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.

Introduction

Transgenic canola (B. napus, AACC, 2n = 38) is cultivated in Canada, Australia, Chile and the USA and the cultivation area has expanded year by year (James 2011). Because transgenic canola plants derived from spilled seeds have been observed growing along roadsides and in vacant and other spaces in Canada (Yoshimura ), Japan (Aono , Mizuguti ) and other countries (Claessen , 2005b), the potential of introgression from transgenic canola into wild relatives has aroused public concern and led to worldwide debate (Aono , Wei , Wilkinson and Tepfer 2009).

B. juncea (AABB, 2n = 36) is cultivated and is also found as a weed and feral plant in Japan (Shimizu ), Asian countries including China (Di ), Europe (Hultén and Fries 1986), Australia (OGTR 2011), Canada and the USA (Bryson and DeFelice 2010). Since B. juncea is considered the second most likely species after B. rapa to be a recipient of B. napus genes by virtue of their crossability and weediness (Di , OGTR 2011, Scheffler and Dale 1994), the risk assessment regarding introgression from B. napus to B. juncea should be carried out carefully. Therefore, persistence of chromosome derived from B. napus should be investigated in hybrid progenies.

Although B. juncea and B. napus are crossable and hybrids can be easily produced by artificial pollination (Bing , Jørgensen , Tsuda ), the highest spontaneous hybridization frequency was only 3% under a mixed planting condition (Bing , Jørgensen , Tsuda ), with the frequency decreasing sharply with distance from B. napus as the pollen source (Tsuda ). Furthermore, the fertility of the F1 hybrid between B. juncea and B. napus tends to be poor and less seeds productivity (Bing , Frello ). However, fertility was restored in backcross progenies between B. juncea and B. napus than in F1 hybrids (Frello , Song ). If backcross progenies carry any genome regions derived from C-chromosome of B. napus, these regions could be introgressions and be inherited to their progeny.

Frello evaluated the persistence of B. napus-specific RAPD markers in the BC1 generation obtained from B. juncea × F1 hybrid, but did not identify the locations of the markers. Distinguishing between A genome chromosomes of B. juncea and B. napus is currently difficult, but C-chromosomes can be identified using specific SSR markers constructed by Piquemal . Then, in order to investigate the introgression of the B. napus genome into B. juncea, we evaluated the persistence of C-chromosome regions in F1 hybrids and their backcross progenies, BC1, BC2 and BC3 generations.

Materials and Methods

Plant materials

B. juncea L. cv. Kikarashina (Takii & Co., Ltd., Kyoto, Japan) and B. napus L. cv. Westar (Genebank of NIAS, JP No. 40734) were used as the maternal and paternal parents, respectively. F1 hybrid plants were obtained by artificial bud pollination in B. juncea × B. napus. Backcrosses to obtain BC1 plants were performed by reciprocal crossings between B. juncea and the F1 hybrid by artificial bud pollination. One seed of BC1 was obtained from backcrossing of B. juncea × F1 and the BC2 and BC3 were produced by backcrossing of Kikarashina × BC1 and Kikarashina × BC2. Twenty-one seeds were randomly selected from 139 of BC2 seeds and we distinguished and treated these 21 BC2 plants as an independent line. A total of 63 BC3 plants from 21 BC2 lines were used for SSR analysis. Numbers of plants used as seed or pollen parents are shown in Table 1. Artificial bud pollination, germination and growth conditions were as described by Tsuda . Seeds per pollinated flowers was calculated from the numbers of pollinated flowers and obtained seeds (Table 1).

Table 1

Cross combinations and seed productivity of F1 hybrid and backcross progenies

Plant type produced	Cross combination	No. of used plants		No. of pollinated flowers	No. of seeds	Seeds per pollinationa	No. of plants for SSR analysis
		♀	♂
B. juncea	B. juncea × B. juncea	9	9	276	1,712	6.2 ± 2.7	5
F1	B. juncea × B. napus	11	10	231	999	4.3 ± 1.3	7
BC1	B. juncea × F1	42	50	624	1	0.0016 ± 0.011	1
BC2	B. juncea × BC1	1	1	25	139	5.6b	21
BC3	B. juncea × BC2	21	21	337	1,955	5.8 ± 3.5	63
BC1	F1 × B. juncea	48	40	698	0	0	–

Seeds per pollination represents the number of obtained seeds per pollinated flower and the standard deviation for seeds per pollination in each pollinated plant.

Means of standard deviation in seeds per pollination among individual plants could not be calculated, because only one plant was used as seed parents.

Chromosome preparations

Meiotic chromosome numbers were counted in pollen mother cells (PMCs) using the 1% acetic orcein smear method and were based on at least 20 cells per plant.

Morphological characteristics

Hybridity of F1 plants was evaluated according to morphological characteristics such as flower organ size, shape of the leaf margin, leaf rugose, leaf fairness, waxy leaf and flowering time as described in Tsuda . Morphological characteristics in backcross progenies were evaluated by the same characteristics.

SSR analysis

Genomic DNA was extracted from young leaves by ISOPLANT II (NIPPON GENE CO., LTD., Toyama, Japan) according to the manufacturer’s instructions. PCR reactions for SSR analysis were carried out under the following conditions. The composition of the reaction mixture by final concentrations was as follows: 0.5 U/μl Taq DNA polymerase (Gene taq: NIPPON GENE CO., LTD.), 1× PCR Buffer for Gene taq, 0.2 mM dNTP, 0.25 μM forward primer, 0.25 μM reverse primer, 2 ng/reaction DNA. PCR was conducted with a GeneAmp PCR System 9700 (Applied Biosystems) and PCR conditions followed Piquemal . The PCR products were electrophoresed on 5% acrylamide gel and visualized by staining with ethidium bromide, and bands were visualized with an ultraviolet illuminator. SSR analyses were performed in duplicate.

We screened applicable 83 C-chromosome specific SSR markers in our research from reported 141 SSR markers located on linkage groups N11–N19 by Piquemal . The stability of the screened markers was checked using total DNA of five independent plants each of B. juncea and B. napus.

Seven F1 hybrids were randomly selected and used for SSR analysis. Twenty-one plants were randomly selected from 139 BC2 seeds for analysis. Three BC3 plants from each of the 21 BC2 plants were used for SSR analysis, for a total of 63 BC3 plants. The 83 selected markers were used for analysis of the F1 and BC1. Twelve of the 83 markers detected in the BC1 were used for analysis of the BC2 and BC3 generations. In addition, five markers (MR025, CB10036A, CB10109B, CB10234 and CB10504B) undetected in BC1 were selected from five linkage groups (N11, N13, N14, N16 and N18) of C-chromosomes and used to confirm the absence of these markers in BC2 and BC3.

Results

Fertility and morphology of F1 plants and backcross progenies

Artificial pollination of B. juncea × B. napus produced 999 seeds and the production efficiency was 4.3 seeds/pollination (Table 1). Fifty putative F1 seeds were randomly selected and hybridity of F1 plants was evaluated by observation of morphological characteristics. These F1 plants showed intermediate characteristics between B. juncea and B. napus in flower organ size, shape of leaf margin, leaf rugose, leaf fairness and waxy leaf (Fig. 1C) and the flowering time of these F1 plants was the same as that of B. napus (Fig. 1G). No seed was obtained by 698 bud pollination in F1 × B. juncea and 1 seed was obtained by 624 bud pollination in B. juncea × F1 and the production efficiency was 0.0016 seeds/pollination (Table 1). One hundred and thirty-nine BC2 seeds were obtained from 25 bud pollinations of B. juncea × BC1 plant (obtained from B. juncea × F1) and the production efficiency was 5.6 seeds/pollination. In total 1955 BC3 seeds were obtained by 337 bud pollinations and the production efficiency was 5.8 seeds/pollination. Although the fertility of F1 plants was extremely low, the production efficiencies in the BC2 and BC3 were very close to that for B. juncea self-pollination (6.2 seeds/pollination) (Table 1). This result was assumed that very low fertility in F1 hybrids can be recovered rapidly during backcrossing.

Fig. 1

Morphological characteristics and chromosomal analysis in B. juncea, B. napus, F1 hybrid and backcross progenies. A: B. juncea plant, B: B. napus plant, C: F1 hybrid plant, D: BC1 plant, E: Plant BC2-5, F: Plant BC3-5-2, G: Plant types of B. juncea, B. napus, F1 hybrid, BC1, BC2 and BC3 plants, H: Chromosomes in PMC of B. juncea (2n = 36), I: Chromosomes in PMC of F1 hybrid (2n = 37), J: Chromosomes in PMC of BC2 (2n = 36), K: Chromosomes in PMC of BC3 (2n = 36). Bars = 3 cm.

BC1 leaves had more rugose and fairness and less waxy than F1 plants (Fig. 1D) and the flowering time of the BC1 was intermediate between F1 and B. juncea (Fig. 1G). BC2 and BC3 had morphological characteristics and flowering time similar to those B. juncea (Fig. 1G).

Chromosome numbers of F1 hybrids and backcross progenies

The F1 hybrids had 37 chromosomes (Fig. 1I). In contrast, 36 chromosomes were observed in BC1 plant (data not shown). BC2 and BC3 plants had also confirmed 36 chromosomes (Fig. 1J, 1K) by observation of 21 plants and 45 plants, respectively. These chromosome numbers in backcross progenies are the same number as B. juncea. Chromosome pairing in BC2 and BC3 exhibited same manner as that of B. juncea and mainly consisted of 18 bivalent (Fig. 1J, 1K).

Screening of SSR markers

To evaluate persistent regions of C-chromosome in this experiment, 83 SSR markers were screened from reported 141 of B. napus C-chromosome specific SSR markers by Piquemal (Table 2). These 83 markers were clearly detected in all control plants of B. napus and they were not detected in B. juncea plants.

Table 2

The list of SSR markersa used

Chromosome number	Marker name
N11	CB10587, CB10208, CB10369, CB10443, MR025, Ol12-F11, CB10277, CB10281, CB10258, Na12-C06, Na10-H06, Ol10-A11, Na10-H03, BRAS074
N12	Na12-A01, CB10316, Na14-H11, CB10350, Ol13-G05, CB10026, Ni2-C12, Ol10-H02
N13	Ol13-D03, CB10036A, CB10569, Ol11-B05, Na12-E02, Ol10-B04, CB10132, CB10057, BRAS051, BRAS087, BRAS005, Na10-D03, CB10415B, Na12-F12, Ol13-A10, MR061A, MR061B, MR049A, MR049B, BRAS068, Ol13-H09, Na10-C01A
N14	Ol13-C03, CB10103, Ra2-F11, Ni4-A07, CB10109B, Na12-G04, CB10122, CB10288
N15	Na10-G08, A48350, MR129, Ol12-F02, Na10-A08, Na10-D11, MR097, CB10487
N16	CB10502, CB10234, CB10343, Na12-A02, CB10544, Ra2-A05
N17	CB10297, CB10528, BRAS019, CB10217, Na10-C01B, Na12-F03, BRAS107, CB10299, CB10268, CB10431
N18	CB10139, CB10504B, CB10373, Ni2-F11, Ol12-G04
N19	CB10344, BRAS002

We screened these available SSR markers for our experiment from reported B. napus C-chromosome specific markers (Piquemal ).

Evaluation for persistent C-chromosome regions by SSR analysis

The segregations of detected markers in each backcross generation are shown in Fig. 2. All 83 SSR markers were detected in F1 hybrids, whereas in BC1 plant 71 of 83 markers (85%) were deleted and 12 markers were detected. These 12 markers were used for analysis in BC2 and BC3 plants. The five markers (MR025, CB10036A, CB10109B, CB10234 and CB10504B) selected for confirmation from the 71 undetected in the BC1 were also not detected in BC2 and BC3 (data not shown).

Fig. 2

Detection of B. napus C-chromosome specific SSR markers in BC2 and BC3 plants. Black cells indicate marker detection.

The 12 markers detected in BC1 segregated in the BC2 generation. Nine of 12 markers vanished from more than half of the analyzed BC2 plants. Other three markers, Na14-H11A, CB10415B and CB10288, remained in 13, 21 and 20 BC2 plants, respectively (Fig. 2).

Of the 12 SSR markers detected in the BC1, only CB10415B and/or CB10288 were detected and segregated in the BC3. Of 63 plants, CB10415B was detected in 22 plants and CB10288 in 29 plants. BC3 plants were classified into four types for persistence pattern of CB10415B and CB10288: both markers were detected in 10 plants of 7 lines, only CB10415B was detected in 12 plants of 7 lines, only CB10288 was detected in 19 plants of 11 lines and neither marker was detected in 22 plants of 12 lines. Both SSR markers were not carried in about one-third of the BC3 plants after backcrossing. But all three tested plants in lines BC3-12, −13 and −16 showed the persistence of CB10415B and all tested plants in lines BC3-1, −6, −7, −9, −12 and −20 carried the CB10288 marker.

Discussion

In B. juncea × B. napus, it is suggested that the possibility of spontaneous hybridization is generally low (Bing , Jørgensen , Tsuda ) and very low fertility of F1 hybrids in B. juncea × B. napus (Bing , Jørgensen ). Our results agreed with those of previous researchers in showing seed sterility of F1 hybrids by reciprocal pollination between F1 hybrids and B. juncea (Table 1). In interspecific and intergeneric hybridization of Brassica genus, low seed fertility has been reported often in such as cross combinations of B. rapa × B. oleracea and Raphanus sativus × B. oleracea (Namai ), B. napus × R. raphanistrum and B. napus × R. sativus (Ammitzbøll and Jørgensen 2006). And also, F1 hybrids sterility is common in many plant species (Grant 1981). This observation suggests that introgression from B. napus to B. juncea is rare in natural environments. However, introgression from transgenic plants to wild relatives through backcrossing is took into account and then many research groups intend to study in Brassica (OGTR 2011) and other crops (Andersson and de Vicente 2010, Stewart ) around the world. Seed productivities of BC2 and BC3 generations recovered to the same level as B. juncea despite the low seed fertility of the F1 hybrid (Table 1). Song also reported restoration of seed fertility in back-cross progenies between B. juncea and B. napus. Moreover, fertility recovery in backcross progenies has been reported in other Brassica species (Hauser , Snow , Song ), coffee (Coulibaly ), wheat (Seefeldt , Wang ) and cotton (Jiang ). Once B. napus genome regions are integrated in chromosomes of B. juncea, the regions must have persisted and transmitted to subsequent progenies. Therefore, we should reveal how C-chromosome regions would be persisted in backcross progenies.

In BC1 plant, 71 markers were deleted and 12 markers were persisted. The chromosome number of the BC1 was 36 and its morphological characteristics were similar to those of B. juncea. The 12 markers located on six C-chromosomes (two on N12, six on N13, one each on N14, N15, N16 and N17) showed that the entire C-chromosome was not added to the hybrid progeny.

In BC3 generation between B. napus and B. carinata, Navabi speculated that a part of C-chromosome of B. carinata was integrated into C-chromosomes of B. napus by homologous recombination. Brassica species have generally high homoeology among A, B and C genomes (McGrath and Quiros 1991, Prakash and Chopra 1990, Quiros , Truco , U 1935) and Mason reported that the homologous pairing frequency of allosyndesis in A–C genome chromosome was higher than that of B–C. Bing also proposed the possibility of intergenomic chromosomal recombination resulting in the introgression of C-chromosome region of B. napus to B. juncea. From these previous reports and our results, we speculated that the persisting C-chromosome regions were integrated into A or B genome chromosomes of B. juncea by homologous recombination.

In contrast, the entire chromosome and a large part of B-chromosome were detected in hybrid progeny, F5 plants (Schelfout ) derived from B. napus × B. juncea and BC3 plants (Navabi ) derived from B. carinata × B. napus. It was considered that homologous recombination may hardly occur due to lower homology between B genome and C genome than between A genome and C genome (Mason ). Therefore, it is speculated that persistent manner of chromosome was affected by homology among A, B and C genomes.

The two SSR markers, CB10415B and CB10288, detected in the BC3 generation (Fig. 2) are mapped on the C-chromosomes N13 and N14, respectively (Piquemal ). Akaba reported that chromosomes N11, N15 and N18 of B. napus did not undergo pairing with A-chromosomes of B. rapa. Our results showed that only one marker, MR129 on N15, was detected in the BC2, whereas SSR markers on N11 and N18 were not detected. This observation supports the ready elimination of markers on N11, N15 and N18 owing to the lower affinity of these chromosomes to A-chromosomes of B. juncea. Thus, studies on chromosomal homology among A, B and C genomes are further progressing (Akaba , Ge and Li 2007, Truco ), at least, N13 and N14 of B. napus C-chromosome did not have lower affinity to A and B genomes of B. juncea (Akaba ). We demonstrated the possibility for persistence of some C-chromosome regions in hybrid progeny. The persisting regions were thought to be fixed and inherited to progenies, although most C-chromosome regions had disappeared. In other words, most chromosomal regions from C genome did not remain in hybrid progenies, and this result may have application for controlling introgression of transgenes. Namely, transgenes should disappear in hybrid progeny if the transgenes are integrated into the C-chromosome region with the lowest affinity by novel plant breeding technology e.g., gene targeting technology.

Di reported that the F1 hybrid from wild B. juncea × transgenic canola showed higher fertility than found in our study (Table 1) and reported previously (Bing , Frello ). Di also discussed that vigorous vegetative and reproductive growth of wild B. juncea allowed the maintenance of higher fertility in F1 hybrid. Given that wild B. juncea in natural environments is thought to comprise multiple genotypes, a discussion of introgression potential should also take into account this genotypic variation.