Molecular Cytology at Meiosis in Allotriploid Nicotiana tabacum（SST）

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

Abstract

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

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.

Figures/Tables 14

References 25

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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

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

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

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

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