Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 183-192.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0668
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WANG Ge-ge1(), QIU Shi-rui2, ZHANG Lin-han1, YANG Guo-wei1, XU Xiao-yun1, WANG Ai-ling1, ZENG Shu-hua1(), LIU Ya-jie1()
Received:
2022-05-30
Online:
2023-02-26
Published:
2023-03-07
WANG Ge-ge, QIU Shi-rui, ZHANG Lin-han, YANG Guo-wei, XU Xiao-yun, WANG Ai-ling, ZENG Shu-hua, LIU Ya-jie. Molecular Cytology at Meiosis in Allotriploid Nicotiana tabacum(SST)[J]. Biotechnology Bulletin, 2023, 39(2): 183-192.
引物Primer | 序列Sequence(5'-3') |
---|---|
ACTIN L | GTACCACCGGTATTGTGTTGGAC |
ACTIN R | CTATCAGTGAGGTCACGACCTG |
DMC1 L | AGGGAACGTTTCGGCCAGATC |
DMC1 R | AGAACAGCTCCTGCGTCCATTC |
CENH3: 11592 L | GCCACACGTTCTGCTGC |
CENH3: 11592 R | TGGCCTGTAACGAGTCTTC |
CDKA: 0239 L | GCGCTTCTTCCAATGTGATTCAAT |
CDKA: 0239 R | CGCACCCCAAACTATAACAAAC |
CDKA: 5319 L | GCGCTGTTATTCTTCCAATGTGATTC |
CDKA: 5319 R | CACCCGGAACTATAACAAAATG |
CYCA: 6720 L | GATGGCGGAGAATAATCACGG |
CYCA: 6720 R | ATGTTGGCAGAACTGAGGAAGC |
CYCA: 0111 L | CAAGAAACGACCAGCCCTCAC |
CYCA: 0111 R | CATCACTTCTTGGAATAGACACAG |
MIS12: 76707 L | GCAAGCGTTGGAGTTGTATCAG |
MIS12: 76707 R | TGCTCTGCGGTGCTCAGTGTC |
Table 1 Primers used in RT-qPCR
引物Primer | 序列Sequence(5'-3') |
---|---|
ACTIN L | GTACCACCGGTATTGTGTTGGAC |
ACTIN R | CTATCAGTGAGGTCACGACCTG |
DMC1 L | AGGGAACGTTTCGGCCAGATC |
DMC1 R | AGAACAGCTCCTGCGTCCATTC |
CENH3: 11592 L | GCCACACGTTCTGCTGC |
CENH3: 11592 R | TGGCCTGTAACGAGTCTTC |
CDKA: 0239 L | GCGCTTCTTCCAATGTGATTCAAT |
CDKA: 0239 R | CGCACCCCAAACTATAACAAAC |
CDKA: 5319 L | GCGCTGTTATTCTTCCAATGTGATTC |
CDKA: 5319 R | CACCCGGAACTATAACAAAATG |
CYCA: 6720 L | GATGGCGGAGAATAATCACGG |
CYCA: 6720 R | ATGTTGGCAGAACTGAGGAAGC |
CYCA: 0111 L | CAAGAAACGACCAGCCCTCAC |
CYCA: 0111 R | CATCACTTCTTGGAATAGACACAG |
MIS12: 76707 L | GCAAGCGTTGGAGTTGTATCAG |
MIS12: 76707 R | TGCTCTGCGGTGCTCAGTGTC |
染色体数目/条 Chromosome number/bar | 统计总量/株 Total amount/plant | 单株数量/株 Amount/plant | 频率 Rate/% |
---|---|---|---|
36 | 117 | 68 | 58.12 |
48 | 15 | 12.82 | |
27-34 nr | 34 | 29.06 |
Table 2 Chromosome number statistics of hybrid off spring
染色体数目/条 Chromosome number/bar | 统计总量/株 Total amount/plant | 单株数量/株 Amount/plant | 频率 Rate/% |
---|---|---|---|
36 | 117 | 68 | 58.12 |
48 | 15 | 12.82 | |
27-34 nr | 34 | 29.06 |
Fig. 2 GISH analysis of allotriploid hybrids a: blue color is from DAPI staining chromosomes. b: Red color is from the biotin-labeled S genome probe. c: Merged. bar=5 μm
Fig. 3 Morphological characteristics of allotriploid hybrids and parents A, B: Parent and triploid seedling stage and flowering stage, bar =10 cm. C: The flower stage of parent and triploid, bars=2 cm. a: The seeding stage of K326. b: The seeding stage of Nicotiana sylvestris. c: The seeding stage of F1. d-f: The flower shape of K326, F1 and Nicotiana sylvestris(from left to right). g: The flowering stage of K326. h: The flowering stage of Nicotiana sylvestris. i: The flowering stage of F1
材料 Material | 株高 Plant height/cm | 叶片数 Leaf number | 最大叶长 The largest leaf length/cm | 最大叶宽 The largest leaf width/cm | 花色 Flower color | 花冠长度 Corolla length/cm |
---|---|---|---|---|---|---|
K326 | 84. 13a | 21c | 27.25a | 10.05a | 浅红色 | 5.28c |
F1 | 74.72b | 23b | 22.56b | 8. 17b | 浅粉色 | 6.65b |
林烟草 | 69.67c | 25a | 19.34c | 6.70c | 白色 | 9.72a |
Table 3 Morphological characteristics comparison between the allotriploid and parents
材料 Material | 株高 Plant height/cm | 叶片数 Leaf number | 最大叶长 The largest leaf length/cm | 最大叶宽 The largest leaf width/cm | 花色 Flower color | 花冠长度 Corolla length/cm |
---|---|---|---|---|---|---|
K326 | 84. 13a | 21c | 27.25a | 10.05a | 浅红色 | 5.28c |
F1 | 74.72b | 23b | 22.56b | 8. 17b | 浅粉色 | 6.65b |
林烟草 | 69.67c | 25a | 19.34c | 6.70c | 白色 | 9.72a |
材料 Material | 可育花粉(粒) Fertile pollen | 不可育花粉(粒) Infertile pollen | 可育性(可育/总数) Fertility/% |
---|---|---|---|
K326 | 2 115 | 261 | 89.02 |
林烟草 | 2 027 | 712 | 74.01 |
F1 | 318 | 2 560 | 11.05 |
Table 4 Pollen fertility of the allotriploid, BC1 and parents
材料 Material | 可育花粉(粒) Fertile pollen | 不可育花粉(粒) Infertile pollen | 可育性(可育/总数) Fertility/% |
---|---|---|---|
K326 | 2 115 | 261 | 89.02 |
林烟草 | 2 027 | 712 | 74.01 |
F1 | 318 | 2 560 | 11.05 |
Fig. 5 Meiosis of the allotriploids hybrid and K326 a: The zygotene stage of K326. b: The zygotene stage of F1. d: Metaphase I of F1. e: Anaphase I of F1. f: Metaphase II of F1. g: Telophase II of F1. l: Metaphase I of K326. m: Anaphase I of K326. n: Metaphase II of K326. o: Anaphase II of K326. The magnified part in the black frame is placed at the bottom right, the black arrows point to segments without synapsis, the white arrows point to segments with synapsis
Fig. 6 GISH analysis of allotriploid hybrids The probe is a biotin-labeled S genome, showing red signals. a-c: Metaphase I. d-f: Anaphase I. g-i: Metaphase II. j-l: Telophase II. The arrows point to lagging chromosomes, bar=5 μm
减数分裂时期 Meiotic phase | 异常现象 Abnormality | 统计细胞数 Total amount | 异常细胞总数 Total abnormal cell | 异常细胞数 Abnormal cell | 异常细胞频率(占异常细胞数量) Abnormal cell/% |
---|---|---|---|---|---|
减Ⅰ中期 | 落后染色体 | 1 285 | 376 | 111 | 29.52 |
减Ⅰ后期 | 不均等分离 | 48 | 12.77 | ||
落后染色体 | 14 | 3.72 | |||
减Ⅱ中期 | 落后染色体 | 136 | 36.17 | ||
减Ⅱ后期 | 不均等分离 | 42 | 11.17 | ||
落后染色体 | 13 | 3.46 | |||
染色体桥 | 5 | 1.33 | |||
减Ⅱ末期 | 微核 | 7 | 1.86 |
Table 5 Abnormal chromosome behavior during the meiosis of the F1 SST
减数分裂时期 Meiotic phase | 异常现象 Abnormality | 统计细胞数 Total amount | 异常细胞总数 Total abnormal cell | 异常细胞数 Abnormal cell | 异常细胞频率(占异常细胞数量) Abnormal cell/% |
---|---|---|---|---|---|
减Ⅰ中期 | 落后染色体 | 1 285 | 376 | 111 | 29.52 |
减Ⅰ后期 | 不均等分离 | 48 | 12.77 | ||
落后染色体 | 14 | 3.72 | |||
减Ⅱ中期 | 落后染色体 | 136 | 36.17 | ||
减Ⅱ后期 | 不均等分离 | 42 | 11.17 | ||
落后染色体 | 13 | 3.46 | |||
染色体桥 | 5 | 1.33 | |||
减Ⅱ末期 | 微核 | 7 | 1.86 |
异常情况Abnormality | 细胞数量Number of cells | 与对应异常细胞总数占比 Proportion in total number of corresponding abnormal cells /% | |
---|---|---|---|
减I中期 | 落后1条 | 38 | 34.23 |
落后2条 | 57 | 51.35 | |
落后3条 | 11 | 9.91 | |
其他 | 5 | 4.5 | |
后I分离比 | 17∶18(落后1条) | 14 | 29.17 |
18∶18 | 9 | 18.75 | |
20∶16 | 3 | 6.25 | |
21∶15 | 11 | 22.92 | |
其他 | 11 | 22.92 | |
减II中期 | 落后1条 | 46 | 33.82 |
落后2条 | 81 | 59.56 | |
其他 | 9 | 6.62 |
Table 6 Chromosome behavior of interspecific hybrids SST during meiosis
异常情况Abnormality | 细胞数量Number of cells | 与对应异常细胞总数占比 Proportion in total number of corresponding abnormal cells /% | |
---|---|---|---|
减I中期 | 落后1条 | 38 | 34.23 |
落后2条 | 57 | 51.35 | |
落后3条 | 11 | 9.91 | |
其他 | 5 | 4.5 | |
后I分离比 | 17∶18(落后1条) | 14 | 29.17 |
18∶18 | 9 | 18.75 | |
20∶16 | 3 | 6.25 | |
21∶15 | 11 | 22.92 | |
其他 | 11 | 22.92 | |
减II中期 | 落后1条 | 46 | 33.82 |
落后2条 | 81 | 59.56 | |
其他 | 9 | 6.62 |
株系 Line | 减数分裂时期 Meiotic phase | 细胞数 Number of cells observed | 落后染色体数量及组成Number and composition of lagging chromosomes | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
S | T | S+T | 2S | 2T | S+2T | 2S+T | 3S | 3T | 其他Others | |||
F1 | 减I中期 | 55 | 4 | 16 | 5 | 9 | 15 | 2 | 1 | 0 | 1 | 2 |
减II中期 | 68 | 10 | 14 | 12 | 7 | 21 | 0 | 0 | 0 | 1 | 3 |
Table 7 GISH statistics of lagging chromosomes composition
株系 Line | 减数分裂时期 Meiotic phase | 细胞数 Number of cells observed | 落后染色体数量及组成Number and composition of lagging chromosomes | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
S | T | S+T | 2S | 2T | S+2T | 2S+T | 3S | 3T | 其他Others | |||
F1 | 减I中期 | 55 | 4 | 16 | 5 | 9 | 15 | 2 | 1 | 0 | 1 | 2 |
减II中期 | 68 | 10 | 14 | 12 | 7 | 21 | 0 | 0 | 0 | 1 | 3 |
Fig. 7 Differential expressions of meiosis related genes in allotriploid and parent tobacco K326 A: The collection time is prophase I. B: The collection time of is after prophase I. (S),(T)refers to the primers designed for genes of S and T chromosome groups respectively. * indicates significant difference(P<0.05), ** indicates extremely significant difference(P<0.01)
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