems on poor and problematic soils. With an aim to determine novel alleles for enhanced phosphorus uptake efficiency in wild species germplasm of rice Oryza rufipogon, we investigated phosphorus uptake1 (Pup1) locus with 11 previously reported SSR markers and sequence characterized the phosphorus-starvation tolerance 1 (PSTOL1) gene. In the present study, we screened 182 accessions of O. rufipogon along with Vandana as a positive control with SSR markers. From the analysis, it was inferred that all of the O. rufipogon accessions undertaken in this study had an insertion of 90 kb region, including Pup1-K46, a diagnostic marker for PSTOL1, however, it was absent among O. sativa cv. PR114, PR121, and PR122. The complete PSTOL1 gene was also sequenced in 67 representative accessions of O. rufipogon and Vandana as a positive control. From comparative sequence analysis, 53 mutations (52 SNPs and 1 nonsense mutation) were found in the PSTOL1 coding region, of which 28 were missense mutations and 10 corresponded to changes in the amino acid polarity. These 53 mutations correspond to 17 haplotypes, of these 6 were shared and 11 were scored only once. A major shared haplotype was observed among 44 accessions of O. rufipogon along with Vandana and Kasalath. Out of 17 haplotypes, accessions representing 8 haplotypes were grown under the phosphorus-deficient conditions in hydroponics for 60 days. Significant differences were observed in the root length and weight among all the genotypes when grown under phosphorus deficiency conditions as compared to the phosphorus sufficient conditions. The O. rufipogon accession IRGC 106506 from Laos performed significantly better, with 2.5 times higher root weight and phosphorus content as compared to the positive control Vandana. In terms of phosphorus uptake efficiency, the O. rufipogon accessions IRGC 104639, 104712, and 105569 also showed nearly two times higher phosphorus content than Vandana. Thus, these O. rufipogon accessions could be used as the potential donor for improving phosphorus uptake efficiency of elite rice cultivars

Red rot resistant transgenic sugarcane developed through expression of β-1,3-glucanase gene.

Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of β-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. β-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving β-1,3-glycosidic bonds and pathogen hyphal lysis

Identification of genomic regions associated with shoot fly resistance in maize and their syntenic relationships in the sorghum genome.

Shoot fly (Atherigona naqvii) is one of the major insects affecting spring maize in North India and can cause yield loss up to 60 per cent. The genetics of insect resistance is complex as influenced by genotypic background, insect population and climatic conditions. Therefore, quantitative trait loci (QTL) mapping is a highly effective approach for studying genetically complex forms of insect resistance. The objective of the present study was to dissect the genetic basis of resistance and identification of genomic regions associated with shoot fly resistance. A total of 107 F2 population derived from the cross CM143 (resistant) x CM144 (susceptible) was genotyped with 120 SSR markers. Phenotypic data were recorded on replicated F2:3 progenies for various component traits imparting resistance to shoot fly at different time intervals. Resistance to shoot fly was observed to be under polygenic control as evidenced by the identification of 19 putative QTLs governed by overdominance to partial dominance and additive gene actions. The major QTLs conditioning shoot fly resistance viz., qDH9.1 (deadheart) and qEC9.1 (oviposition) explaining 15.03 and 18.89 per cent phenotypic variance, respectively were colocalized on chromosome 9. These QTLs are syntenic to regions of chromosome 10 of sorghum which were also accounted for deadheart and oviposition suggesting that the same gene block may be responsible for shoot fly resistance. The candidate genes such as cysteine protease, subtilisin-chymotrypsin inhibitor, cytochrome P450 involved in synthesis of alleochemicals, receptor kinases, glossy15 and ubiquitin-proteasome degradation pathway were identified within the predicted QTL regions. This is the first reported mapping of QTLs conferring resistance to shoot fly in maize, and the markers identified here will be a valuable resource for developing elite maize cultivars with resistance to shoot fly

Structural model showing protein-ligand interaction.

<p>β-1,3-glucanase protein (GenBank Accession No. AIC32930) docked with D-glucose.</p

Quantitative RT-PCR analysis.

<p>Relative <i>β-1</i>,<i>3-glucanase</i> expression of RT-PCR positive CG₁ plants. Bars represent range of 2^{- ΔΔC}_T.</p

Scanning electron micrographs of stalk sections from CG₁ transgenic and NT plants following inoculation with <i>C</i>. <i>falcatum</i>.

<p><b>A</b> Sucrose-filled parenchyma cells of non-inoculated NT plant (control). Arrow indicates the characteristic turgid cell. Bar represents 20 μm. <b>B</b> Presence of normal Cf 09 fungal hyphae in parenchyma cells of susceptible NT plant. Arrow indicates the sucrose depleted cell. Bar represents 20 μm. <b>C</b> Parenchyma cells of transgenic plant moderately resistant to Cf 09. Arrow indicates abnormal fungal hypha and amorphous debris. Bar represents 100 μm. <b>D</b> Sucrose-filled parenchyma cells of transgenic plant resistant to Cf 08 showing absence of hyphae. Arrow indicates the turgid cell. Bar represents 100 μm.</p