Intraspecific variations of the cytoplasmic male sterility genes orf108 and orf117 in Brassica maurorum and Moricandia arvensis, and the specificity of the mRNA processing.

The mitochondrial gene orf108, which is co-transcribed with atp1 and causes cytoplasmic male sterility in Brassica crops, is widely distributed across wild species and genera of Brassicaceae. However, to date, intraspecific variations in the presence of orf108 have not yet been studied, and the mechanisms underlying the wide distribution of the gene remain unclear. We analyzed the presence and sequence variations of orf108 in two wild species, Brassica maurorum and Moricandia arvensis. After polymerase chain reaction amplification of the 5' region of atp1 and the coding sequence of orf108, we determined the DNA sequences. Brassica maurorum and M. arvensis showed variations in the presence of orf108 or orf117 (orf108V117) both between and within accessions and were not fixed to the mitochondrial type with the male sterile genes. Sequencing of the amplicons showed that B. maurorum had orf108V117 instead of orf108. Sequencing also indicated mitochondrial heteroplasmy in the two species; in particular, in B. maurorum, one plant possessed both orf108 and orf108V117 sequences. These results suggest that substoichiometric shifting of mitochondrial genomes leads to the acquisition or loss of orf108. Furthermore, fertility restorer genes of the two species were involved in the processing of the mRNA of male sterility genes at different sites.

Characterization of cytoplasmic female sterility in an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Diplotaxis tenuifolia.

Alloplasmic plants exhibit various phenotypic changes such as cytoplasmic male sterility (CMS). We have been attempting to produce an alloplasmic Brassica rapa CMS line (2n = 20) carrying Diplotaxis tenuifolia cytoplasm (cyt-Dt) for several years, but a single extra chromosome always remained in all lines produced. We confirmed a D. tenuifolia-specific band in the alloplasmic line carrying D. tenuifolia cytoplasm by RAPD analysis, indicating that the additional chromosome was derived from D. tenuifolia. Here, we observed the phenotypic characteristics of the alloplasmic B. rapa monosomic addition line, named (cyt-Dt) B. rapa MAL, and investigated why a single extra chromosome is required in its genetic background for viability. When the (cyt-Dt) B. rapa MALs were crossed with pollen of several B. rapa lines, approximately 50% of the ovules attracted pollen tubes, and all the progeny had the additional chromosome. These results suggested that only the female gametes with n = 11 rather than n = 10 were fertilized and developed into mature seeds, and that cytoplasmic female sterility was overcome by nuclear restorer gene(s) derived from the cytoplasmic donor species.

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.

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.

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.

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.