Assessment of genetic relationships among cultivated and wild Rubus accessions using AFLP markers

The genus Rubus comprises more than 740 species divided into 12 subgenera and contains the raspberries, blackberries, arctic fruits, and flowering raspberries, all of which have been utilized in breeding programs. The objective of this study was to evaluate the phylogenetic relationships among wild and cultivated Rubus species mainly collected in Japan. To evaluate genetic resources in Rubus, 81 accessions were analyzed with three amplified fragment length polymorphism (AFLP) primer pairs and data were analyzed with the neighbor-joining and unweighted pair group methods with arithmetic mean. Two of the generated phylogenetic trees grouped subgenera Anoplobatus, Eubatus, Idaeobatus, and Malachobatus into different clusters. Accessions of Rubus idaeus L. var. aculeatissimus collected from four regions in Hokkaido formed distinct clusters reflecting sampling sites. Four hybrid accessions between raspberry cultivars (R. idaeus L.) and R. idaeus L. var. aculeatissimus, and one hybrid accession between a raspberry cultivar and Rubus spectabilis Pursh were clearly distinguished from parental accessions. These results indicated that AFLP markers are a reliable technique for assessing genetic diversity and studying phylogenetic relationships in Rubus. Data from diversity and phylogenetic studies revealed valuable information on the availability of unique fragments in different accessions that would be useful for the development of improved genotypes through conventional breeding and marker-assisted selection

Evaluating histone modification analysis of individual preimplantation embryos

ウシ受精卵の新しい遺伝子解析技術を開発 --遺伝子のヒストン修飾を簡易に診断--. 京都大学プレスリリース. 2024-01-23.[Background] We previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT). [Results] WOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing. [Conclusions] These results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis

Early Start of Chemotherapy after Resection of Primary Colon Cancer with Synchronous Multiple Liver Metastases: A Case Report

The start of chemotherapy treatment usually requires a delay of about 4 weeks after surgical resection in patients with primary colorectal cancer and synchronous distant metastasis. However, there is no evidence to indicate the required length of this delay interval. In addition, there is a chance that a patient may die because postoperative chemotherapy was not started soon enough and a metastatic tumor was able to develop rapidly. Here, we present a case in which combination chemotherapy with capecitabine and oxaliplatin (XELOX) was started within 1 week after a right hemicolectomy for synchronous multiple liver metastases. To our knowledge, this is the first report of the start of chemotherapy, involving treatments such as folinic acid, fluorouracil, and oxaliplatin (FOLFOX); folinic acid, fluorouracil, and irinotecan (FOLFIRI); and XELOX, within 1 week after a colorectal cancer operation with anastomosis. The findings suggest possible changes in the start time of chemotherapy after surgery in the future

Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

Advances in Alstroemeria Biotechnology

The genus Alstroemeria belongs to the family Alstroemeriaceae and comprises many ornamental species. This genus, including more than 60 species, is indigenous to South America. Thus far, numerous cultivars, which are used as cut flowers and potted plants worldwide, have been produced by interspecific hybridization and mutation breeding. Recently, biotechnological approaches are being applied in order to improve Alstroemeria strains. Interspecific hybrid plants have been produced by ovule cultures. By improving certain culture techniques, sexual incompatibility was overcome in some cross combinations using ovule cultures. Plant regeneration systems that involved the use of explants, immature ovules, leaves, etc., through callus cultures have been reported. Isolation of protoplasts and cultures resulting in plant regeneration were achieved by using the embryogenic callus. Particle bombardment and Agrobacterium-mediated procedures were applied for genetic transformation, and some transformed plants with marker genes were produced. The procedure of in vitro fertilization using single isolated gametes has been developed to study fertilization and early zygotic embryogenesis in higher plants. This technique will also be utilized as a novel strategy in plant breeding for inducing the fusion of gametes obtained from distantly-related incompatible species and for achieving direct gene transfer into isolated zygotes. In the case of Alstroemeria, isolation of egg cells and zygotes from ovules has been attempted in order to develop an in vitro fertilization technique

An overview of the current research program for haskap (Lonicera caerulea), a useful genetic resource in Hokkaido, Japan

There are many useful genetic resources as berry crop candidates in Hokkaido, Japan. Representative plant genetic resources are haskap ( Lonicera caerulea; Caprifoliaceae), Rubus idaeus (Rosaceae), Actinidia arguta (Actinidiaceae), and so on. Among them, this manuscript focuses on haskap, showing attractive features for commercial production in the Hokkaido regions. In this manuscript, characterization of the wild genetic resources is described. Both diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) were found in Hokkaido. Our study showed that tetraploid haskap populations are widely distributed in Hokkaido, while diploid populations are distributed only in eastern Hokkaido. The factors affecting this distribution pattern are now analyzed. As an attempt to improve haskap accessions, interspecific hybridization has been applied between haskap and Miyama -uguiskagura (Lonicera gracilipes), belonging to the same genus as haskap. The interspecific hybrids were fertile and could produce fruits. To characterize the fruit contents of the parental species and interspecific hybrids, liquid chromatography/tandem mass spectrometry (LC/MS) and imaging mass spectrometry were used for detailed analyses

Interploid and intraploid hybridizations to produce polyploid Haskap (Lonicera caerulea var. emphyllocalyx) plants

We produced polyploid Haskap (Lonicera caerulea var. emphyllocalyx) plants by performing interploid and intraploid crosses of wild accessions. Embryo rescue in a tetraploid (4x) × diploid (2x) cross produced triploid plants; reciprocal 2x × 4x cross failed to produce viable seeds. Intraploidy crosses of 4x × 4x produced mostly tetraploids but also several hexaploid (6x) and octoploid (8x) plants. Using hexaploids obtained from this cross, we examined reciprocal 4x–6x crosses and found that both produced pentaploid plants. An octoploid was produced by applying colchicine to a tetraploid; a 4x × 8x cross using this plant and aided by embryo rescue culture produced three hexaploid plants, with an aneuploid number of chromosomes. Several plants obtained in this study flowered and set fruits. We discuss the overall efficiency of producing polyploid plants in interploid and intraploid crosses

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry of pollen tubes at different developmental stages that were cultured in vitro revealed changes in the nuclear phase at 9 h and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cell showed a tendency to increase. The association was suggested as male germ unit. When sperm cells, vegetative nuclei, and partial male germ units were separately collected from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial male germ units was counted as 2C. Therefore, the second change in the nuclear phase, which resulted in an increase in 2C nuclei, was possibly related to the formation of male germ units