Seed colour loci, homoeology and linkage groups of the C genome chromosomes revealed in Brassica rapa-B. oleracea monosomic alien addition lines

Abstract

Background and aims: Brassica rapa and B. oleracea are the progenitors of oilseed rape B. napus. The addition of each chromosome of B. oleracea to the chromosome complement of B. rapa results in a series of monosomic alien addition lines (MAALs). Analysis of MAALs determines which B. oleracea chromosomes carry genes controlling specific phenotypic traits, such as seed colour. Yellow-seeded oilseed rape is a desirable breeding goal both for food and livestock feed end-uses that relate to oil, protein and fibre contents. The aims of this study included developing a missing MAAL to complement an available series, for studies on seed colour control, chromosome homoeology and assignment of linkage groups to B. oleracea chromosomes.

Methods: A new batch of B. rapa-B. oleracea aneuploids was produced to generate the missing MAAL. Seed colour and other plant morphological features relevant to differentiation of MAALs were recorded. For chromosome characterization, Snow's carmine, fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used.

Key results: The final MAAL was developed. Morphological traits that differentiated the MAALs comprised cotyledon number, leaf morphology, flower colour and seed colour. Seed colour was controlled by major genes on two B. oleracea chromosomes and minor genes on five other chromosomes of this species. Homoeologous pairing was largely between chromosomes with similar centromeric positions. FISH, GISH and a parallel microsatellite marker analysis defined the chromosomes in terms of their linkage groups. Conclusions A complete set of MAALs is now available for genetic, genomic, evolutionary and breeding perspectives. Defining chromosomes that carry specific genes, physical localization of DNA markers and access to established genetic linkage maps contribute to the integration of these approaches, manifested in the confirmed correspondence of linkage groups with specific chromosomes. Applications include marker-assisted selection and breeding for yellow seeds.

Fig. 1.

Scheme showing the origin of the developed Brassica MAALs.

Fig. 2.

Morphological features of relevance for the differentiation of plants that carry the alien C chromosome. (A) General small size of siliques exemplified by siliques of a C2-carrier plant and large siliques of the parental Brassica rapa species. (B) Yellow flower of B. rapa, the colour characteristic of all MAALs except the MAAL for C4. (C) White flower colour characteristic of B. oleracea var. alboglabra and of C4 carriers. (D, E) C1 carriers: (D) three cotyledons; (E) two cotyledons, one almost double the size of the other. (F) Two white-flowered C4 carriers to the left and two smaller yellow-flowered C4d carriers to the right. (G) Eight brown seeds and 14 yellow seeds in a silique of a C4 carrier. (H) Puckered surface of a leaf of a C5 carrier.

Fig. 3.

Seed colour of the Brassica material studied. (A) Yellow in B. rapa var. trilocularis (K-151); (B) black in B. oleracea var. alboglabra (no. 4003); (C) black, dark grey and brown in resynthesized B. napus (no. 7406); (D) brown seeds harvested from a C1 carrier; (E) selected seeds with brown spots/patches from a C2 carrier; (F) bulk seeds harvested from a C3d carrier; seeds with faint brown dots are difficult to detect by the naked eye; (G, H) mixtures of easily distinguishable yellow and brown seeds originating from C4 and C4d carriers, respectively; the brown seeds are slightly lighter in colour in the case of C4d; (I–K) selected seeds with brown spots/patches from plants carrying C5, C6 and C7, respectively; (L, M) yellow seeds harvested from plants with C8 and C9, respectively.

Fig. 4.

Diakinesis chromosomes. (A, C) Brassica rapa (AA); differential condensation and staining marking the heavily stained heterochromatic pericentric regions, numeric designations of chromosomes with median/sub-median centromeres (A1–A5), sub-median/sub-terminal centromeres (A6–A8 and A10) and the nucleolar pair (A9) are according to Cheng et al. (1995). (B) B. oleracea var. alboglabra (CC); chromosomes are generally homogenously stained. Scale bars = 10 µm.

Fig. 5.

The behaviour of the C chromosome during diakinesis in the MAALs. It either remains unpaired as a univalent (I), or pairs with one A chromosome forming a heteromorphic bivalent, while the other A chromosome remains as a univalent (II + I), or appears as part of a trivalent when pairing with a pair of A chromosomes with median/sub-median centromeres (III¹), sub-median/sub-terminal centromeres (III²), or the nucleolar organizers (III³), or appears as part of a pentavalent (V) when pairing with two pairs of A chromosomes. In the case of C4d and C8, two trivalents representing short and long arm associations of the C chromosome with the nucleolar A pair are depicted. Scale bar = 10 µm.

Fig. 6.

Identification of the A and C chromosomes after applying two rounds of multiple target FISH according to Xiong and Pires (2011). (A and B) Diakinesis chromosomes of B. rapa (K-151), using in the first round (A) probes for 5S rDNA (yellow), 45S rDNA (white), repeated DNA sequences in eight chromosome pairs of B. rapa by using BAC KBrB072L17 (green), and repeated DNA sequences specific to two pairs of B. rapa by using BAC KBrH092N24 (red), and applying in the second round (B) probes for the repetitive centromeric DNA sequences CentBr1 (white) and CentBr2 (green) and for repetitive DNA sequences that are C genome specific by using the BAC BNIH 123L05 (red). (C–F) Identification of the C chromosome in MAALs; (C, D) diakinesis chromosomes with C8 (LG-C7); (E, F) mitotic chromosomes with C1 (LG-C9). Scale bars = 10 µm.

Fig. 7.

Identification of the C chromosome in MAALs after applying chromosome-specific BAC probes (A–C), the 5S rDNA probe followed by GISH with labelled C genome DNA hybridizing to pericentric regions of A chromosomes and to C chromatin (D, E), or only GISH (F–H), and detection of possible intergenomic introgression (H). (A) Diakinesis chromosomes with C4 (LG-C3) labelled with BAC KBrH117M18 specific for LG-C3 and LG-A3 (green) and BAC BNIH 123L05 specific for C genome chromosomes (red), and 45S rDNA probe (white); (B, C) mitotic chromosomes with C5 (LG-C4) labelled with BAC BoB004H11 probe (green) followed by GISH (red), arrows; (D, E) C5 labelled by 5S rDNA (red) and GISH (red) marked by arrows. (F–H) Mitotic chromosomes; (F) C3d mainly composed of one arm (arrow); (G) C5 (arrow); (H) C4 (arrow) whose distal region of the short arm is unlabelled possibly denoting introgressed A chromatin, also labelling of intercalary sites in two A chromosomes (arrowheads) possibly reflecting introgressed C chromatin. Asterisks denote satellites of the nucleolar A pair. Scale bars = 10 µm.