Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F₂ progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.     

Tevaluation of transgenic event CBH 351 (StarLink) corn in pig

This study examined the influence of transgenic event CBH (StarLink™; SL)-derived hybrid corn on growth, health and physiological functions of pigs, as well as the possibility of transferring the cry9C gene or Cry9C protein to the blood, liver or muscles, in comparison with pigs fed a diet with non-transgenic (isogenic) corn (non-SL). The diet for the SL group was composed of 70% SL corn, and the diet for the non-SL group was composed of 70% non-SL corn. Forty pigs approximately 3 months in age were used in the current experiment. After the pigs were acclimatized to their environment for 7 days, they were fed piglet diets for 7 weeks, and afterwards fed growing-finishing diets until the end of the experiment. There were no significant differences in bodyweight gain, feed intake or feed conversion ratio between the pigs fed SL diet and those of non-SL diet. No abnormalities were observed in the health conditions of either the SL or the non-SL group. Moreover, no significant differences were observed between the two groups in hematological values, histopathological examination and necropsy findings. Although the serum biochemical values within each group were normal, the blood urea nitrogen values of the SL group showed a tendency to be slightly higher than those of the non-SL group. Also, the blood glucose values of the SL group were significantly lower than those of the non-SL group. However, the cause of the significant differences in the blood glucose values between the two groups is unknown. The PCR and ELISA did not detect the cry9C gene and Cry9C protein in the blood, liver or muscles of the pigs at the end of the experiment.     

Common nucleotide sequence of structural gene encoding fibroblast growth factor 4 in eight cattle derived from three breeds

Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for the proper development of bovine embryos. However, the complete nucleotide sequences of the structural genes encoding FGF4 in identified breeds are still unknown. In the present study, direct sequencing of PCR products derived from genomic DNA samples obtained from three Japanese Black, two Japanese Shorthorn and three Holstein cattle, revealed that the nucleotide sequences of the structural gene encoding FGF4 matched completely among these eight cattle. On the other hand, differences in the nucleotide sequences, leading to substitutions, insertions or deletions of amino acid residues were detected when compared with the already reported sequence from unidentified breeds. We cannot rule out a possibility that the structural gene elucidated in the present study is widely distributed in cattle. To the best of our knowledge, this is the first determination of the complete nucleotide sequence of the structural gene encoding bovine FGF4 in identified breeds.     

Comparison of the functions of the barley nicotianamine synthase gene HvNAS1 and rice nicotianamine synthase gene OsNAS1 promoters in response to iron deficiency in transgenic tobacco

Barley ( Hordeum vulgare L.) nicotianamine synthase gene ( HvNAS1 ) expression in barley is strongly induced by Fe deficiency in the roots and rice ( Oryza sativa L.) nicotianamine synthase gene ( OsNAS1 ) expression in rice is induced by Fe deficiency both in the roots and in the shoots. In dicots, NAS genes are not strongly induced by Fe deficiency, and they function to maintain Fe homeostasis. Rice OsNAS1promoter::GUS or barley HvNAS1promoter::GUS was introduced into tobacco ( Nicotiana tabacum L.) and tissue specificities and systemic regulation of their expression were compared. A split-root experiment revealed that the HvNAS1 promoter exhibited functions similar to those of Fe-acquisition-related genes in tobacco roots, suggesting that this promoter responded to certain Fe-deficiency systemic signals and to the Fe concentration in the rhizosphere. The HvNAS1 promoter might harbor a type of universal system of gene expression for Fe acquisition. However, the OsNAS1 promoter did not respond to local application of Fe to the roots and induced GUS activities in mature leaves in response to Fe deficiency. This promoter might possess numerous types of cis -acting sequences that are involved in Fe metabolism.     

Down-regulation of endogenous Wt1 expression by Sry transgene in the murine embryonic mesonephros-derived M15 cell line

Wt1 is one of numerous candidate genes comprising the hypothetical chain of gene expression essential for male sex differentiation of the bipotential indifferent gonads during embryogenesis. However, the evidence in the literature is ambivalent regarding the position of Wt1 relative to Sry in this scheme; Wt1 might act either upstream or downstream of Sry. In the present study, the effects of Sry expression upon Wt1 were investigated using M15 cells (XX karyotype), which are derived from murine embryonic mesonephros and express endogenous Wt1. In 3 stably-transformed Sry-expressing M15 cell lines, we showed that the expression levels of the mRNAs coding for all 4 isoforms of the WT1 proteins were down-regulated. Similarly, Wnt 4 expression was down-regulated in these cell lines. Silencing of Sry in the transformed cell lines using ribozymes or short hairpin RNAs (shRNAs) resulted in elevated levels of Wt1 and Wnt4 expression. These results strongly indicate that Wt1 might be under the control of Sry during gonadal differentiation in the mouse. In electrophoretic mobility shift assays (EMSA), we demonstrated that the 3.7 kb 5'-upstream DNA stretch of Wt1 containing potential Sry binding sites was capable of forming molecular complexes with nuclear protein(s) from Sry expressing cells but not with those from control non-Sry expressing cells. In summary, our present results support the notion that Wt1 is located downstream of Sry and down-regulated by the sex determining gene. Although the precise biological meaning of the present findings have yet to be clarified, it is possible that Wt1 plays a dual role during gonadal differentiation, i. e., turning on Sry expression on one hand, and being down-regulated by its product, Sry, on the other, possibly forming a type of negative feed-back mechanism. Further work is needed to substantiate this view.     

Influence of repeated ochratoxin A ingestion on milk production and its carry‐over into the milk,blood and tissues of lactating cows

An experiment was conducted to investigate the influence of repeated ingestion of ochratoxin A (OTA) on milk production of lactating Holstein cows over 28 days, and the carry‐over of OTA from the diets into the milk and tissues of the cows. Nine cows were divided into three groups, labeled OTA5, OTA50 and OTA100, and fed a diet containing 5, 50 and 100 µg OTA/kg of dry matter, respectively. Body weight, feed intake and daily milk yield in cows were not different among the three groups during the OTA‐intake period. OTA residues were neither detected in the tissues, such as liver, kidney, muscles, fat and jejunoileum, nor in the milk of any cows in the OTA intake groups. In contrast, a small amount of OTA (0.1 µg/kg) was detected in the blood plasma of one sample in the OTA50 group and multiple samples in the OTA100 group. The results of this study show that the ingestion of diets containing up to 100 µg/kg of OTA over 28 days does not affect feed intake or milk production of cows, and the dietary OTA is not carried over into milk and edible tissues such as the liver, muscles and fat. © 2015 Japanese Society of Animal Science     

Mitigation of aluminum toxicity by calcium and magnesium in Japanese cedar (Cryptomeria japonica)

We investigated the short-term and long-term mitigation of Al toxicity by Ca and Mg in pot trials of Japanese cedar (Cryptomeria japonica D. Don). We found that in the initial stages of treatment, Al toxicity at high Al concentration (5mM) was mitigated by Ca and Mg through the stimulation of antioxidant enzyme activities in needles. However, growth reduction occurred after 11 months exposure to Al despite the coexistence of Ca and Mg. Growth reduction was related to Al³⁺ activity in solution rather than the concentration of Ca and Mg. Therefore, when considering the influence of soil acidification on Al toxicity in forest ecosystems, it is important to consider not only the potential for mitigation of Al toxicity by base cations, but also the potential for factors in the soil solution to change the chemical form of Al.