Interspecific and intergeneric hybridization and chromosomal engineering of Brassicaceae crops.

In Brassicaceae crop breeding programs, wild relatives have been evaluated as genetic resources to develop new cultivars with biotic and abiotic stress resistance. This has become necessary because of the diversification of ecotypes of diseases and pests, changing food preferences, advances in production technology, the use of new approaches such as in vitro breeding programs, and the need for economical production of F1 seed. To produce potential new cultivars, interspecific and intergeneric hybridizations have been performed between cultivated species and between cultivated species and their wild relatives. Furthermore, interspecific and intergeneric hybrids have been successfully produced using embryo rescue techniques. In this paper, we review the interspecific and intergeneric incompatibilities between Brassicaceae crops and their wild relatives, and the production, characterization, and improvement of synthetic amphidiploid lines, alien gene introgression lines, alloplasmic lines, monosomic alien chromosome addition lines, and monosomic alien chromosome substitution lines. The goal is to provide useful materials to support practical breeding strategies and to study the genetic effects of individual chromosomes on plant traits, the number of genes that control a trait, their linkage relationships, and genetic improvement in Brassicaceae crops.

High-throughput capture of nucleotide sequence polymorphisms in three Brassica species (Brassicaceae) using DNA microarrays.

PREMISE OF THE STUDY: To capture molecular markers that are applicable to environmental risk assessment of genetically modified oilseed rape, and to streamline their development, we screened variations in nucleotide sequences of three Brassica species by DNA microarray analysis. METHODS AND RESULTS: Using the Affymetrix GeneChip Arabidopsis ATH1 Genome Array, we monitored gene expression at 22810 loci among the Brassica species and picked out 192 putative polymorphic loci. We sequenced 25 of these and successfully aligned them among all three species. All 25 loci possessed some interspecific and at times intraspecific nucleotide variation. CONCLUSIONS: DNA microarray analysis effectively detected a large number of nucleotide sequence variations among closely related Brassica species. The polymorphic regions will allow the subsequent development of functional gene markers.

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus.

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer's fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F(1) hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC(1) obtained from B. juncea × F(1) hybrid, and this BC(1) exhibited a plant type like that of B. juncea. Most markers were deleted in BC(2) and BC(3) plants, with only two markers persisting in the BC(3). These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.

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.

Occurrence of metaxenia and false hybrids in Brassica juncea L. cv. Kikarashina × B. napus.

Imported genetically modified (GM) canola (Brassica napus) is approved by Japanese law. Some GM canola varieties have been found around importation sites, and there is public concern that these may have any harmful effects on related species such as reduction of wild relatives. Because B. juncea is distributed throughout Japan and is known to be high crossability with B. napus, it is assumed to be a recipient of B. napus. However, there are few reports for introgression of cross-combination in B. juncea × B. napus. To assess crossability, we artificially pollinated B. juncea with B. napus. After harvesting a large number of progeny seeds, we observed false hybrids and metaxenia of seed coats. Seed coat color was classified into four categories and false hybrids were confirmed by morphological characteristics and random amplified polymorphic DNA (RAPD) markers. Furthermore, the occurrence of false hybrids was affected by varietal differences in B. napus, whereas that of metaxenia was related to hybridity. Therefore, we suggest that metaxenia can be used as a marker for hybrid identification in B. juncea L. cv. Kikarashina × B. napus. Our results suggest that hybrid productivity in B. juncea × B. napus should not be evaluated by only seed productivity, crossability ought to be assessed the detection of true hybrids.

Production and characterization of an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Moricandia arvensis.

Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F(1) hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC(1) plants were generated from a single cross combination of amphidipolid × B. rapa 'Ko1-303' through embryo rescue. One BC(2) and three BC(3) plants were obtained from successive backcrossing with B. rapa 'Ko1-303' employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC(3) plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.

B1-control receive array coil (B-RAC) for reducing B1+ inhomogeneity in abdominal imaging at 3T-MRI.

B1+ inhomogeneity in the human body increases as the nuclear magnetic resonance (NMR) frequency increases. Various methods have thus been developed to reduce B1+ inhomogeneity, such as a dielectric pad, a coupling coil, parallel transmit, and radio-frequency (RF) shimming. However, B1+ inhomogeneity still remains in some cases of abdominal imaging. In this study, we developed a B1-control receive array coil (B-RAC). Unlike the conventional receive array coil, B-RAC reduces B1+ inhomogeneity by using additional PIN diodes to generate the inductive loop during the RF transmit period. The inductive loop can generate dense and sparse regions of the magnetic flux, which can be used to compensate for B1+ inhomogeneity. First, B-RAC is modeled in the numerical simulation, and the spatial distributions of B1+ in a phantom and a human model were analyzed. Next, we fabricated a 12-channel B-RAC and measured receive sensitivity and B1+ maps in a 3T-MRI experiment. It was demonstrated that B-RAC can reduce B1+ inhomogeneity in the phantom and human model without increasing the maximum local specific absorption rate (SAR) in the body. B-RAC was also found to have almost the same the receive sensitivity as the conventional receive coil. Using RF shimming combined with B-RAC was revealed to more effectively reduce B1+ inhomogeneity than using only RF shimming. Therefore, B-RAC can reduce B1+ inhomogeneity while maintaining the receive sensitivity.

Microbubble-induced increase in ablation of liver tumors by high-intensity focused ultrasound.

We studied the possibility of using high-intensity focused ultrasound (HIFU) together with a microbubble agent to treat hepatocellular carcinoma. Development of liver tumors in rats was induced by administration of Dimethylnitrosamin (100ppm). Rats with liver tumors were anesthetized, underwent laparotomy, and were given the microbubble agent Levovist or saline intravenously. After the injection, the liver was exposed to HIFU for 30s (2.18MHz, 600W/cm(2), 40mm in diameter). Immediately after HIFU exposure, ultrasound images of the HIFU area were evaluated. Then the liver was excised and the volume of coagulated tissue was measured. The mean volumes of hyperechoic areas after HIFU were as follows (mm(3), Levovist versus saline: 355.3+/-180.7 versus 47.4+/-35.6, P<0.001, n=13). The volumes of liver tissue coagulated by HIFU were as follows (mm(3), Levovist versus saline: 275.3+/-120.0 versus 60.1+/-23.6, P<0.001, n=13). On microscopic examination of areas exposed to HIFU, implosion cysts were seen, and many cancer cells were found to have been destroyed completely (loss of cell membranes or nuclei). In conclusion, the microbubble agent Levovist can increase the volume of tissue coagulated by HIFU.

Erythrocytes, as well as microbubble contrast agents, are important factors in improving thermal and therapeutic effects of high-intensity focused ultrasound.

Erythrocytes, as well as microbubble contrast agents, are important factors in improving thermal effect of high-intensity focused ultrasound (US), or HIFU, and increasing the coagulation volume produced by HIFU irradiation. In vitro experiments used human plasma with various concentrations of human erythrocytes in combination with or without Levovist. In vivo experiments used eight Japan white rabbits with three degrees of anemia. Using a 2.17-MHz transducer, HIFU was applied for 60 s, and the temperature rise and the volume of coagulation necrosis was evaluated. There was a significant correlation between the HIFU-induced temperature rise and hematocrit, with a correlation coefficient of 0.998 (p=0.0001). Although the temperature rise was smaller at low hematocrit, it was significantly increased by adding Levovist in the suspension (p<0.01). The mean volume of coagulation necrosis was significantly greater in the rabbits with higher hematocrits (p<0.01), and that in the moderate anemia group was significantly increased by using Levovist (p<0.01).

Polyacrylamide gel containing egg white as new model for irradiation experiments using focused ultrasound.

A polyacrylamide (PAA) gel containing egg white as a new model for irradiation experiments in high-intensity focused ultrasound HIFU is introduced. The gel is transparent except in thermally necrosed regions which are white. The model is similar to, and has the same benefits as, gels containing bovine serum albumin (BSA) protein, but egg is less expensive than BSA. The acoustic properties of the gel are very favorable, similar to those of soft tissues; density 1.0 g/cm(3), sound speed 1540 m/s and acoustic attenuation 0.4 dB/cm at 2 MHz. We illustrate the usefulness of the gel in visualizing HIFU lesions and introduce a new model of gastric submucosal tumor in a rabbit stomach, showing that this gel can be formed into any desired shape. Our PAA gel containing egg white is an attractive candidate for a disposable acoustic material suitable for experimental applications of HIFU.

Appraisal of bone maturity age derived from broadband ultrasonic attenuation in Japanese children and adolescents.

It is clinically important to evaluate the level of skeletal maturation in juveniles to determine the appropriate timing for orthodontic treatment. The purpose of this study was to assess the age of bone maturity by using an ultrasonic bone analyzer (Cuba Clinical, McCue Ultrasonics Ltd., Winchester, U.K.), Broadband ultrasonic attenuation (BUA:dB/MHz) was measured at the left calcaneus as an effective indicator of the age of bone maturity. The subjects consisted of 249 males and 304 females aged 12 to 29 years who had not suffered constitutional bone disease or a disease of the endocrine system. The peak value of BUA considered as bone maturity was 104.44 at the age of 19 years in males and 77.80 at the age of 16 years in females, and the peak age range was indicated as 18-19 years in males and 13-16 years in females by statistical evaluation. The peak age range indicated by BUA was wider in females than that in males. The present results can be used as reference ages for maturity in growth prediction for orthodontic treatment of Japanese children and adolescents. The heights and weights of the subjects were also collected as basic data. A significant positive correlation was observed between BUA values and weight (r = 0.34 p < 0.01 in females, r = 0.52 p < 0.01 in males). BUA is known to describe the quality of bone because the calcaneus is a loading bone. The relation between bone quality and growth has not been discussed. Further research is required to investigate this relationship.

Broadband ultrasonic attenuation of children and young adults in Japan.

The purpose of this study was to establish broadband ultrasonic attenuation (BUA: dB/MHz) as bone mineral density (BMD) norms for healthy young Japanese and to evaluate the standard values for an ultrasonic bone analyzer (Cuba Clinical, McCue Ultrasonics Ltd., Winchester, England), which facilitates BMD measurement without exposure to radiation. The subjects were 472 healthy young individuals with no endocrine or skeletal disorders, 197 males (mean age 16 y 5 m) and 275 females (mean age 15 y 7 m) aged from 5 to 29 years. BUA was measured at the left calcaneus. The subjects were divided into five age-stratified different age groups of five years intervals. The mean BUA values (dB/MHz) obtained were 40.6, 60.9, 78.0, 90.4 and 86.0 for males, and 41.9, 61.0, 73.4, 68.4 and 70.8 for females in the 5-9, 10-14, 15-19, 20-24 and 25-29 age groups, respectively. A significant positive correlation was observed between BUA and age in both males and females except in the male 25-29 age group and the female 20-24 and 25-29 age groups. A significantly different BUA between males and females was found in the 20-24 and 25-29 age groups (p < 0.001). The BUA values obtained in this study may serve as BMD norms for children and young adults. It might be thought that measuring BUA from childhood through early adulthood made it possible to determine peak values and peak periods of BMD, providing useful information for assessment of growth and development.

Anthocyanins from the flowers of Nagai line of Japanese garden Iris (Iris ensata).

Six anthocyanins were isolated from the flowers of the Nagai line of Iris ensata Thunb. They were identified as petunidin and malvidin 3-O-beta-[(4"'-Z-p-coumaroyl-alpha-rhamnopyranosyl)-(1-->6)-beta-glucopyranoside]-5-O-beta-glucopyranosides (1 and 3) and their E-forms (2 and 4), and petunidin and malvidin 3-O-rutinoside-5-O-glucosides (5 and 6). Though the E-form of petunidin 3-O-[(4"'-p-coumaroylrhamnosyl)-(1-->6)-glucoside]-5-O-glucoside has been reported, its Z-form was found for the first time. The presence of Z- and E-forms of malvidin 3-O-[(4'''-p-coumaroylrhamnosyl)-(1-->6)-glucoside]-5-O-glucoside is also reported for the first time. Fifty-one cultivars of Nagai line and their wild form (I. ensata var. spontanea) were divided into four anthocyanin patterns, i.e. 1) the presence of 1-4, 2) the presence of 2 and 4, 3) the presence of 5 and 6, and 4) no anthocyanin.

Seeds of a possible natural hybrid between herbicide-resistant Brassica napus and Brassica rapa detected on a riverbank in Japan.

Transgenic herbicide-resistant varieties of Brassica napus, or oilseed rape, from which canola oil is obtained, are imported into Japan, where this plant is not commercially cultivated to a large extent. This study aimed to examine the distribution of herbicide-resistant B. napus and transgene flow to escaped populations of its closely related species, B. rapa and B. juncea. Samples were collected from 12 areas near major ports through which oilseed rape imports into Japan passed--Kashima, Chiba, Yokohama, Shimizu, Nagoya, Yokkaichi, Sakai-Senboku, Kobe, Uno, Mizushima, Kita-Kyushu, and Hakata--and the presence of glyphosate- and/or glufosinate-resistant B. napus was confirmed in all areas except Yokohama, Sakai-Senboku, Uno, and Kita-Kyushu. The Yokkaichi area was the focus because several herbicide-resistant B. napus plants were detected not only on the roadside where oilseed rape spilled during transportation but also on the riverbanks, where escaped populations of B. rapa and B. juncea grew. Samples of B. napus that were tolerant to both herbicides were detected in four continuous years (2005-2008) in this area, suggesting the possibility of intraspecific transgene flow within the escaped B. napus populations. Moreover, in 2008, seeds of a possible natural hybrid between herbicide-tolerant B. napus (2n = 38) and B. rapa (2n = 20) were detected; some seedlings derived from the seeds collected at a Yokkaichi site showed glyphosate resistance and had 2n = 29 chromosomes. This observation strongly suggests the occurrence of hybridization between herbicide-resistant B. napus and escaped B. rapa and the probability of introgression of a herbicide-resistance gene into related escaped species.

Cephalometric study of elderly with nearly intact dental arches.

OBJECTIVE: The purpose of this study was to investigate the craniofacial morphology of elderly people with many remaining teeth using cephalometric analysis. SUBJECTS AND METHODS: The subjects were 30 Japanese elderly who participated in the '8020 campaign 2001' in Bunkyo Ward, Tokyo, organised by The Dental Association of Tokyo, as well as 30 Japanese young adults with normal occlusion. Lateral cephalograms of all subjects were analysed using the Coben method. RESULTS: In the female elderly group, the lower face depth was smaller than in the younger adults. In the male elderly group, the height and depth of both the total face and the lower face were longer than in the younger group. In comparing the 8020 achievers with the younger group, the proportion of the lower facial height was greater than the upper facial height, and this finding was more pronounced in women than in men. CONCLUSION: For the lateral facial pattern of the elderly, a reduction of lower facial height because of tooth occlusal reduction was not apparent. It was clear that there are age differences for males and females; in addition, differences in the total face and lower face area of the elderly group were due to their having many remaining teeth over a long time period. Also, these changes were more apparent in women than in men, and it is clear that there is a male-female difference in ageing.

Heating and coagulation volume obtained with high-intensity focused ultrasound therapy: comparison of perflutren protein-type A microspheres and MRX-133 in rabbits.

PURPOSE: To compare the efficiency of different microbubble contrast agents in the heating and coagulation of rabbit liver tissue by using high-intensity focused ultrasound therapy. MATERIALS AND METHODS: The use of and protocols for Japan white rabbits were approved by the University of Tokyo Committee on Animal Resources. In vitro experiments were conducted in a 1-mL cylindric space of polyacrylamide gel, which contained different microbubble contrast agents (MRX-133 or perflutren protein-type A microspheres). In vivo experiments were performed in six Japan white rabbits (two for the perflutren protein-type A microsphere group, two for the MRX-133 group, and two for the control group). In each rabbit, the liver was directly subjected to high-intensity focused ultrasound after intravenous injection of different microbubble contrast agents. Natural saline was used as a control. High-intensity focused ultrasound was applied for 30 seconds by using a 2.18-MHz transducer, and the temperature increase and volume of coagulation necrosis were evaluated. Statistical analysis was performed by using an analysis of variance test, which was followed by a Student-Newman-Keuls test; a P value of <.05 was considered to indicate a statistically significant difference. RESULTS: In the MRX-133 group, the mean temperature increase was significantly greater and faster than that in the control group (P < .01) or in the perflutren protein-type A microsphere group (P < .05). In the perflutren protein-type A microsphere group, the mean temperature increase was greater and faster than that of the control group (P < .01) for both in vitro and in vivo experiments. The mean volume of the coagulation necrosis lesion was 117.9 mm3 +/- 48.4 (+/-standard deviation) for the MRX-133 group, 45.4 mm3 +/- 24.9 for the perflutren protein-type A microsphere group, and 17.7 mm3 +/- 9.0 for the control group. In the MRX-133 group, the mean volume of coagulation necrosis was significantly greater than that of the control or the perflutren protein-type A microsphere group (P < .01), and in the perflutren protein-type A microsphere group, the volume of coagulation necrosis was greater than that of the control group (P < .05). CONCLUSION: MRX-133 had greater efficiency than perflutren protein-type A microspheres in high-intensity focused ultrasound-induced heating and coagulation of rabbit liver tissue.

Use of a microbubble agent to increase the effects of high intensity focused ultrasound on liver tissue.

In order to find out whether high intensity focused ultrasound (HIFU) might be useful against hepatocellular carcinoma, we analyzed the effect of a microbubble agent (Levovist) on the temperature rise and tissue necrosis induced by HIFU. Rabbits were given 7 ml Levovist (300 mg/ml) or saline intravenously. Up to six areas per rabbit liver were exposed to HIFU for 60 s (2.18 MHz, I(SPTA)=400 W/cm(2)). The volume of the tissue coagulated by HIFU was measured 10 min after the start of HIFU. HIFU-induced lesions were larger in the animals given Levovist: (mm(3), Levovist versus saline) 371+/-104 versus 166+/-71 (P<0.001). Temperatures in the animals given Levovist were also higher 60 s after the start of exposure: ( degrees C, Levovist versus saline) 20.3+/-3.5 versus 13.2+/-3.8 (P<0.001). The amount of damage differed greatly, but the pathological changes caused by HIFU with Levovist were the same as those caused by HIFU with saline. Hemorrhagic areas and implosion cysts were seen, and many cells had been disrupted or destroyed. Microbubble agents developed for diagnostic uses could also be used in anticancer therapy.

Structural and biochemical dissection of photorespiration in hybrids differing in genome constitution between Diplotaxis tenuifolia (C3-C4) and radish (C3).

We compared the structural, biochemical, and physiological characteristics involved in photorespiration of intergeneric hybrids differing in genome constitution (DtDtR, DtDtRR, and DtRR) between the C(3)-C(4) intermediate species Diplotaxis tenuifolia (DtDt) and the C(3) species radish (Raphanus sativus; RR). The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of radish had only a few chloroplasts and mitochondria. In the hybrids, the numbers of chloroplasts and mitochondria, the ratio of centripetally located organelles to total organelles, and the mitochondrial size in the BS cells increased with an increase in the constitution ratio of the Dt:R genome. The P-protein of glycine decarboxylase (GDC) was confined to the BS mitochondria in D. tenuifolia, whereas in radish, it accumulated more densely in the mesophyll than in the BS mitochondria. In the hybrids, more intense accumulation of GDC in the BS relative to the mesophyll mitochondria occurred with an increase in the Dt:R ratio. These structural and biochemical features in the hybrids were reflected in the gas exchange characteristics of leaves, such as the CO(2) compensation point. Our data indicate that the leaf structure, the intercellular pattern of GDC expression, and the gas exchange characteristics of C(3)-C(4) intermediate photosynthesis are inherited in the hybrids depending on the constitution ratio of the parent genomes. Our findings also demonstrate that the apparent reduced photorespiration in C(3)-C(4) intermediate plants is mainly due to the structural differentiation of mitochondria and chloroplasts in the BS cells combined with the BS-dominant expression of GDC.