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.     

Antioxidative and antimutagenic activities of 4-vinyl-2,6-dimethoxyphenol (canolol) isolated from canola oil

A potent antioxidative compound in crude canola oil, canolol, was recently identified, and reported herein are studies of its scavenging capacity against the endogenous mutagen peroxynitrite (ONOO(-)). ONOO(-) is generated by the reaction between superoxide anion radical and nitric oxide, both of which are produced by inflammatory leukocytes. Among various antioxidative substances of natural or synthetic origin, canolol was one of the most potent antimutagenic compounds when Salmonella typhimurium TA102 was used in the modified Ames test. Its potency was higher than that of flavonoids (e.g., rutin) and alpha-tocopherol and was equivalent to that of ebselen. Canolol suppressed ONOO(-)-induced bactericidal action. It also reduced intracellular oxidative stress and apoptosis in human cancer SW480 cells when used at a concentration below 20 microM under H(2)O(2)-induced oxidative stress. In addition, canolol suppressed plasmid DNA (pUC19) strand breakage induced by ONOO(-), as revealed by agarose gel electrophoresis.     

Characteristics of an aminopeptidase from Japanese cedar (Cryptomeria japonica) pollen

A peptidase from Japanese cedar pollen, Jc-peptidase, was clarified to preferentially hydrolyze an MCA substrate of Phe-MCA (L-phenylalanyl-4-methylcoumaryl-7-amide). This study examined substrate specificities of Jc-peptidase using oligopeptides. Jc-peptidase hydrolyzed Phe-Phe and Tyr-Phe effectively and hydrolyzed Leu-Phe, Met-Phe, and Arg-Phe moderately. Other substrates such as Ala-Phe, Asp-Phe, and Pro-Phe were not hydrolyzed with the peptidase. Results obtained with a series of aminoacyl-Phe peptides were compatible with the facts obtained for MCA substrates except for Arg-MCA. Effects of amino acid residues in the P1' position were also examined using Phe-amino acids. An N-terminal phenylalanine residue was actually released from bioactive peptides such as molluscan cardioexcitatory neuropeptide (FMRF-NH(2)). Because the activity was inhibited with Zn(2+) and EDTA, Jc-peptidase was inferred to belong to the metalloproteases. The N-terminal amino acid sequence was determined to be APIGVQLEIEENYVHMYNGF and an internal sequence to be EIFAATFNVDEETEA, but no homology with other proteins was found.     

Pacific Ocean and Japan Sea ecotypes of Japanese beech (Fagus crenata) differ in photosystem responses to continuous high light

Two ecotypes of Japanese beech (Fagus crenata Blume), the Pacific Ocean type (PAO) and the Japan Sea type (JAS), show different responses to high solar irradiance. When PAO and JAS saplings were grown in continuous high-light (H), leaves of JAS became pale green. To elucidate this phenomenon, we investigated in vivo photochemistry based on pigment concentrations of Photosystem (PS) I and PS II and Western blot analysis. In JAS-H leaves, the amount of D1-protein decreased, resulting in decreases in the maximal quantum yield of PS II (F(v)/F(m)) and electron transport rate, whereas PAO-H leaves maintained high activities. The PS I photochemistry determined by measurement of P-700 photo-oxidation showed that the intersystem electron pool size was 1.4 times greater in JAS-H leaves than in PAO-H leaves. Furthermore, the re-reduction kinetics of P-700(+) showed that cyclic electron transport around PS I was 1.2 times faster in PAO-H leaves than in JAS-H leaves. Analysis of the area over the fluorescence induction kinetics indicated that the relative abundance of the PS IIalpha center increased in PAO-H leaves, whereas JAS leaves were observed to have low acclimation capacity to high light. These results demonstrate that PAO leaves possess acclimation mechanisms to continuous high light, whereas JAS leaves are more vulnerable to continuous high light, resulting in reduced leaf longevity owing to photoinhibition caused by increases in the intersystem electron pool size and suppression of photochemistry at the level of PS I and PS II.     

Immunohistochemical localization of steroidogenic enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca largha) during late pregnancy

To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3betaHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3betaHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3betaHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.     

Insect antifeedant activity of flavones and chromones against Spodoptera litura

The antifeedant polymethylated flavones 5-hydroxy-3,6,7,8,4'-heptamethoxyflavone, 5-hydroxy-3,6,7,8-tetramethoxyflavone, and 5,6-dihydroxy-3,7-dimethoxyflavone have been isolated from the cudweed, Gnaphalium affine D. Don (Compositae). These flavonoids and authentic analogues showed insect antifeedant activity against the common cutworm (Spodoptera litura F.). In a previous paper, it was suggested that there was no substituent on the B-ring of the flavonoid for the beneficial antifeedant activity against the common cutworm. These flavonoids having a phenyl group as the B-ring and the chromone as elimination of the B-ring from the flavonoids were used to test the hypothesis of the previously described B-ring effect. The known fact is that Sculletaria baicarensis (Rutaceae) produced the 2-phenyl flavone. Test compounds and their methylated derivatives were prepared from this material for the structure-activity relationship (SAR) study of insect antifeedant activity. In spite of the 2-phenyl flavonoids, some tested compounds did not show any insect antifeedant activity against the common cutworm, although these inactive flavonoids were deficient in the 6-substituent group on the A-ring of the flavonoid. This 6-position-substituted derivative almost showed strong insect antifeedant activity against common cutworm. Moreover, the tested flavonoids having a hydroxyl group as a substituent on any of the positions tended to increase the activity. These results suggested the importance of the 6-position substitution on the flavonoid; however, hydrophilic substituents decreased the activity. Baicalein (5,6,7-trihydroxyflavone) derivatives did not show any activity despite having the 6-substituent derivative. Although the activity of some chromones increased the activity of the flavone, the bulky B-ring was a disadvantage for the antifeedant activity. It was suggested that the charge on C(3) and C(5) of the flavonoid was important for the biological activity. Additionally, an adequate hydrogen bonding property, which is different from lipophilicity, was an advantage for the activity on the basis of a QSAR analysis.     

Relationship between hybridization frequency of Brassica juncea × B. napus and distance from pollen source (B. napus) to recipient (B. juncea) under field conditions in Japan

Several imported transgenic canola (Brassica napus) seeds have been spilled and have grown along roadsides around import ports. B. juncea, a relative of B. napus with which it has high interspecific crossability, is widely distributed throughout Japan. There is public concern about the harmful impacts of feral B. napus plants on biodiversity, but spontaneous hybridization between spilled B. napus and weedy B. juncea populations is hardly revealed. We evaluated the relationship between the hybridization frequency of B. juncea × B. napus and their planting distance in field experiments using the mutagenic herbicide-tolerant B. napus cv. Bn0861 as a pollen source for hybrid screening. The recipient B. juncea cv. Kikarashina was planted in an experimental field with Bn0861 planted in the center. No hybrids were detected under natural flowering conditions in 2009. However, the flowering period was artificially kept overlapping in 2010, leading to a hybridization frequency of 1.62% in the mixed planting area. The hybridization frequency decreased drastically with distance from the pollen source, and was lower under field conditions than estimated from the high crossability, implying that spontaneous hybridization between spilled B. napus and weedy B. juncea is unlikely in the natural environment.     

Estimation for the timing of the internal developmental processes of acorns from fruit size in <Emphasis Type="Italic">Quercus serrata</Emphasis> Thunb. ex Murray

To evaluate the relative importance of the factors affecting the premature abortion of acorns in Quercus serrata Thunb. ex Murray during the course of their development, inner developmental processes of acorns were observed by microscope, and applicability of the diameter of fruits (acorns with cupules) as an indicator of the inner developmental processes was discussed. Based on the observations using microscope, the developmental processes of acorns were classified into five stages; one prezygotic and four postzygotic stages (stages I to V). The diameter of sound fruits increased exponentially until 14 weeks after pollination, and the variance of diameter in each tree was fairly small (CV = 6%) during the process. Diameter of fruits can be used as a good indicator of developmental stages of acorns at individual level, and effective for the evaluation of the ecological factors affecting the premature abortion of acorns.