陆地棉与瑟伯氏棉远缘杂交后代性状鉴定与遗传分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0539

摘要/Abstract

摘要：

远缘杂交及多倍化是物种形成和种质创新的基础，将陆地棉与野生瑟伯氏棉进行远缘杂交及染色体加倍获得可育杂种。旨在将瑟伯氏棉的有益农艺性状转移到陆地棉中，拓宽陆地棉的遗传基础。以陆地棉为母本，瑟伯氏作父本进行远缘杂交合成杂种F₁，并进行染色体加倍，获得异源六倍体，对F₁及异源六倍体进行形态学、细胞学、生理生化及SSR分子标记鉴定。形态学鉴定结果表明，异源六倍体植株整体与杂种三倍体相似，介于双亲之间，异源六倍体的雄蕊多于亲本及F₁，有淡红色的花斑，与亲本及F₁都不同。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；杂种三倍体（F₁）和异源六倍体的减数分裂行为表明，二者的减数分裂均有正常和异常行为，三倍体中的多分体较多，而六倍体正常四分体明显增多。三倍体的多分体中，正常四分体占比为24.33%；六倍体的多分体中，正常四分体占比为82.17%，表明六倍体的育性在恢复。生理生化结果表明，酶的活性随不同世代倍性的增长而升高。SSR鉴定结果表明，三倍体杂种和异源六倍体既扩增出了父母本的条带，同时也扩增出了新的特异性条带，表明远缘杂交过程中产生了染色体重组现象。通过远缘杂交成功合成陆瑟杂种，通过染色体加倍成功获得了可育的异源六倍体。

关键词: 瑟伯氏棉, 远缘杂交, 异源六倍体, 形态性状鉴定, 生理生化鉴定, 细胞学鉴定, SSR

Abstract:

Distant hybridization and polyploidization are fundamental processes for the development of new species and germplasm innovation. In this study, we obtained fertile offsprings by interspecific distant hybridization and chromosome doubling between Gossypium hirsutum and Gossypium thurberi. The objective is to penetrate beneficial agronomic traits of G. thurberi into G. hirsutum and expand the genetic diversity of G. hirsutum. In this study, we obtained hybrid F₁ by synthesizing the female parent G. hirsutum with the male parent G. thurberi, and had allohexaploid cotton by chromosome doubling. And then we examined the morphology, cytology, physiology, biochemistry and SSR molecular markers of the F₁ and allohexaploid. The results of morphological identification showed that the allohexaploid plant was similar to the heterotriploid, with mid-parents'characters. The allohexaploid had more stamens than the parent and F₁had, with reddish spots, which were different from the parents and F₁. Cytological identification showed that stomatal density was in a decreasing trend with the increase of ploidy; while stomatal length, chloroplast number and pollen grain diameter all showed an increasing trend. The meiotic behavior of heterotriploids and allohexaploids revealed that both normal and abnormal tendencies, with more polyploids in triploids and significantly more normal tetrads in hexaploids. In triploids, the percentage of normal tetrads was 24.33%, while the percentage of normal tetrads was 82.17% in the polyploid hexaploids, indicating the recovery of hexaploid fertility. Physiological and biochemical results showed that the activities of enzymes increased with the increase of ploidy in different generations. The SSR molecular marker identification results revealed that distant hybridization led to chromosomal recombination in triploid hybrids and heterohexaploids, resulting in the amplification of both parental bands and new specific bands. The comprehensive results show that hybrids are successfully synthesized by distant hybridization and fertile heterohexaploids are successfully obtained by chromosome doubling, which provides valuable materials for cotton germplasm innovation and further genetic research.

Key words: G. thurberi, distant hybridization, allohexaploid, character identification, physiological and biochemical index, cytology identification, SSR

候林慧, 郑赟, 荣二花, 吴玉香. 陆地棉与瑟伯氏棉远缘杂交后代性状鉴定与遗传分析[J]. 生物技术通报, 2023, 39(12): 128-135.

HOU Lin-hui, ZHENG Yun, RONG Er-hua, WU Yu-xiang. Character Identification and Genetic Analysis of Progeny from Distant Hybrid Between Gossypium hirsutum and G. thurberi[J]. Biotechnology Bulletin, 2023, 39(12): 128-135.

图/表 13

表1 研究材料基本信息

Table 1 Basic information of research materials

类名 Classific name	染色体组 Genome	染色体数Number of chromosomes	来源 Source
陆地棉 G. hirsutum	AD₁	2n=4X=52	中国农科院棉花研究所 Cotton Research Institute, CAAS
瑟伯氏棉 G. thurberi	D₁	2n=2X=26	国家种质三亚野生棉圃 Sanya National Germplasm Wild Cotton Garden

表2 SSR引物序列信息

Table 2 SSR primers sequence information

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'）
BNL4108-F	TCCACCATTCCCGTAAATGT	BNL4108-R	TGGCCAAGTCATTAGGCTTT
BNL4053-F	TGAAGGCTTTGAAGCAAACA	BNL4053-R	AAGCAAGCACCAAGTTAGCC
NAU2026-F	GAATCTCGAAAACCCCATCT	NAU2026-R	ATTTGGAAGCGAAGTACCAG
NAU1355-F	ATCTGTTTACGCCACTCTCC	NAU1355-R	CCAGCCTTTGACATTTTTCT
NAU1169-F	GGGTAGTAGCTTTTATGATAGGG	NAU1169-R	CCATTCCTTCCCCTAATTCT
NAU1157-F	GAGTTTGGTTCTGGGTTGAG	NAU1157-R	GATCCTTTTCATCTCCTCCA
NAU1052-F	CGCAGATAAAGGATGGATTT	NAU1052-R	AGAGCTGGAGGACATAACAAA
NAU1042-F	CATGCAAATCCATGCTAGAG	NAU1042-R	GGTTTCTTTGGTGGTGAAAC
NAU1164-F	CCAACGCTAATTCTACCTCCT	NAU1164-R	GCGGGTAATTGTAGTACATGC
NBRI_GI1015-F	GAGTTTGGCTCCGAGTTGAG	NBRI_GI1015-R	GCGAGTGAACTCACCGATAA
NBRI_GM7-F	GTGATACCTGGAAAGATGTGA	NBRI_GM7-R	TCCTGATGCTGTTTTAGTCTT
NBRI_Gpd_21-F	GCAATTGTGGAAGAGAAGTAA	NBRI_Gpd_21-R	GGACCTCTGAGTCCATTTATT
NBRI_Gpd_85-F	CTCAAAAACCCCTTTAGTCTC	NBRI_Gpd_85-R	GAGTGAAAACCCGCAAAG

图1 不同世代花的解剖图和花蕊的比较 P1：陆地棉母本；P2：瑟伯氏棉父本；F1：杂种三倍体；M1：异源六倍体。下同

Fig. 1 Comparison of flower anatomy and stamens of different generations P1: G. hirsutum; P2: G. thurber; F1: hybrid; M1: allohexaploid. The same below

图2 不同世代花的形态比较

Fig. 2 Comparison of flower morphologies in different generations

图3 不同世代叶片的比较

Fig. 3 Comparison of the leaves in different generations

图4 不同世代减数分裂行为的对比 A-E为F1（A：单分体；B：二分体和2个微核；C：三分体；D：四分体；E：多分体）；F-J为M1（F：单分体；G：三分体；H：异常四分体；I：正常四分体；J：多分体）

Fig. 4 A comparison of meiosis behaviors in different generations（Bar=20 μm） A-E are F1（A: Monad. B: Dyad and two Micronucleus. C: Triad. D: Tetrad.E: Polyad）. F-J are M1（F: Monad. G: Triad. H: Abnormal Tetrad. I: Normal Tetrad.J: Polyad

表3 各世代减数分裂的对比

Table 3 Comparison of meiosis in different generations

倍性 Ploidy	单分体 Monad	二分体 Dyad	三分体 Triad	四分体 Tetrad	多分体 Polyad	总数 Total
F₁(3X)	73(12.17)	25(4.17)	38(6.33)	146(24.33)	318(53.00)	600
M₁(6X)	63(10.50)	15(2.50)	17(2.83)	493(82.17)	12(2.00)	600

图5 不同世代的气孔的比较

Fig. 5 Comparison of stomatas in different generations（Bar=20 μm）

表4 各世代气孔的比较

Table 4 Comparison of stomatas in different generations

材料（倍性） Material（Ploidy）	气孔密度 Stomatal density	气孔比 Porosity ratio	叶绿体数Number of chloroplasts
F₁（3X）	36.35±1.55a	1.28±0.17a	16.45±2.54a
M₁（6X）	26.45±1.89b	1.40±0.12b	26.31±2.30b

图6 各世代酶活性的比较小写字母表示在0.05水平差异显著，大写字母表示在0.01水平差异显著。下同

Fig. 6 Comparison of enzyme activities in different generations Lowercase letters indicate a significant difference at the 0.05 level, the capital letters indicate a significant difference at the 0.01 level. The same below

图7 各世代代谢产物含量的比较

Fig. 7 Comparison of metabolite contents in different generations

表5 不同世代的 SSR 多态性分析

Table 5 Analysis of SSR polymorphism in different generations

材料Material	3X			6X
材料Material	母本 Female parent	父本 Male parent	重组片段Recombination	母本 Female parent	父本 Male parent	重组片段Recombination
SSR条带SSR bands	34	21	4	34	21	6
遗传比例Genetic ratio/%	57.62	35.59	6.79	55.73	34.42	9.85

图8 SSR 分子标记在不同世代的扩增结果

Fig. 8 Amplification results of SSR markers in different generations

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