生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 64-73.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1288
杨亚杰(), 李昱樱, 申状状, 陈天, 荣二花, 吴玉香()
收稿日期:
2021-10-10
出版日期:
2022-05-26
发布日期:
2022-06-10
作者简介:
杨亚杰,女,硕士研究生,研究方向:种质创新与遗传工程;E-mail: 基金资助:
YANG Ya-jie(), LI Yu-ying, SHEN Zhuang-zhuang, CHEN Tian, RONG Er-hua, WU Yu-xiang()
Received:
2021-10-10
Published:
2022-05-26
Online:
2022-06-10
摘要:
多倍化是物种形成及植物育种的重要途径。以实验室前期合成的草棉同源多倍体后代S1为材料,对其进行流式细胞术、形态学、细胞学和SRAP分子标记鉴定。流式细胞倍性鉴定表明,以二倍体的荧光峰值作为参照,8株后代中有5株三倍体和3株四倍体。形态学鉴定表明,二倍体表现为株高、茎细、叶薄、叶片较小、叶色翠绿、花朵较小,三倍体和四倍体则表现为植株矮小、茎秆粗壮、叶片增厚和皱缩、叶片较大、叶色浓绿、花朵较大。细胞学鉴定表明,随着倍性的增加,气孔密度呈下降趋势,而气孔长度、叶绿体数和花粉粒直径均呈上升趋势;二、三和四倍体草棉在花粉母细胞减数分裂过程中均出现正常和异常行为,包括单分体(0.17%,2.50%和2.17%)、二分体(0.33%,0.67%和0)、三分体(4.17%,10.00%和2.33%)、正常四分体(94.83%,54.00%和73.67%)、异常四分体(0.17%,0.33%和0.83%)和多分体(0.33%,32.50%和21.00%);异常多分体均不能形成正常花粉粒,所以三者的正常花粉粒所占比例分别为95.33%、58.83%和77.67%。SRAP分子标记鉴定表明,三倍体和四倍体不仅扩增出与二倍体完全一致的条带,还出现了条带的缺失与新的特异性条带,其中三倍体的遗传比例分别为80.81%、6.73%和12.46%,四倍体的遗传比例分别为76.59%、10.37%和13.04%,从分子水平证明了多倍体的真实性。
杨亚杰, 李昱樱, 申状状, 陈天, 荣二花, 吴玉香. 草棉同源多倍体后代筛选及性状鉴定[J]. 生物技术通报, 2022, 38(5): 64-73.
YANG Ya-jie, LI Yu-ying, SHEN Zhuang-zhuang, CHEN Tian, RONG Er-hua, WU Yu-xiang. Selection and Character Identification for Autopolyploid Progenies of Gossypium herbaceum[J]. Biotechnology Bulletin, 2022, 38(5): 64-73.
正向引物 Forward primer | 引物序列 Primer sequence(5'-3') | 反向引物 Reverse primer | 引物序列 Primer sequence(5'-3') |
---|---|---|---|
Me 1 | TGAGTCCAAACCGGATA | Em 1 | GACTGCGTACGAATTAAT |
Me 2 | TGAGTCCAAACCGGAGC | Em 2 | GACTGCGTACGAATTTGC |
Me 3 | TGAGTCCAAACCGGAAT | Em 3 | GACTGCGTACGAATTGAC |
Me 4 | TGAGTCCAAACCGGTGC | Em 4 | GACTGCGTACGAATTAAC |
Me 5 | TGAGTCCAAACCGGAAG | Em 5 | GACTGCGTACGAATTGCA |
表1 SRAP扩增引物
Table1 SRAP amplification primers
正向引物 Forward primer | 引物序列 Primer sequence(5'-3') | 反向引物 Reverse primer | 引物序列 Primer sequence(5'-3') |
---|---|---|---|
Me 1 | TGAGTCCAAACCGGATA | Em 1 | GACTGCGTACGAATTAAT |
Me 2 | TGAGTCCAAACCGGAGC | Em 2 | GACTGCGTACGAATTTGC |
Me 3 | TGAGTCCAAACCGGAAT | Em 3 | GACTGCGTACGAATTGAC |
Me 4 | TGAGTCCAAACCGGTGC | Em 4 | GACTGCGTACGAATTAAC |
Me 5 | TGAGTCCAAACCGGAAG | Em 5 | GACTGCGTACGAATTGCA |
材料Material | 平均荧光强度X-mean | 倍性Ploidy |
---|---|---|
1 | 189.25 | 2X |
2 | 401.62 | 4X |
3 | 381.03 | 4X |
4 | 347.04 | 4X |
5 | 313.78 | 3X |
6 | 271.03 | 3X |
7 | 314.07 | 3X |
8 | 302.32 | 3X |
9 | 307.86 | 3X |
表2 不同倍性材料的荧光峰值
Table 2 Fluorescence peaks of different ploidy materials
材料Material | 平均荧光强度X-mean | 倍性Ploidy |
---|---|---|
1 | 189.25 | 2X |
2 | 401.62 | 4X |
3 | 381.03 | 4X |
4 | 347.04 | 4X |
5 | 313.78 | 3X |
6 | 271.03 | 3X |
7 | 314.07 | 3X |
8 | 302.32 | 3X |
9 | 307.86 | 3X |
倍性 Ploidy | 气孔密度 Stomatal density | 气孔长度 Stomatal length/cm | 叶绿体数 Chloroplast number |
---|---|---|---|
2X | 44.20±1.75a | 2.64±0.13c | 11.80±1.32c |
3X | 31.80±1.32b | 2.93±0.11b | 18.90±1.20b |
4X | 23.80±1.32c | 3.44±0.12a | 24.20±1.32a |
表3 不同倍性草棉的叶片气孔性状统计
Table 3 Stomatal character statistics of leaves among diffe-rent ploidy G. herbaceum
倍性 Ploidy | 气孔密度 Stomatal density | 气孔长度 Stomatal length/cm | 叶绿体数 Chloroplast number |
---|---|---|---|
2X | 44.20±1.75a | 2.64±0.13c | 11.80±1.32c |
3X | 31.80±1.32b | 2.93±0.11b | 18.90±1.20b |
4X | 23.80±1.32c | 3.44±0.12a | 24.20±1.32a |
图7 不同倍性草棉的正常和异常减数分裂行为(标尺:20 μm) A-E为2X,A:单分体;B:二分体;C:三分体:D:正常四分体;E:多分体。F-J为3X,F:单分体;G:二分体;H:三分体;I:正常四分体;J:多分体。K-O为4X,K:单分体;L:三分体;M:正常四分体;N:异常四分体;O:多分体
Fig. 7 Normal and abnormal meiosis behavior of different ploidy G. herbaceum(Ruler:20 μm) A-E are 2X. A:Monad. B:Dyad. C:Triad. D:Normal tetrad. E:Polyad. F-J are 3X. F:Monad. G:Dyad. H:Triad. I:Normal tetrad. J:Polyad. K-O are 4X. K:Monad. L:Triad. M:Normal tetrad. N:Abnormal tetrad. O:Polyad
倍性 Ploidy | 单分体 Monad | 二分体 Dyad | 三分体 Triad | 正常四分体 Normal tetrad | 异常四分体 Abnormal tetrad | 多分体 Polyad | 总数 Total |
---|---|---|---|---|---|---|---|
2X | 1(0.17%) | 2(0.33%) | 25(4.17%) | 569(94.83%) | 1(0.17%) | 2(0.33%) | 600 |
3X | 15(2.50%) | 4(0.67%) | 60(10.00%) | 324(54.00%) | 2(0.33%) | 195(32.50%) | 600 |
4X | 13(2.17%) | 0(-) | 14(2.33%) | 442(73.67%) | 5(0.83%) | 126(21.00%) | 600 |
表4 不同倍性草棉减数第二次分裂末期的多分体数量及比例
Table 4 Number of multispores in telophase II of different ploidy G. herbaceum
倍性 Ploidy | 单分体 Monad | 二分体 Dyad | 三分体 Triad | 正常四分体 Normal tetrad | 异常四分体 Abnormal tetrad | 多分体 Polyad | 总数 Total |
---|---|---|---|---|---|---|---|
2X | 1(0.17%) | 2(0.33%) | 25(4.17%) | 569(94.83%) | 1(0.17%) | 2(0.33%) | 600 |
3X | 15(2.50%) | 4(0.67%) | 60(10.00%) | 324(54.00%) | 2(0.33%) | 195(32.50%) | 600 |
4X | 13(2.17%) | 0(-) | 14(2.33%) | 442(73.67%) | 5(0.83%) | 126(21.00%) | 600 |
图8 不同倍性草棉的正常和异常花粉粒(标尺:20 μm) A-E为2X,A:正常花粉粒;B:破裂的花粉粒;C:变形的花粉粒;D:一个正常和一个发育不良形花粉粒;E:两个不均等分裂的花粉粒。F-J为3X,F:正常花粉粒;G:畸形的花粉粒;H:大椭圆形花粉粒;I:不规则形花粉粒;J:一个正常和一个未发育完全花粉粒。K-O为4X,K:正常花粉粒;L:变形的花粉粒;M:破裂的花粉粒;N:两个发育不良形花粉粒:O:一个正常和一个未发育完全花粉粒
Fig. 8 Normal and abnormal pollen grains of different ploidy G. herbaceum(Ruler:20 μm) A-E are 2X. A:Normal pollen grains. B:Cracked pollen grains. C:Deformed pollen grains. D:One normal and one small dysplastic pollen grains. E:Two unequal division pollen grains. F-J are 3X. F:Normal pollen grains. G:Deformed pollen grains. H:Large oval pollen grains. I:Irregular pollen grains. J:One normal and one small under-developed pollen grains. K-O are 4X. K:Normal pollen grains. L:Deformed pollen grains. M:Cracked pollen grains. N:Two dysplastic pollen grains. O:One normal and one small under-developed pollen grain
倍性 Ploidy | 花粉粒直径 Pollen grains diameter/cm | 正常花粉粒 Normal pollen grains | 异常花粉粒 Abnormal pollen grains | 正常花粉粒所占比例 Proportion of normal pollen grains /% |
---|---|---|---|---|
2X | 3.64±0.14c | 572 | 28 | 95.33 |
3X | 4.22±0.17b | 353 | 247 | 58.83 |
4X | 4.41±0.14a | 466 | 134 | 77.67 |
表5 不同倍性草棉的花粉粒统计
Table 5 Pollen grains statistics among different ploidy G. herbaceum
倍性 Ploidy | 花粉粒直径 Pollen grains diameter/cm | 正常花粉粒 Normal pollen grains | 异常花粉粒 Abnormal pollen grains | 正常花粉粒所占比例 Proportion of normal pollen grains /% |
---|---|---|---|---|
2X | 3.64±0.14c | 572 | 28 | 95.33 |
3X | 4.22±0.17b | 353 | 247 | 58.83 |
4X | 4.41±0.14a | 466 | 134 | 77.67 |
材料 Material | 3X | 4X | |||||||
---|---|---|---|---|---|---|---|---|---|
总计 Total | 相同 Same | 缺失 Missing | 新增 New | 总计 Total | 相同 Same | 缺失 Missing | 新增 New | ||
SRAP条带 SRAP bands | 297 | 240 | 20 | 37 | 299 | 229 | 31 | 39 | |
遗传比例 Genetic ratio/% | - | 80.81 | 6.73 | 12.46 | - | 76.59 | 10.37 | 13.04 |
表6 不同倍性草棉的SRAP多态性来源
Table 6 Resources of SRAP polymorphism among different ploidy G. herbaceum
材料 Material | 3X | 4X | |||||||
---|---|---|---|---|---|---|---|---|---|
总计 Total | 相同 Same | 缺失 Missing | 新增 New | 总计 Total | 相同 Same | 缺失 Missing | 新增 New | ||
SRAP条带 SRAP bands | 297 | 240 | 20 | 37 | 299 | 229 | 31 | 39 | |
遗传比例 Genetic ratio/% | - | 80.81 | 6.73 | 12.46 | - | 76.59 | 10.37 | 13.04 |
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