Development and Genetic Analysis of Two Nullisomic Lines（NC1 and NC2）in Natural Brassica napus

doi:10.13560/j.cnki.biotech.bull.1985.2022-0577

Abstract

Abstract:

The nullisomic（2（n-1））line losing a pair of chromosome from the normal organism chromosome set has been recognized as a rare and important genetic resources for revealing the missing chromosome. Herein, after screening the offsprings from the cross and backcross between Brassica napus and the restituted B. rapa（RBR, 2n =20, AⁿAⁿ）, we used the integration of specific C chromosome molecular markers and fluorescence in situ hybridization technology to screen two nullisomic lines of chromosome C1（NC1）and chromosome C2（NC2）in natural B. napus（2n =38, AⁿAⁿCⁿCⁿ）for the first time. Both two nullisomic lines had 18 bivalents at diakinesis and a dominant segregation of 18:18 at meiosis anaphase I（MA I）in the pollen mother cells. However, some lagging chromosomes were observed at MA I and MA II in both two lines. Compared to their parental B. napus, the two lines presented significantly smaller phenotype at biomass, pollen stainability, and seed setting, also showed some specific features. For example, the NC1 had the leaves with obvious burrs, and NC2 showed significantly early flowering time of two months earlier than its parental B. napus, indicating some genes inhibited such characters were locating on the missing chromosome. The two nullisomic lines effectively reduce the complexity of B. napus genome, which can be used to map the target traits, or to verify the gene function, and thus to provide convenience for future genetic analysis of B. napus.

Key words: nullisomic, Brassica napus, fluorescence in situ hybridization, germplasm resources

CAI Meng-xian, GAO Zuo-min, HU Li-juan, FENG Qun, WANG Hong-cheng, ZHU Bin. Development and Genetic Analysis of Two Nullisomic Lines（NC1 and NC2）in Natural Brassica napus[J]. Biotechnology Bulletin, 2023, 39(3): 81-88.

Figures/Tables 5

Fig. 1 Construction process of nullisomic of C chromosome in natural B. napus

Fig. 2 Identification of C chromosome nullisomic-specific markers in NC1 and NC2 M: Marker. The mark sizes were 2 000, 1 500, 1 000, 750, 500, 250 and 100 bp. Oro is B. napus. NC1 and NC2 refer to the nullisomic of chromosome C in B. napus. C1c, C2g, …C9e are chromosomal specific primers for C genome

Fig. 3 FISH analysis of somatic cells and pollen mother cells of B. napus “Oro”，NC1，and NC2 A: “Oro” mitotic cells with 38 chromosomes（blue）, including 18 C subgenomic chromosomes（red）. B-C: NC1, NC2 mitotic cells with 36 chromosomes（blue）, including 16 C subgenomic chromosomes（red）. D: C2 showing 8 red hybridization signals at diakinesis. E-F: The PMC of NC2 and NC1 show equal separation of 16 hybridization signals at anaphase I. Blue indicates DAPI staining and red indicates BAC BoB014O06 probe signal. Bar: 10 μm

Fig. 4 Phenotype and pollen fertility of parental B. napus “Oro”，NC1，and NC2 A: Parental B. napus “Oro”. B: NC1 plants. The enlargement shows that blade, vein, and leaf margin cover with burrs. C: NC2 plants. The enlargement shows that NC2 plant has early squaring stage. E-F: The pollen fertility of “Oro”, NC1, and NC2

Fig. 5 Observation of chromosome behavior of at meiosis of NC1 and NC2 in B. napus A-D: Somatic chromosome number（2n=36）of NC1, PMCs of NC1 at DK, AP I, and AP II, respectively. E-H: Somatic chromosome number（2n=36）of NC2, PMCs of NC2 at DK, AP I, and AP II, respectively. I: the PMC of nullisomic show normal pairing at metaphase I. J, K: The PMCs of NC1 and NC2 with delaying chromosome at AP I. L: The PMC of NC2 with delaying chromosome at AP II. The arrows show the delaying chromosomes. Bar: 10 μm

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