Ploidy analysis
The offspring of a hybrid between high-ploidy and low-ploidy that are prone to intermediate ploidy [19]. In order to understand whether there were differences in ploidy level before and after induction, we selected different Brassica napus induced F1, and tetraploid Brassica napus ZS11, which was used as control. The detection results are as follows (Fig 1, Additional file 1, Additional file 2). The fluorescence intensity of F1 generation obtained by pollination of Y3560 and four maternal parents was about 409.5~510.5D thousand lines. The fluorescence intensity of F1 generation of Y3380 pollinated with five maternal parents was about 398.9~521.1D thousand lines, and the detection results were almost the same as peak value of 423.1~ 487.7D thousand lines in control hybrid offsprings, so they were all tetraploid plants, too. At the same time, we tested the ploidy of pollinated maternal parents and DH inducer. The fluorescence intensity of the maternal parent plants ranged from 406.9~ 502.1D thousand lines, all of which were tetraploid plants. The fluorescence intensity of inducer was about 753.2~852.5D thousand lines, which was two times ZS11 as control, so all of them were octoploid plants. However, in this study we selected octoploid rapeseed DH lines as the paternal parent of the tetraploid Brassica napus. The offspring ploidy were consistent their maternal parent ploidy (tetraploid), the preliminary explanation of octoploid and tetraploid rape crossing does not make sense but the tetraploid Bassica napus were pollinated by octoploid rape, which has played an induced or partial chromosomal hybrid effect.
Plant morphology and fertility investigation
Fertility and plant morphology investigations (Fig 2, Additional file 3) of F1 showed that most of plant were steriles, while only a few were fertile. Which sterile lines were pollinated by the DH inducer lines (used as paternal parent), while those of the fertile plants have shown no obvious difference from the maternal parents except for the flower morphology. Fertility identification results of paternal parent Y3560, Y3380 and induced F1 were shown as follows Additional file 3. The homozygous and stable pol CMS of 0068A, 0933A, and D717A that were used as the maternal parent crossed with Y3560 and six, two, and one fertile plant were appeared in their offspring, respectively. Only one fertile plant was appeared in F1, when Y3380 was used as the paternal parent and homozygous pol CMS 0933A was used as maternal parent; fertile plants were not found in F1, when Y3380 was crossed with L0068A and L0933A as maternal parent. At same time, the hybrid progeny ZS11 was also used as paternal parent for crossing with different maternal parents, and all resulting progeny produced were semi-sterile or sterile plants. Results of fertility identification revealed that among the induced offspring from paternal parents Y3560 and Y3380, the fertile plants appeared in the offspring of pol CMS, when it was used as maternal parent with a probability of 2.22% to 30.00%. Among them, the probability of offspring (0068A×Y3560) was highest (30.00%) while lowest (2.22%) was found in the offspring (D717A×Y3560). The induction of fertile plants in pol CMS maternal parent may be attributed to the hybridization compatibility of Y3380 and Y3560 with pol cytoplasmic restoration genes. According to the probability of fertile plants, the induction rate of Y3380 and Y3560 was estimated to vary from 70% to 100%.
Screening results of SSR molecular markers and SNP chips for induction line F1
600 pairs of SSR primers were used to differentiate the polymorphism between parental and maternal parents and their progeny (Fig 3, Additional file 4, Additional file 5). When Y3560 was used the parental parent, three pairs of specific primers were amplified with 0068A; four pairs of specific primers were amplified with 0933A and L0933A. Those specific primers were also amplified in their offsprings. Amplification of SSR analysis revealed that induced F1 generation plants were consistent with maternal band type, without paternal or heterozygous band type. At the same time, combined with the field fertility survey, the M1-1, M1-8 and M1-9, the progeny of 0068A×Y3560 were tentatively judged as the induced plants. While that of Y3380, three pairs of specific primers were amplified with L0068A, five pairs of specific primers were amplified with 0933A and L0933A, and amplification results showed that in addition to L0068A× Y3380 offspring M4-5, M4-9 revealed heterozygous band type, while remaining offspring and maternal band type. ZS11 was used a parental parent, and 2-3 pairs of specific primers were amplified in each of maternal parent. The amplification of primers revealed that the progeny contained the band type of both parents, hence all were hybrid plants. Since the polymorphic primers were not enough, so 6K SNP chip analysis covering more marker sites in whole of genome should be used to verify whether the progeny of each combination is homozygous or heterozygous induced plant? In view of the fact that ploidy, morphology and SSR molecular marker identification would not produce accurate results, 62 plants were selected for 6K SNP chip analysis (Additional files 6). The results of SNP homozygosity and genetic similarity are given in Additional file 7 and the 7-materials (Additional files 8) used this study showed a homozygosity rate that ranges from 97.93% ~ 99.29%. Such higher homozygosity rate was close to ZS11 as control (99.16%), suggesting that these seven maternal parents were homozygous. The homozygosity rate of F1 hybrid was 63.73%~ 68.74%, and genotyping (Fig 4d, Additional file 9c, Additional file 10e) also indicated that these offspring were F1 hybrids produced by ZS11 as the paternal parent. At the same time, the homozygosity rate of some F1 plants after induction ranged from 56.39% ~ 78.87%, and genotyping (Fig 4a-c, Additional file 9a-b, Additional file 10a-d) was used to identify the F1 as hybrid or partial chromosome hybrid offspring. The rate of homozygous SNP sites in the remaining single plants ranged from 98.48% ~ 99.33%. Genotyping (Fig 4a-c, Additional file 9a-b, Additional file 10a-d) confirmed that these F1 were homozygous plants. Therefore, the analysis of the homozygous rate between different individual plants showed that used the DH lines as the paternal parent, the offspring produced by the homozygous rate of about 60% were hybrid offspring, and the homozygous rate greater than 95% was the induced offspring. Subsequently, we analyzed the genetic distance between the maternal parent and 62 materials, and calculated the genetic similarity rate between the maternal parents and their F1 offspring. The genetic similarity rate between hybrid offspring and the maternal parent was 64.64% ~ 68.74%, while that of genetic similarity rate between the induced homozygous individual plant and the maternal parent was as high as 99.33%.
Genotyping revealed paternal chromosome hybridization and infiltration during induction process
In order to better understand the paternal chromosome hybridization and infiltration in induction process, homozygous SNP sites with parental differences were screened and genotyping revealed SNP sites in the offspring (Fig 4, Additional file 9, Additional file 10, Additional file 11). The number of homozygous SNP sites in parental differences ranged from 873-1227 (Table 1, Fig 5a, Additional file 11). Taking the maternal parent 0933 as an example, compared with the hybrid combination 0933A× ZS11 (Fig 4d), 97.91% heterozygosity, when 0933B (Fig 4a) and 0933A (Fig 4b) were the maternal parent, genotype of the induced offspring was consistent with those of the maternal parent. Hybridization of 33.84% ~ 44.26% paternal genes and paternal infiltration rate of 0.09~0.19% were observed in the hybrid offspring. When L0933A (Fig 4c) was used as maternal parent, the hybrid offspring revealed 37.87% ~ 53.03% (Table 1) paternal genes heterozygosity and 0.09% ~ 0.18% (Table 1) paternal introgression. These results indicated that the induction efficiency was influenced by cytoplasm genotype, and the hybridization of ogu cytoplasmic genotype was more prone to occur in the maternal parent when the karyogene was same. When the cytoplasmic genotype was pol (0068A, 0933A, D717A), the offspring of 0068A (33.82% ~ 47.10%) (Table 1) were more likely to cross with the paternal parent; and the cytoplasmic type was nap (0933B, D717B), the offspring of D717B (24.84% ~ 42.86%) (Table 1) were most likely to infiltrate the paternal gene; while the cytoplasm genotype was ogu (L0068A, L0933A), the L0933A was easier to cross or exchange with the paternal parent, and was more likely to be on the C-genome (Fig 5b), indicating that the cytoplasm genotype was the same, the induction effect was affected by the maternal parent nuclear genotype. In conclusion, the induction efficiency is influenced by both the maternal karyogene and the cytoplasm genotype, and karyogene > cytoplasmic genotype.
Table 1 Statistics of genotyping SNP sites.
Sample source
|
sample name
|
SNP number
|
Heterozygous number
|
Heterozygosity rate(%)
|
maternal number
|
maternal infiltration rate(%)
|
paternal number
|
paternal infiltration rate(%)
|
0068A×Y3560
|
M1-1
|
1174
|
551
|
46.93
|
622
|
52.98
|
1
|
0.09
|
M1-2
|
1174
|
553
|
47.10
|
619
|
52.73
|
2
|
0.17
|
M1-3
|
1174
|
539
|
45.91
|
634
|
54.00
|
1
|
0.09
|
M1-8
|
1174
|
0
|
0.00
|
1174
|
100.00
|
0
|
0.00
|
M1-9
|
1174
|
2
|
0.17
|
1172
|
99.83
|
0
|
0.00
|
M1-10
|
1174
|
397
|
33.82
|
777
|
66.18
|
0
|
0.00
|
L0068A×Y3380
|
M4-3
|
1088
|
324
|
29.78
|
762
|
70.04
|
2
|
0.18
|
M4-4
|
1088
|
0
|
0.00
|
1088
|
100.00
|
0
|
0.00
|
M4-5
|
1088
|
626
|
57.54
|
460
|
42.28
|
2
|
0.18
|
M4-9
|
1088
|
702
|
64.52
|
385
|
35.39
|
1
|
0.09
|
0068A×ZS11
|
M5-1
|
1115
|
1106
|
99.19
|
3
|
0.27
|
6
|
0.54
|
0933A×Y3560
|
M31-1
|
1071
|
1
|
0.09
|
1070
|
99.91
|
0
|
0.00
|
M31-2
|
1071
|
3
|
0.28
|
1068
|
99.72
|
0
|
0.00
|
M31-5
|
1071
|
416
|
38.84
|
654
|
61.06
|
1
|
0.09
|
M31-7
|
1071
|
1
|
0.09
|
1070
|
99.91
|
0
|
0.00
|
M31-8
|
1071
|
474
|
44.26
|
595
|
55.56
|
2
|
0.19
|
M31-12
|
1071
|
0
|
0.00
|
1071
|
100.00
|
0
|
0.00
|
0933A×Y3380
|
M30-1
|
1038
|
2
|
0.19
|
1036
|
99.81
|
0
|
0.00
|
M30-2
|
1038
|
438
|
42.20
|
599
|
57.71
|
1
|
0.10
|
M30-3
|
1038
|
2
|
0.19
|
1036
|
99.81
|
0
|
0.00
|
M30-6
|
1038
|
0
|
0.00
|
1038
|
100.00
|
0
|
0.00
|
M30-10
|
1038
|
1
|
0.10
|
1037
|
99.90
|
0
|
0.00
|
M30-11
|
1038
|
0
|
0.00
|
1038
|
100.00
|
0
|
0.00
|
0093B×Y3380
|
M28-2
|
1062
|
0
|
0.00
|
1062
|
100.00
|
0
|
0.00
|
M28-6
|
1062
|
1
|
0.09
|
1061
|
99.91
|
0
|
0.00
|
M28-7
|
1062
|
1
|
0.09
|
1061
|
99.91
|
0
|
0.00
|
M28-9
|
1062
|
1
|
0.09
|
1061
|
99.91
|
0
|
0.00
|
L0933A×Y3560
|
M35-1
|
1088
|
18
|
1.65
|
1070
|
98.35
|
0
|
0.00
|
M35-7
|
1088
|
17
|
1.56
|
1071
|
98.44
|
0
|
0.00
|
M35-9
|
1088
|
577
|
53.03
|
475
|
43.66
|
36
|
3.31
|
M35-10
|
1088
|
33
|
3.03
|
1055
|
96.97
|
0
|
0.00
|
M35-12
|
1088
|
17
|
1.56
|
1071
|
98.44
|
0
|
0.00
|
M35-14
|
1088
|
102
|
9.38
|
984
|
90.44
|
2
|
0.18
|
L0933A×Y3380
|
M34-1
|
1062
|
531
|
50.00
|
529
|
49.81
|
2
|
0.19
|
M34-2
|
1062
|
470
|
44.26
|
513
|
48.31
|
79
|
7.44
|
M34-3
|
1062
|
509
|
47.93
|
500
|
47.08
|
53
|
4.99
|
M34-5
|
1062
|
539
|
50.75
|
522
|
49.15
|
1
|
0.09
|
M34-6
|
1062
|
402
|
37.85
|
659
|
62.05
|
1
|
0.09
|
M34-9
|
1062
|
79
|
7.44
|
982
|
92.47
|
1
|
0.09
|
0933A×ZS11
|
M36-1
|
1053
|
1031
|
97.91
|
2
|
0.19
|
20
|
1.90
|
L0933A×ZS11
|
M36-1
|
1079
|
1041
|
96.48
|
2
|
0.19
|
36
|
3.34
|
D717A×Y3560
|
M39-2
|
1071
|
465
|
43.42
|
604
|
56.40
|
2
|
0.19
|
M39-4
|
1071
|
2
|
0.19
|
1069
|
99.81
|
0
|
0.00
|
M39-6
|
1071
|
1
|
0.09
|
1070
|
99.91
|
0
|
0.00
|
M39-8
|
1071
|
460
|
42.95
|
610
|
56.96
|
1
|
0.09
|
M39-13
|
1071
|
480
|
44.82
|
588
|
54.90
|
3
|
0.28
|
D717A×Y3380
|
M38-1
|
873
|
403
|
46.16
|
470
|
53.84
|
0
|
0.00
|
M38-2
|
873
|
396
|
45.36
|
477
|
54.64
|
0
|
0.00
|
M38-5
|
873
|
388
|
44.44
|
485
|
55.56
|
0
|
0.00
|
M38-9
|
873
|
390
|
44.67
|
478
|
54.75
|
5
|
0.57
|
D717B×Y3560
|
M10-1
|
1071
|
408
|
38.10
|
397
|
37.07
|
266
|
24.84
|
M10-2
|
1071
|
7
|
0.65
|
605
|
56.49
|
459
|
42.86
|
D717B×Y3380
|
M9-1
|
873
|
43
|
4.93
|
830
|
95.07
|
0
|
0.00
|
M9-2
|
873
|
1
|
0.11
|
872
|
99.89
|
0
|
0.00
|
M9-3
|
873
|
2
|
0.23
|
871
|
99.77
|
0
|
0.00
|
D717A×ZS11
|
M42-1
|
1227
|
1208
|
98.45
|
3
|
0.24
|
16
|
1.30
|
Bold text represents induced offspring. Italic text is the hybrid offspring of ZS11 as the paternal parent.
Analysis of the interaction effect between the inducer line and the maternal karyogene
Since maternal karyogene and cytoplasm genotype jointly affects the induction efficiency, it is not clear whether the inducer hybridization and infiltration of the sites is random. Based on the comparison of SNP in genotyping (Table 1, Fig 5a), it was found that the SNP sites of normal hybrid offspring with ZS11 as the paternal parents were heterozygous with the exception of individual sites infiltrated by the paternal parents, and the heterozygous rate were 96.48%~ 99.19% (Table 1), and the paternal infiltration rate was 0.54% ~ 3.34% (Table 1). While using the inducer as the paternal parent, although hybrid progeny will be produced, the heterozygous rate of these hybrid progeny is only 29.78% ~ 53.03% (Table 1), and most of the sites are the same as the maternal parent. Subsequent analysis of the chromosomes in which these hybridization sites are located found that when ZS11 is the paternal parent, hybridization mainly occurs on the C03 and C04 chromosomes of the C genome, and the heterozygosity rate is as high as 99.2% ~ 100% (Table 1); When the inducer was used as the paternal parent, although the same as the normal hybridization, it mainly occurred on the C03 and C04 chromosomes of the C genome, but the heterozygosity rate on C03 and C04 in the hybrid progeny was 28.1% ~ 68.14% and 30.71% ~ 86.2% (Table 1), which were significantly lower than the normal hybridization level. Therefore, the cross produced by using the inducer as the paternal parent is not an ordinary cross.
At the same time, the chromosome where the paternal infiltration site is located is analyzed (Table 1, Fig 5b-c). 0068 and 0933 have similar genetic background (from part of one same parent), when used as maternal parent, the inducer was the paternal parent, the infiltration of the paternal parent gene was more likely to be on the C04 and C06 chromosomes of the C genome; while ZS11 was the paternal parent, the sites of paternal parent infiltration were mainly on the C05 and C06 chromosomes of the C-genome, and the paternal parent of the inducer is more likely to cause the infiltration (Fig 5b). However, since the site on chromosome C06 mostly the same site, thus eliminating C06 penetration site on the chromosome, and presumed DH lines is more likely to cause parent gene penetration on the C04 chromosome of the C genome, and the paternal infiltration caused by normal hybridization is more likely to occur on the C05 chromosome. When the maternal parent was D717, the paternal parent infiltration sites of the induced was more likely to appear on the C07 chromosome of the C genome, while the parent of ZS11 was mainly on the A06 chromosome of the A genome and the C06 chromosome of the C genome. In summary, the analysis of the paternal cross and infiltration sites, it is indicated that the DH inducer has an interaction effect with the maternal karyogene, and has the biases on different genomes.