美洲黑杨种质材料倍性鉴定

doi:10.3969/j.issn.1000-2006.201812022

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南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (5): 51-58.doi: 10.3969/j.issn.1000-2006.201812022

美洲黑杨种质材料倍性鉴定

李伟强(), 戴晓港, 李小平, 李淑娴^*()

南京林业大学,南方现代林业协同创新中心, 江苏省杨树品种改良与种质创新重点实验室, 江苏南京 210037

收稿日期:2018-12-12 修回日期:2019-04-13 出版日期:2019-10-08 发布日期:2019-10-08
通讯作者: 李淑娴
基金资助:
国家自然科学基金面上项目(31570662);江苏高校优势学科建设工程资助项目(PAPD)

Assessment of ploidy levels in the Populus deltoides Marsh. germplasm

LI Weiqiang(), DAI Xiaogang, LI Xiaoping, LI Shuxian^*()

Co-Innovation Center for the Sustainable Forestry in Southern China, Jiangsu Key Laboratory for Poplar Germplasm Enhancement and Variety Improvement,Nanjing Forestry University, Nanjing 210037, China

Received:2018-12-12 Revised:2019-04-13 Online:2019-10-08 Published:2019-10-08
Contact: LI Shuxian

摘要/Abstract

摘要：

【目的】对美洲黑杨种质资源库保存的种质材料进行倍性评估,并对候选多倍体进行倍性鉴定。【方法】利用9对用于多倍体鉴定的SSR引物检测448份美洲黑杨种质材料,根据每个引物扩增位点的等位基因数目对种质材料的倍性进行评估,基于初步鉴定结果筛选出候选多倍体材料。在此基础上,使用流式细胞仪(FCM)对候选多倍体种质材料的倍性进行鉴定。【结果】分子标记基因型分型统计结果显示,9个SSR位点共扩增出129条多态性条带,每个位点的等位基因数为5(Ploidp-10)~25(Ploidp-07)个,平均每个位点的等位基因数为14.33个。9个SSR位点的多态性信息含量(PIC)变动范围为0.46~0.93,平均PIC值为0.76。在检测的448份美洲黑杨种质材料中,440份美洲黑杨种质材料在9个引物位点上扩增出的等位基因数不多于2个;有8份种质材料在2个引物位点上扩增出了3个不同的等位基因,其中编号为1346、16-42、17-49、18-25的4份种质材料在Ploidp-02引物位点扩增出了3个等位基因,而编号为5-43、5-45、15-14、19-37的另外4份种质材料在Ploidp-04引物位点上也扩增出了3个等位基因。分子检测结果表明:该8份种质材料在对应的染色体区域发生了遗传物质增加现象,为候选三倍体种质材料。用FCM对8份三倍体候选种质材料进行倍性鉴定发现,编号为1346的种质材料为三倍体,其余7份种质材料疑似为发生局部染色片段增加的候选非整倍体。【结论】9对SSR分子标记均具有较高的多态性信息含量,可用于美洲黑杨种质资源多倍体初步筛选,结合FCM检测,可在大量样品中快速筛选出多倍体种质材料。

关键词: 美洲黑杨, 微卫星标记, 流式细胞仪, 倍性, 多倍体

Abstract:

【Objective】 Polyploid breeding is an important approach to generate elite varieties of forest trees. In this study, we assessed ploidy levels in germplasm collections of Populus deltoides Marsh. to identify candidate polyploids for breeding.【Method】 In total, 448 P. deltoides stands were examined with 9 SSR primer pairs, and the ploidy levels of these stands were assessed based on the number of alleles detected per locus. Subsequently, the polyploid candidates based on the preliminary identification results were examined by flow cytometry.【Result】 By SSR genotyping, 129 polymorphic bands were generated at 9 SSR loci, ranging from 5 (Ploidp-10) to 25 (Ploidp-07) alleles per locus, with a mean of 14.33. The polymorphic information content (PIC) of the 9 microsatellite markers ranged from 0.46 to 0.93, with an average value of 0.76. Among the 448 stands evaluated, no more than two alleles were amplified at each of the 9 microsatellite loci in 440 stands. However, for eight stands, three alleles were obtained at two of the microsatellite loci. In particular, for clones 1346, 16-42, 17-49 and 18-25, three alleles were obtained using the Ploidp-02 primer, and for clones 5-43, 5-45, 15-14 and 19-37, three alleles were obtained using the Ploidp-04 primer. An additional chromosomal segment was detected at the corresponding genetic loci in these eight stands. Therefore, these stands were candidate triploids. A flow cytometry analysis confirmed that clone 1346 was triploid and the remaining seven stands were suspected aneuploid candidates with additional local chromosome segments.【Conclusion】 The nine microsatellite markers employed in this study have high polymorphic information content and are suitable for screening polyploid candidates fromP. deltoides germplasms, and, combined with flow cytometry, we can rapidly identify polyploids from a large number of individuals.

Key words: Populus deltoides Marsh., microsatellite marker, flow cytometry, ploidy level, polyploid

中图分类号:

S722
S792

李伟强,戴晓港,李小平,等. 美洲黑杨种质材料倍性鉴定[J]. 南京林业大学学报（自然科学版）, 2019, 43(5): 51-58.

LI Weiqiang, DAI Xiaogang, LI Xiaoping, LI Shuxian. Assessment of ploidy levels in the Populus deltoides Marsh. germplasm[J].Journal of Nanjing Forestry University (Natural Science Edition), 2019, 43(5): 51-58.DOI: 10.3969/j.issn.1000-2006.201812022.

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地点 location	家系编号 family No.	纬度 latitude (N)	经度 longitude (W)	无性系数量 number of clones
德克萨斯州 State of Texas	1	30°40'	96°30'	2
	7	30°40'	96°30'	1
	22	30°20'	96°30'	3
	24	30°20'	96°30'	2
	28	30°00'	96°10'	2
	31	29°50'	96°00'	5
	35	29°30'	96°00'	1
	36	29°10'	96°10'	2
	40	29°10'	96°10'	5
	47	29°30'	95°40'	3
	48	29°30'	95°40'	4
路易斯安那州 State of Louisiana	52	30°00'	91°30'	1
	60	30°00'	91°30'	3
	62	30°10'	91°30'	1
	63	30°10'	91°30'	16
	66	30°30'	91°30'	24
	70	30°30'	91°30'	3
	72	30°40'	91°30'	3
	74	31°00'	91°00'	1
	77	30°50'	91°00'	6
	78	30°50'	91°00'	6
	80	30°50'	91°00'	5
	81	30°00'	91°50'	37
	86	32°00'	91°20'	36
	87	32°00'	91°20'	7
密西西比州 State of Mississippi	88	32°50'	91°00'	8
	89	32°50'	91°00'	19
	90	32°50'	91°00'	2
	93	33°00'	91°00'	12
	94	33°00'	91°00'	45
	95	33°00'	91°00'	48
	96	33°00'	91°00'	21
	97	33°00'	91°00'	1
	100	33°00'	91°00'	3
	101	33°00'	91°00'	4
	102	33°00'	91°00'	39
	103	33°00'	91°00'	65
	104	33°00'	91°00'	2

SSR引物 SSR primer	检测到的等位基因数 the detected allele number	区分的基因型数 the identified genotype number	多态性信息含量 PIC
Ploidp-01	6.00	15.00	0.52
Ploidp-02	14.00	54.00	0.78
Ploidp-03	11.00	40.00	0.79
Ploidp-04	23.00	123.00	0.93
Ploidp-05	14.00	47.00	0.79
Ploidp-06	8.00	23.00	0.74
Ploidp-07	25.00	123.00	0.93
Ploidp-08	23.00	93.00	0.91
Ploidp-10	5.00	9.00	0.46
平均mean	14.33	58.56	0.76

样品编号 sample No.	C_G0+ C_G1	比率^* ratio	倍性 ploidy level	变异系数/% CV
美洲黑杨2-2(CK)	14 511	1.00	2	4.83
1346(104)	22 067	1.52	3	3.81
16-42(102)	14 651	1.00	2	4.91
17-49(89)	14 528	1.00	2	4.28
18-25(95)	14 550	1.00	2	4.76
5-43(103)	14 618	1.01	2	3.58
5-45(86)	14 571	1.00	2	4.46
15-14(47)	14 782	1.02	2	4.93
19-37(104)	14 432	0.99	2	4.80

样品编号 sample No.	C_G0/C_G1	比率 ratio	倍性 ploidy level	变异系数/% CV	样品编号 sample No.	C_G0/C_G1	比率 ratio	倍性 ploidy level	变异系数/% CV
美洲黑杨2-2(CK)	14 521	1.00	2	4.90	10-19(103)	14 640	1.01	2	4.84
1-12(52)	14 613	1.01	2	4.66	10-28(103)	14 532	1.00	2	3.79
1-19(100)	14 637	1.01	2	3.52	10-44(88)	14 535	1.00	2	4.98
1-30(70)	14 549	1.00	2	4.27	10-50(86)	14 575	1.00	2	4.79
1-37(80)	14 527	1.00	2	4.93	10-58(94)	14 449	1.00	2	3.57
1-40(36)	14 618	1.01	2	4.88	11-6(102)	14 647	1.01	2	4.96
1-49(78)	14 601	1.01	2	4.48	1-56(31)	14 578	1.00	2	4.28
样品编号 sample No.	C_G0/C_G1	比率 ratio	倍性 ploidy level	变异系数/% CV	样品编号 sample No.	C_G0/C_G1	比率 ratio	倍性 ploidy level	变异系数/% CV
2-9(47)	14 530	1.00	2	4.31	11-20(103)	14 538	1.00	2	4.17
2-11(101)	14 569	1.00	2	4.15	11-45(94)	14 516	1.00	2	4.83
2-28(62)	14 763	1.02	2	4.54	12-18(102)	14 571	1.00	2	3.82
2-40(22)	14 774	1.02	2	4.95	12-29(96)	14 432	0.99	2	4.21
4-26(95)	14 782	1.02	2	4.07	12-45(103)	14 636	1.01	2	4.75
5-26(95)	14 562	1.00	2	4.22	12-56(86)	14 541	1.00	2	4.464
6-2(102)	14 625	1.01	2	4.55	13-18(81)	14 548	1.00	2	4.97
6-46(103)	14 499	1.00	2	3.71	13-27(94)	14 775	1.02	2	4.30
6-60(94)	14 683	1.01	2	4.86	13-32(94)	14 415	0.99	2	3.89
7-37(95)	14 649	1.01	2	3.88	13-44(66)	14 535	1.00	2	4.91
7-55(81)	14 524	1.00	2	4.35	13-60(81)	14 640	1.01	2	4.70
8-12(89)	14 617	1.01	2	4.71	15-6(96)	14 611	1.01	2	3.56
8-21(86)	14 791	1.01	2	4.82	15-11(28)	14 713	1.01	2	4.81
8-34(93)	14 435	0.99	2	3.91	15-18(86)	14 589	1.00	2	4.65
8-41(95)	14 635	1.01	2	4.93	15-24(95)	14 497	1.00	2	4.33
8-49(96)	14 523	1.00	2	4.71	15-40(87)	14 615	1.01	2	3.99
9-1(72)	14 624	1.01	2	4.43	15-48(95)	14 601	1.01	2	4.74
9-35(86)	14 589	1.00	2	4.94	15-54(96)	14 471	1.00	2	4.23

美洲黑杨种质材料倍性鉴定

Assessment of ploidy levels in the Populus deltoides Marsh. germplasm

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