Elucidation of the Origin of the Monumental Olive Tree of Vouves in Crete, Greece

The olive tree of Vouves in Crete, is considered the oldest producing olive tree in the world with an estimated age exceeding 4000 years. In the present study, we sequenced two samples (from the bottom and the top of the tree) to elucidate the genetic relation of this ancient tree with other olive cvs as well as to gain some insights about its origin. Our results showed that both samples have different genetic origins, proving that this ancient tree has been grafted at least one time. On the basis of whole genome sequences the sample from the top of the Vouves tree showed relation of the same order than half-siblings to one accession corresponding to the present-day Greek cv ‘Mastoidis’. Nevertheless, in the framework of a microsatellite analysis it was found to cluster with the ‘Mastoidis’ samples. The Vouves rootstock (bottom sample) showed a clear grouping with the oleaster samples in a similar way to that of ‘Megaritiki’ Greek cv although it does not show any signal of introgression from them. The genomic analyses did not show a strong relation of this sample with the present-day Greek cvs analyzed in this study so it cannot be proved that it has been used as a source for cultivated olive tree populations represented by available genome sequences. Nevertheless, on the basis of microsatellite analyses, the Vouves rootstock showed affinity with two present-day Greek cvs, one “ancient” rootstock from continental Greece as well as monumental trees from Cyprus. The analysis of the impact of the variants in the gene space revealed an enrichment of genes associated to pathways related with carbohydrate and amino acid metabolism. This is in agreement with what has been found before in the sweep regions related with the process of domestication. The absence of oleaster gene flow, its old age and its variant profile, similar to other cultivated populations, makes it an excellent reference point for domestication studies.

The Streptochaeta Genome and the Evolution of the Grasses

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

The Streptochaeta genome and the evolution of the grasses

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size, capturing most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

Comparative genomics plastomes of the Amaryllidaceae family species

The genus Allium covers more than 800 species, signaling among the largest among monocotyledons. The genus contains many economically important species, including garlic, leeks, onions, chives and Chinese chives. Due to the high conservation of chloroplast genomes compared to nuclear genomes and mitochondrial genome, sequence of chloroplasts in Amaryllidaceae have been consistently used for species identification and various in silico programs and strategies have been used to identify, characterize and compare plastid genome regions. Plastome from 15 species of the Amaryllidaceae family revealed similarity in both sequences and in the organization of their gene regions. The base pairs (bp) number ranged from 145,819 (A. paradoxum) to 159,125 (A. ursinum). In respect the GC content, the species presented a variation between 36.7% (A. schoenoprasum and A. sativum) and 37.5% (A. coddii) and the gene space ranged from 84.760 (A. paradoxum) to 94.766 (A. sativum). The genes that encode proteins showed values between 78 (A. paradoxum) to 89 (A. cepa). Phylogenetic trees acquired through alignment of complete plastomas and the plastidial matK gene revealed similarity to the proposed classification for the family. For the genus Allium, there was the formation of three clades with perfect correspondence of the clusters to the three evolutionary lines of the genus.

A draft genome of grass pea (Lathyrus sativus), a resilient diploid legume

We have sequenced the genome of grass pea (Lathyrus sativus), a resilient diploid (2n=14) legume closely related to pea (Pisum sativum). We determined the genome size of the sequenced European accession (LS007) as 6.3 Gbp. We generated two assemblies of this genome, i) EIv1 using Illumina PCR-free paired-end sequencing and assembly followed by long-mate-pair scaffolding and ii) Rbp using Oxford Nanopore Technologies long-read sequencing and assembly followed by polishing with Illumina paired-end data. EIv1 has a total length of 8.12 Gbp (including 1.9 billion Ns) and scaffold N50 59,7 kbp. Annotation has identified 33,819 high confidence genes in the assembly. Rbp has a total length of 6.2 Gbp (with no Ns) and a contig N50 of 155.7 kbp. Gene space assessment using the eukaryote BUSCO database showed completeness scores of 82.8 % and 89.8%, respectively.

Amplicon-sequencing of BCL11a-edited stem cells used to treat beta thalassemia and sickle cell disease suffices to demonstrate that CRISPR is a chromosome shredder

The quanta of ’genome vandalism’ CRISPR does is easily observed by amplicon sequencing of the target gene. The algorithm chooses reads that match the targeted gene GENE, but also another gene (OFFGENE) - meaning there has been a translocation/integration. This translocation/integration is possible if and only if OFFGENE has also been edited - an off-target. Larger the number of reads with GENE-OFFGENE, larger the confidence. For example, the stem-cell study, which edits BCL11a to increase fetal hemoglobin, has 111 BCL11a-RNA45SN5 reads [1]. Analysis of the edit location in RNA45SN5 shows 8 mismatches with the gRNA, which falls within the permissible limits [2]. There are 20 such genes which have integrated with the BCL11a gene with high confidence. This is a very conservative estimate for two reasons. First, amplicon-sequencing only looks at GENE. If two OFFGENE’s were integrating, the data would not show that. Secondly, I have only looked the gene space - so only 2% of the genome. Since we find 20 genes integrating with BCL11a after such constraints, it is fair to assume that CRISPR is literally shredding the chromosome - a possible reason for significant fatalities in clinical trials [3].