The progress of intersubgenomic heterosis studies in Brassica napus

The new nomenclature of Brassica has been suggested in a previous study by same authours where the symbols of A, A and A represented the A genome in the Brassica rapa, Brassica juncea and Brassica napus, B, B and B for the B genome of Brassica nigra (black mustard), B. juncea and Brassica carinata, C, C and C for the C genome of Brassica oleracea, B. napus and B. carinata. Numerous efforts have focused on exploring novel B. napus (AACC) breeding stocks by the hybridization between Brassica species. Thereafter, most interspecific hybrids in Brassicas could be considered as intersubgenomic hybrids. In this review, examples are shown from recent studies on the method for construction of new-typed B. napus with genome composition of AACC and AACC, the meiosis and embryo sac development of new-typed B. napus, the appearance of intersubgenomic (AACC and AACC) heterosis and the mechanism for production of intersubgenomic heterosis were described.


INTRODUCTION
In Brassicas, three diploid species, that is, Brassica rapa (AA, 2n = 20), Brassica nigra (BB, 2n = 16), Brassica oleracea (CC, 2n = 18) and three natural spontaneous amphidiploids species of Brassica napus (AACC,2n = 38), Brassica juncea (BBCC,2n = 36) and Brassica carinata (AABB, 2n = 34) have been on existence.Lots of research revealed that the three amphidiploids species of Brassicas were derived from the interspecific crosses between three diploids (Morinaga, 1933(Morinaga, , 1934;;UN, 1935;Snowdon, 2007).B. napus was the most important oilseed Brassica crop in the world due to good production potential and resistances.B. napus accounts for about 85% of oilseed rapeseed in China (Fu, 2000).B. napus was one of the specie that the heterosis was widely used, the first CMS male sterile line with practical value and the first hybrid variety that was successfully cultivated in China (Fan and Stefansson, 1986;Downey and Röbbelen, 1989;Fu, 2000).Lots of research revealed that the heterosis has a relationship with the genetic *Corresponding author.*E-mail: limaoteng426@163.com.**jmeng@mail.hzau.edu.cndiversity of the parents (Diers et al., 1996;Riaz et al., 2001;Liu et al., 2002;Qian et al., 2005).The germplasm of B. napus was rather narrow compared with other species of B. rapa, B. oleracea and B. carinata for only about 400 years of domestication (Gómez-Campo, 1999).The narrow genetic basis limiting its potential for improving seed yield, otherwise, the A genome of B. rapa and C genome of B. carinata is rather different from A and C genome of B. napus (Prakash and Hinata, 1980;Hoenecke and Chyi, 1991;Song et al., 1995;Li et al., 2005Li et al., , 2006)).Introgression of A genome of B. rapa and C genome of B. carinata into B. napus would explore the genetic bases of B. napus.Recently, some efforts have been made to widen the germplasm of B. napus by introgressions of genomic components from the parental species (Chen and Heneen, 1989;Seyis et al., 2003;Qian et al., 2005).C genome in B. oleracea and B. carinata (Inomata, 1985;Song et al., 1988).To distinguish the difference, the concept of subgenome was introduced to genus Brassicas.(Qian et al., 2005;Li et al., 2004Li et al., , 2006Li et al., , 2007) ) (Meng et al., 1998;Bing et al., 1996;Rahman, 2001;Li et al., 2004)  n were obtained (Liu et al., 2002;Qian et al., 2003Qian et al., , 2005)).The chromosome number of the trigenomic hybrids of A r A n C n was 29 chromosomes, which had abnormal meiosis behavior, so the chromosome of the seeds obtained by self-crossing of trigenomic hybrids might varied.The plants with 38 chromosomes were obtained by chromosome checking of all the plants that were derived from self-crossing of trigenomic hybrids of

THE CONCEPT OF SUBGENOME OF
The plants with high ratio of A r /A n were obtained by using amplified fragment length polymorphisms (AFLP) and simple sequence repeat (SSR) molecular markers (Figure 2I).Materials with 38 chromosome and high ratio of A r /A n were also obtained by backcrossing between A r A n C n and B. rapa n × A r A r and BC 1 F 2 varied from 28.2 to 69.6%, with an average of 60.5%.

The procedure for producing of new-typed B. napus with genome composition of
In order to obtain the new-typed B. napus with genome composition of A r A r C c C c , the breeding procedure shown in Figure 2II was conducted in previous studies (Li et al., 2004(Li et al., , 2005(Li et al., , 2005(Li et al., , 2006(Li et al., , 2007)).Firstly, trigenomic ) with 46 chromosomes (Figure 3d).The A r A n B c C c C n hybrids were preferred self-crossed and the laggards appeared in profusion at anaphase I and anaphase II.GISH analysis showed that the B c chromosomes could be lost in meiosis (Figure 3e).It indicated that the materials with 38 chromosomes without B c chromosomes could be produced (Figure 3f).
Thousands of the plants with 38 chromosomes were performed molecular analysis.The results revealed that about 50% of the genomic components in new-typed B. napus were replaced by A r and C c subgenome of B. rapa and B. carinata (Li et al., 2007).The molecular marker analysis also showed that different material from the same combination of B c B c C c C c × A r A r had different genetic background (Li et al., 2007).The hybridization was again made between different materials with different genetic background and the ratio of A r and C c was increased to 80% in new-typed B. napus as expected, that is, the new typed B. napus was almost with the genome composition of A r A r C c C c .The new-typed B. napus lines with normal meiosis behavior, normal embryo sac development process and good pollen fertility (Figure 4) indicated that those new-typed B. napus had balanced genetic basis (Li et al., 2006(Li et al., , 2007)).n and by surveying hybrids between 20 lines of the new-typed of B. napus in BC 1 F 5 .The heterosis was from 29.17 to 95.83% and the amount of mid-parental heterosis varied from 21.73 to 86.50%, with an average of 43.15% for seed yield (Qian et al., 2005).

Hhybridization was made between
As for intersubgenomic hybrids of A ), were grown vigorously from the seeding stage to the flowering stage (Figure 5).Seed yield of intersubgenomic hybrids was better than the control and obviously, overstandard heterosis on average were observed.Three lines of new-typed B. napus was selected from the F 5 generation to hybridized to five tester cultivars in order to test the potential of intersubgenomic hybrids on seed production.About 50% of intersubgenomic hybrids showed high parent heterosis (HPH) of 11.98% on the average and HPH values of two combinations was over 40%.Chen et al. (2008) revealed that the mid-parent heterosis value for seed yield exceeded 40% in their studies and the high-parent heterosis value for seed yield was over 50% in some intersubgenomic hybrids.Chen et al. (2008) also observed that eight out of nine tested hybrids showed significant higher seed yield than that of their parents.
The above mentioned phenomena suggested the strong heterosis potential of the intersubgenomic hybrids of

THE POSSIBLE MECHANISM FOR PRODUCTION OF HETEROSIS IN INTERSUBGENOMIC HYBRIDS
Interaction between different genomes of Brassicas might be the reason for production of heterosis.Liu et al. (2002) revealed that some DNA fragments of A r were significantly associated with biomass production in trigenomic hybrids (A  Allelic combinations present in hybrids might result in the alteration of allele expression profiles, production of novel allelic interactions, genesis of beneficial adaptations in the hybrids and give rise to heterotic phenotypes (Springer and Stupar, 2007;Chen et al., 2008).Chen et al. (2008)  ) compared with their parents.By comparing with the additive effects that appeared in rice and wheat and the dominance and overdominance effects that appeared in maize (Xiong et al., 1998;Tian and Dai, 2004;Sun et al., 2004), Chen et al. (2008) considered that dominance and overdominance effects were prominent in the intersubgenomic hybrids.About 15.04 and 0.66% of the transcript-derived fragments (TDFs) that differentially expressed between the intersubgenomic hybrids and their parents showed significant correlation with at least one or over two of analyzed traits of yield.This indicated that allelic variation introduced from A r /C c subgenome may lead to many positive allelic combinations in the intersubgenomic hybrids (Chen et al., 2008).Some TDFs, such as Copia-like TDF, were activated in new-typed B. napus.It indicated that the DNA methylation and chromatin remodeling might be involved in the production of intersubgenomic heterosis (Hirochika et al., 2000;Zilberman et al., 2007;Chen et al., 2008).Further research revealed that 12 TDF-markers were mapped to 12 different linkage groups within the one DH population constructed by Qiu et al. (2006),.Four of these TDFs were located within the confidence intervals of eight quantitative trait loci (QTLs) for yield-related traits, which could explain the phenotype variation from 4.41 to 13.45% in the TN DH population (Figure 6).The genes or ESTs (TDFs) were also mapped within the confidence intervals of QTLs for the target traits in rice, rapeseed and maize (Mao et al., 2004;Liu et BRASSICA Long years of evolution and artificial selection have made the A genome and C genome in B. napus somewhat different from the A genome in B. rapa and B. juncea, the

Figure 1 .
Figure 1.The letters of subgenome of Brassicas and their relationship.
to represent the A genome in B. rapa, B. napus and B. juncea, B b , B j and B c for the B genome of B. nigra (black mustard), B. juncea and B. carinata whilefor the C genome of B. oleracea, B. napus and B. carinata

Figure 2 .
Figure 2. The procedure for producing the new-typed B. napus.
yield heterosis was observed among partial intesubgenomic hybrids.Qian et al. (2005) revealed that about 90% of 129 intesubgenomic hybrids of A r A n C n C n exceeded their respective tester lines, whereas 75 and 25% of combinations surpassed Zhongyou 821 and Huaza 4 of two widely cultivated cultivars in China, respectively.The

Figure 3 .
Figure 3.The chromosome identification of trigenomic hybrid, hexaploid, pentaploid and tetraploid by GISH.The DNA from the B. nigra (B b B b ) was used as the probe.a and b represent the chromosome constitution from somatic and pollen mother cells of trigenomic hybrids (A r B c C c ), respectively; c and d represent the hexaploid (A r A r B c B c C c C c ) and pentaploid (A r A n B c C c C n ), respectively; e represent the meiosis at anaphase I of pentaploid, indicating that the B c chromosome might be lost during meiosis; f represent the plants without the B c chromosomes.Image fromLi et al. (2005 and 2004).
the hybridization between newtyped B. napus (A r A r C c C c ) with natural B. napus ( those DNA fragment had no direct relationship with the hetsrosis of yield.Qian et al. (2005)

Figure 4 .
Figure 4. Observation of the embryo sac development and the pollen tube elongation in new-typed B. napus.a−d represents the development of embryo sac in one partial new-typed B. napus.a = 1-nucleate aposporous embryo sac, b = 2-nucleate aposporous embryo sac, c = 4-nucleate embryo sac, d = 8-nucleate embryo sac.e−f represents pollen germination and pollen tube elongation in one new-typed B. napus, e = the pollen grains germinated normally, f = the pollen tube passes through the pistillar chord.Arrow shows the pollen tube; g = pollen tube reaches the ovary and releases the contents (arrow).Image from Li et al. (2007).
lead to considerable differences in the gene expression profiles of the partial new-typed B. napus (

Figure 5 .
Figure 5. Intersubgenomic hybrids growing at different developmental stages.a represent the seed setting of new-typed B. napus, b, c and d represent the intersubgenomic hybrids growing at seeding, flowering and maturing stages, respectively (arrow and arrow heads represent the control and intersubgenomic hybrids, respectively).Image from Li et al. (2006).

Figure 6 .
Figure 6.The TDF-markers map to yield-related QTL regions on linkage maps.Markers showed in bold are the TDF-derived markers.TN, RIL and BC represent the TN DH population, the HT RIL population and its derived RIL-BC1 population, respectively.The bars (or ellipses) and their label indicated the QTL and their corresponding confidence intervals in different population and environments.''DL'' signifies Dali county of Shanxi province, ''DY'' corresponds to Daye City, ''JZ'' Jingzhou City, ''WH'' Wuhan City of Hubei Province in China and the numbers following these abbreviations show the seeding year.bn represents the first branch number per plant; ft, the flowering time; ph plant height; mt mature time; sn seed number per pod.Image from Chen et al. (2008).