异源三倍体普通烟草（SST）减数分裂期的分子细胞学研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0668

摘要/Abstract

摘要：

烟草属野生种是栽培烟草品种改良的重要种质来源，通过对烟草种间杂种的创建及其减数分裂期分子细胞学分析，为后续优异野生种质的有效转移和利用提供基础。以林烟草Q67（Nicotiana sylvestris）和普通烟草K326（Nicotiana tabacum L. cv. K326）为父母本创建异源三倍体种间杂种，采取常规细胞学观察、基因组原位杂交及实时定量PCR等方法对其染色体组成、减数分裂时期染色体行为及相关基因的表达进行调查。结果表明，68株种间杂种体染色体条数为36条，染色体组成为SST，其整体表型兼具双亲特征。种间杂种的花粉母细胞减数分裂期异常细胞占比为29.26%，主要异常行为为减数分裂I和II中期的T染色体组来源的染色体滞后，落后染色体条数为1-3条，其中，减数分裂中期I和中期II落后类型最多的分别为落后1条T染色体和落后2条T染色体的细胞，分别占该时期携带落后染色体细胞总量的29.09%和30.88%；减数分裂I后期和减数分裂II后期及末期的异常类型主要为不均等分离、落后染色体和微核，其中，减数分裂I后期统计到多种异常分离比，但分离后染色体组成仍接近2∶1（S∶T）。相较于对照K326，在减数分裂I前期，来源于S染色体组的CDKA:5319表达显著上调和CYCA:6720表达极显著上调；在减数分裂I中期至减数分裂II末期，CENH3和来源于T基因组的CYCA:0111表达显著上调，来源于S基因组的CDKA:5319和CYCA:6720均呈极显著上调。表明转录水平上减数分裂过程的紊乱，相关基因的表达变化可能是减数分裂异常的原因。

关键词: 烟草, 种间杂种, 育性, 减数分裂, 基因组原位杂交

Abstract:

The genus Nicotiana is an important germplasm source for cultivated tobacco varieties improvement, the creation of tobacco interspecific hybrids and their molecular cytological analysis during the meiotic phase will provide the basis for the subsequent efficient transfer and utilization of excellent wild germplasm. Allotriploid interspecific hybrids were created from the parents of Nicotiana sylvestris Q67 and common Nicotiana tabacum L. cv. K326. Their chromosome composition, chromosomal behavior during the meiosis and expression of related genes were investigated by routine cytological observation, genomic in situ hybridization and real-time quantitative PCR. In this study, 68 allotriploid hybrids（SST）with 36 chromosomes were identified by phenotypic and cytological analysis from crossing Nicotiana sylvestris with Nicotiana tabacum. The results of general cytology and genomic in situ hybridization showed that 29.26% of the pollen mother cells of interspecific hybrids were abnormal in meiosis. The main abnormal behavior was the backward chromosomes from the T chromosome group in metaphase I and II, with 1-3 lagging chromosomes. The cells with the most lagging types in metaphase I and II were the cells with 1 and 2 lagging T chromosomes respectively. They accounted for 29.09% and 30.88% of the total number of cells carrying backward chromosomes, respectively. The abnormal types of anaphase I, anaphase II and telophase II were mainly unequal segregation, backward chromosomes and micronucleus. In anaphase I, a variety of abnormal segregation ratios were counted, but the chromosome composition after segregation was still close to 2∶1（S∶T）. The results of RT-qPCR showed that in prophase I, except for the significant up-regulation of CDKA:5319 and CYCA:6720 from S chromosome group, the expressions of other genes were significantly down regulated. From the metaphase I to the telophase II, compared with the control, except that MIS12 and CDKA:0239 from T chromosome group had no significant changes, other genes（CENH3, CDKA:5319, CYCA:0111 and CYCA:6720）were significantly or extremely significantly up-regulated, indicating that the disorder expression of meiosis related genes may be causing the abnormality of meiosis.

Key words: tobacco, interspecific hybrids, fertility, meiosis, genomic in situ hybridization

汪格格, 邱诗蕊, 张琳晗, 杨国伟, 徐小云, 汪爱羚, 曾淑华, 刘雅洁. 异源三倍体普通烟草（SST）减数分裂期的分子细胞学研究[J]. 生物技术通报, 2023, 39(2): 183-192.

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.

图/表 14

表1 RT-qPCR所用引物

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

表2 杂交后代染色体条数统计

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

图1 种间杂种染色体计数

Fig. 1 Chromosome number of the allotriploid hybrids Bar=1 μm

图2 种间杂种体细胞原位杂交 a：蓝色为 DAPI染色；b：红色信号来自生物素标记的S基因组探针；c：杂交结果合成图。Bars=5 μm

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

图3 种间杂种及其亲本形态学比较 A、B：亲本及三倍体苗期、花期，bars=10 cm；C：亲本及三倍体花的形态，bars=2 cm。a：K326苗期；b：林烟草苗期；c：F1苗期；d-f：图中所示花的形态从左到右依次为K326、F1、林烟草；g：K326花期；h：林烟草花期；i：F1花期

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

表3 种间杂种及其亲本植株形态特征比较

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

表4 种间杂种及其亲本花粉育性统计

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

图4 种间杂种及其亲本花粉育性 a：F1；b：K326；c：林烟草；bars=100 μm

Fig. 4 Pollen fertility of the allotriploid and parents a: F1; b: K326; c: Nicotiana sylvestris; bars=100 μm

图5 种间杂种、K326减数分裂 a：K326偶线期；b：F1偶线期；d：F1中I；e：F1后I；f：F1中II；g：F1末期II；l：K326中I；m：K326后I；n：K326中II；o：K326后期II。黑框内放大部分置于该图右下，黑色箭头指向未联会染色体，白色箭头指向正常联会染色体

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

图6 种间杂种减数分裂原位杂交探针为生物素标记的S基因组，呈现红色信号。a-c：中期I；d-f：后期I；g-i：中期 II；j-l：末期II。箭头指向落后染色体，bars=5 μm

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

表5 种间杂种SST减数分裂进程的异常染色体行为

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

表6 种间杂种SST减数分裂进程的染色体具体行为

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

表7 落后染色体来源的 GISH分析结果

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
株系 Line	减数分裂时期 Meiotic phase	细胞数 Number of cells observed	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
F₁	减II中期	68	10	14	12	7	21	0	0	0	1	3

图7 减数分裂相关基因在异源三倍体及亲本K326中的差异表达 A：表示取材时间为减数第一次分裂前期的基因表达；B：表示取材时期为减数分裂I中期至减数分裂II末期的基因表达；S、T表示分别对S和T染色体组的基因设计的引物；*表示差异显著（P<0.05），**表示差异极显著（P<0.01）

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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