RNA-seq based SNPs for mapping in Brassica juncea (AABB): synteny analysis between the two constituent genomes A (from B. rapa) and B (from B. nigra) shows highly divergent gene block arrangement and unique block fragmentation patterns

Abstract

Background: Brassica juncea (AABB) is an allotetraploid species containing genomes of B. rapa (AA) and B. nigra (BB). It is a major oilseed crop in South Asia, and grown on approximately 6-7 million hectares of land in India during the winter season under dryland conditions. B. juncea has two well defined gene pools--Indian and east European. Hybrids between the two gene pools are heterotic for yield. A large number of qualitative and quantitative traits need to be introgressed from one gene pool into the other. This study explores the availability of SNPs in RNA-seq generated contigs, and their use for general mapping, fine mapping of selected regions, and comparative arrangement of gene blocks on B. juncea A and B genomes.

Results: RNA isolated from two lines of B. juncea--Varuna (Indian type) and Heera (east European type)--was sequenced using Illumina paired end sequencing technology, and assembled using the Velvet de novo programme. A and B genome specific contigs were identified in two steps. First, by aligning contigs against the B. rapa protein database (available at BRAD), and second by comparing percentage identity at the nucleotide level with B. rapa CDS and B. nigra transcriptome. 135,693 SNPs were recorded in the assembled partial gene models of Varuna and Heera, 85,473 in the A genome and 50,236 in the B. Using KASpar technology, 999 markers were added to an earlier intron polymorphism marker based map of a B. juncea Varuna x Heera DH population. Many new gene blocks were identified in the B genome. A number of SNP markers covered single copy homoeologues of the A and B genomes, and these were used to identify homoeologous blocks between the two genomes. Comparison of the block architecture of A and B genomes revealed extensive differences in gene block associations and block fragmentation patterns.

Conclusions: Sufficient SNP markers are available for general and specific -region fine mapping of crosses between lines of two diverse B. juncea gene pools. Comparative gene block arrangement and block fragmentation patterns between A and B genomes support the hypothesis that the two genomes evolved from independent hexaploidy events.

Figure 1

Coverage of the assembled gene models in the Heera and Varuna genomes. Assembled gene model coverage in the Heera A, Heera B, Varuna A, and Varuna B genomes compared with gene models described for B. rapa in the BRAD database. More than 70% of identified gene models could be assembled at greater than 60% coverage.

Figure 2

Number of gene models obtained after each step of marker development. A larger number of A genome gene models were identified with SNP differences than B genome gene models. At each step of marker development, gene models not meeting the criteria for marker development were removed.

Figure 3

Comparative block arrangement in the LGs of the A and B genomes of B. juncea . (a) LGs A1*–A10* (present study); A1–A10 (reference A genome [25]) (b) B1*–B8* (present study); B1–B8 (consensus of B. juncea B genome map [32] and B. nigra map [37]). For the A genome, blocks that could not be identified in relation to the reference genome are highlighted by darker shading. For the B genome, newly identified blocks are shown in bold, and blocks identified in only one of the two maps being compared are highlighted by darker shading.

Figure 4

Block homoeology between the A and B genomes of B. juncea. Gene blocks on the eight LGs of the B genome (B1-B8) that show homoeology to corresponding blocks on the A genome LGs are shown by the connecting lines. Blocks showing variations in their fragmentation pattern in the B genome are shown in bold with an asterisk. Genomic blocks assigned to the subgenomes LF (red), MF1 (green) and MF2 (blue) are colour coded as in the previous B. rapa study [25].

Figure 5

Chromosome collinearity comparison among A, B, and C genomes. A and C genome collinearity is based on a B. napus study [52]. LF (red), MF1 (green), and MF2 (blue) blocks are colour coded as in the previous B. rapa study [25]. The figure shows that the block arrangement pattern between the A and B genomes is much more complex than that between the A and C genomes.