Selection and Character Identification for Autopolyploid Progenies of Gossypium herbaceum

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

Abstract

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

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.

Figures/Tables 15

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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