草棉同源多倍体后代筛选及性状鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-1288

摘要/Abstract

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。

关键词: 草棉, 同源多倍体, 性状鉴定, 分子标记

Abstract:

Polyploidization is an important way of speciation and plant breeding. In this study,the autopolyploid of Gossypium herbaceum progenies S₁in the early stage were used as materials,and flow cytometry,morphological,cytological and SRAP molecular marker were used to identify them. The results of flow cytometry for ploidy identification indicated that the fluorescence peak value of diploids was used as a reference,among eight polyploid progenies,there were 5 triploid plants and 3 tetraploid plants,respectively. The results of morphological identification showed that compared with polyploids,diploid plants were taller with thinner stalks,as well as thinner,smaller and emerald green leaves,and flowers were smaller. Compared with diploids,triploids and tetraploids all presented shorter and thicker stalks,as well as thicker,shrunken,larger and dark green leaves,and flowers were larger. The results of cytological identification demonstrated that with the increase of ploidy level,it showed a downward trend for stomatal density,while it all showed an upward trend for the length of stomatal,the number of chloroplasts and the diameter of pollen grains. There were normal and abnormal behaviors during meiosis in pollen mother cell for all diploids,triploids and tetraploids,including monad（0.17%,2.50% and 2.17%）,dyad（0.33%,0.67% and 0）,triad（4.17%,10.00% and 2.33%）,normal tetrad（94.83%,54.00% and 73.67%）,abnormal tetrad（0.17%,0.33% and 0.83%）and polyad（0.33%,32.50% and 21.00%）respectively. The abnormal polyad could not produce normal pollen grains,thus the proportion of normal pollen grains in the diploids,triploids and tetraploids were 95.33%,58.83% and 77.67% respectively. The results of SRAP molecular marker identification revealed that there were three polymorphism bands amplified in triploids and tetraploids：including completely consistent with diploids bands,lost bands and new specific bands when comparing with diploids. The genetic proportion of three band types in the triploids were 80.81%,6.73% and 12.46%,and that of the tetraploids were 76.59%,10.37% and 13.04%,respectively. This confirmed the authenticity of polyploidy at the molecular level.

Key words: Gossypium herbaceum, autopolyploid, character identification, molecular marker

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.

图/表 15

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

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

图1 草棉二倍体（A）、三倍体（B）、四倍体（C）植株比较

Fig. 1 Comparison of plant among diploid（A）,triploid（B）and tetraploid（C）G. herbaceum

图2 不同倍性草棉的叶片形态比较（标尺：20 mm）

Fig. 2 Leaf morphology comparison among different ploidy G. herbaceum（Ruler：20 mm）

图3 草棉二倍体（A）、三倍体（B）、四倍体（C）花的形态比较

Fig. 3 Comparison of flower morphology among diploid（A）,triploid（B）and tetraploid（C）G. herba-ceum

图4 二倍体和四倍体草棉的花朵及雌雄蕊比较（标尺：20 mm）

Fig. 4 Flowers,pistil and stamens comparison between diploid and tetraploid G. herbaceum（Ruler：20 mm）

图5 不同倍性草棉的花的分解图（标尺：20 mm）

Fig. 5 Decomposition map of flowers among different ploidy G. herbaceum（Ruler：20 mm）

图6 不同倍性草棉的叶片气孔比较（标尺：20 μm）

Fig. 6 Leaf stomatal comparison among different ploidy G. herbaceum（Ruler：20 μm）

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

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

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

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
材料 Material	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

图9 SRAP引物在不同倍性草棉中的扩增结果

Fig. 9 Amplification results of SRAP primers among diff-erent ploidy G. herbaceum A：Me 2/Em 2;B：Me 4/Em 1;C：Me 4/Em 2

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