Exploitation and Genetic Identification of Triploid Plants from Seedling Populations of Orah Mandarin（Citrus reticulata）

doi:10.16420/j.issn.0513-353x.2023-0522

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (10): 2231-2242.doi: 10.16420/j.issn.0513-353x.2023-0522

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles

Exploitation and Genetic Identification of Triploid Plants from Seedling Populations of Orah Mandarin（Citrus reticulata）

FENG Yisi¹, TIAN Xiaoyu¹, YANG Chongshan¹, CHEN Xiangling³, DENG Xiuxin¹^,², XIE Kaidong¹, GUO Wenwu¹^,², XIE Zongzhou¹, CHAI Lijun¹, YE Junli¹^,^**()

¹ National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops，College of Horticulture and Forestry，Huazhong Agricultural University，Wuhan 430070，China
² Hubei Hongshan Laboratory，Wuhan 430070，China
³ Horticultural Research Institute，Guangxi Academy of Agricultural Sciences，Nanning 530007，China

Received:2024-07-26 Revised:2024-09-04 Online:2024-10-25 Published:2024-10-21
Contact: YE Junli

Abstract

Abstract:

Discovering natural triploids from diploid seedling offspring is an effective mehod to cultivate new seedless citrus cultivar. In this study，the small plump seeds of Orah were germinated and sowed，and the ploidy of seedling offsprings was identified by morphological screening，flow cytometry and chromosome counting. A total of 98 triploid lines of Orah seedlings were obtained. Two pairs of SSR primers with polymorphism between the female parent and its candidate male parent were used to analyze the hybrid origin of triploid progenies. It was found that only the amplification product of Orah mandarin could be detected in the triploid progenies. Further genetic analysis was performed using 36 pairs of S-RNase primers. Results showed that the S genotype S₁₁/S_Mof Orah mandarin was detected in all triploid progenies，and the S genotype S₂₂ of the candidate male parent Shatangju occurred in two lines，presumed that these two lines were triploid progenies produced by the hybridization of Orah and Shatangju，and were formed by fertilization of 2n female gametes. The remaining lines might be autotriploids produced by selfing of Orah. Compared with the diploid seedlings，the triploid seedlings of the same age grew slowly，the internodes became shorter，the dwarfing was obvious，the main roots were short and thick，the fibrous roots were less，the leaves were dark green and thicker. Under the same cultivation and management conditions，the expression of multiple stress response related genes（CitCOMT，CitGRAS，CitERF4 and CitERF9）in the leaves of allotriploid plants was significantly up-regulated compared with diploid and autotriploid plants.

Key words: Orah, seedling progeny, triploid, genetic analysis

FENG Yisi, TIAN Xiaoyu, YANG Chongshan, CHEN Xiangling, DENG Xiuxin, XIE Kaidong, GUO Wenwu, XIE Zongzhou, CHAI Lijun, YE Junli. Exploitation and Genetic Identification of Triploid Plants from Seedling Populations of Orah Mandarin（Citrus reticulata）[J]. Acta Horticulturae Sinica, 2024, 51(10): 2231-2242.

Figures/Tables 8

Table 1 Primer sequences for qPCR analysis

基因 Gene	NCBI编号 Accession ID	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
COMT	XM_052435554	F：TTGAAACCAGCAAGGGATGAG	R：GCGCCGGAAGCGAGTT
PUB19	XM_006483767	F：GGGTCGCCGGGTACTGA	R：TTGACGGAGAAATGGATTCACA
ERF4	XM_006464742	F：TCCCTCCTCCACCTGAAGTG	R：CGCCGTACACGTCAGCATTA
ERF9	XM_006467686	F：TGGTGCCGGTGGAGGAT	R：CCACAGGCTGGAAGGGAAA
TCF1	XM_052439239	F：GGCTCTATGGACAGTGTGGG	R：GTCACCATCGCTGGAAATGC
ERF6	XM_006477063	F：AGGCGATAACGGCGACA	R：AAAGCCAAGGCGGTGAA
GRAS	XM_006475224	F：ATGTCGCTCCACCCTCT	R：TACTGCCGCATTCCTCC
Actin	GU911361	F：CCAAGCAGCATGAAGATCAA	R：ATCTGCTGGAAGGTGCTGAG

Fig. 1 Fruit（a），seed（b）and seedling（c）of Orah mandarin

Fig. 2 Leaf（a），root（b）and leaf oil gland（c，d）of diploid（2x）and suspected triploid（3x）of Orah mandarin

Fig. 3 Ploidy identification by flow cytometry（a，b）and root tip chromosome count（c）of suspected triploid seedlings of Orah mandarin a：Diploid；b：Triploid；c：Triploid root tip chromosome number 2n = 3x = 27.

Fig. 4 Capillary electrophoresis nucleic acid analysis of female parent Orah mandarin（♀），candidate male parent（♂）and triploid plant（3x） a：Primer Csin0342；b：Primer Csin0087. M：Orah mandarin；G：Murcott；H：Hongjiangcheng；B：Baixincheng.

Fig. 5 The specific amplification of S-RNase gene of Orah mandarin（♀，M），triploid plants（3x，1-93）and their candidate male parent（♂）

Fig. 6 Comparison of leaf，roots，branch thorns and internode of triploid（3x）and diploid（2x）plants of Orah mandarin t-test，* P < 0.05，** P < 0.01.

Fig. 7 Differential expression analysis of stress related genes in leaves of Orah mandarin diploid，autotriploid and allotriploid by qPCR One-way ANOVA. n = 3，P < 0.05.

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Exploitation and Genetic Identification of Triploid Plants from Seedling Populations of Orah Mandarin（Citrus reticulata）

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