Characterization of the Q.Ymym region on wheat chromosome 2D associated with wheat yellow mosaic virus resistance

Yellow mosaic disease, caused by wheat yellow mosaic virus (WYMV), is one of the most serious diseases of winter wheat in Japan and China. A single major QTL for WYMV resistance in the Japanese wheat variety 'Yumechikara', designated Q.Ymym, has been mapped on a 43.6 cM linkage block between the two markers Xcfd233 and Xgwm349 on chromosome 2D. We were able to obtain two recombinants within the block, which facilitated reducing the size of the linkage block. The pseudomolecule sequence of 'Chinese Spring' (CS) indicated that the original Q.Ymym region of 43.6 cM corresponded to 68.5 Mb and the narrowed Q.Ymym region represents a size of 27.3 Mb. The sequence features of the Q.Ymym region were unique in comparison with CS sequences, which may have led to the low recombination rate within the block. The Q.Ymym haplotype block was detected in other WYMV-resistant varieties but not in the susceptible varieties used in this study. The unique sequence structure of the Q.Ymym region allowed the development of co-dominant markers for use in marker-assisted selection.     

Quantification of relative flying fish paste content in the processed seafood ago-noyaki using real-time PCR

Real-time polymerase chain reaction (PCR) analysis of the 3′-portion of the mitochondrial 16S RNA gene (rDNA) coding sequence was used to determine flying fish paste in ago-noyaki. We quantified the amount of flying fish paste in ago-noyaki samples using flying fish-specific primers (Tobi16SF3/Tobi16SR) and universal primers (Univ16SF2/Univ16SR2). Using real-time PCR of standard ago-noyaki, a standard equation was obtained (y = 1.08x − 3.20; R ² = 0.977). This equation was then used to estimate the relative flying fish paste contents of eight commercially available ago-noyaki and two similar products. These results verified that the ago-noyaki products that had already been labeled with the E-mark deserved this status.     

Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0–2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.     

Identification and validation of a quantitative trait locus associated with wheat yellow mosaic virus pathotype I resistance in a Japanese wheat variety

Yellow mosaic disease, caused by wheat yellow mosaic virus (WYMV), is one of the most serious diseases of winter wheat (Triticum aestivum L.) in Japan. The three pathotypes of WYMV are distributed in different geographical areas: pathotype I is found mainly in western and central Japan (Kanto), pathotype II in northern Japan (Tohoku and Hokkaido) and pathotype III on the southern island of Japan (Kyushu). A total of 246 doubled‐haploid (DH) lines, derived from a cross between ‘Yumechikara’ (resistant) and ‘Kitahonami’ (susceptible), were evaluated for 2 years for their resistance to WYMV pathotype I. A single major quantitative trait locus, Q.Ymym, mapping to chromosome 2D was associated with resistance to pathotype I in ‘Yumechikara’. This is the first time a QTL responsible for pathotype I resistance has been identified. Fine mapping of Q.Ymym indicated that it was on a tight linkage block originating from ‘Yumechikara’, and the markers associated with this block will accelerate the development of varieties resistant to WYMV pathotype I.     

Abnormal functions of Leydig cells in crossbred cattle–yak showing infertility

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.     

Formation of an Antral Follicle–like Structure of Bovine Cumulus–Oocyte Complexes Embedded Individually or in Groups in Collagen Gels

Culture techniques of antral follicle–like structure (AFLS) derived from cumulus–oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4–5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day = 0), irrespective of the culture methods. The mean diameters of AFLS during Days 4–14 using the individual culture method were significantly higher (p < 0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p < 0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.     

Effect of cellooligosaccharide or synbiotic feeding on growth performance,fecal condition and hormone concentrations in Holstein calves

We investigated the effect of cellooligosaccharide (CE) or a combination of dextran and Lactobacillus casei ssp. casei strain JCM1134^T (synbiotic; SB) feeding on growth performance, fecal condition and hormone concentrations in Holstein calves. Fifty‐two female Holstein calves were randomly assigned to three treatment groups: CE feeding group (n = 16), SB feeding group (n = 18), and control group (n = 18). Body weight at 90 days of age, as well as daily body weight gain (DG) and feed efficiency after weaning to 90 days of age were greater (P < 0.05) in the CE feeding group than in the control group. The total fecal score tended to be lower (P < 0.1) in the SB feeding group than in the control group. Plasma insulin concentration was higher (P < 0.05) in the CE feeding group than in the control group at 90 days of age. Our results indicate that CE feeding improved DG and feed efficiency in calves. On the other hand, there was less effect on growth performance and fecal Escherichia coli counts in calves fed SB.     

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long‐term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5‐SM and 15‐SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5‐SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5‐SM and 15‐SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5‐SM group showed similar rates of fertilization and blastocyst formation in the fresh non‐stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5‐SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.     

Characterization of a mini core collection of Japanese wheat varieties using single-nucleotide polymorphisms generated by genotyping-by-sequencing

A core collection of Japanese wheat varieties (JWC) consisting of 96 accessions was established based on their passport data and breeding pedigrees. To clarify the molecular basis of the JWC collection, genome-wide single-nucleotide polymorphism (SNP) genotyping was performed using the genotyping-by-sequencing (GBS) approach. Phylogenetic tree and population structure analyses using these SNP data revealed the genetic diversity and relationships among the JWC accessions, classifying them into four groups; “varieties in the Hokkaido area”, “modern varieties in the northeast part of Japan”, “modern varieties in the southwest part of Japan” and “classical varieties including landraces”. This clustering closely reflected the history of wheat breeding in Japan. Furthermore, to demonstrate the utility of the JWC collection, we performed a genome-wide association study (GWAS) for three traits, namely, “days to heading in autumn sowing”, “days to heading in spring sowing” and “culm length”. We found significantly associated SNP markers with each trait, and some of these were closely linked to known major genes for heading date or culm length on the genetic map. Our study indicates that this JWC collection is a useful set of germplasm for basic and applied research aimed at understanding and utilizing the genetic diversity among Japanese wheat varieties.