Growth and genetic diversity analysis of clones screened by phenotypical resistant to pine wilt disease in Pinus massoniana

doi:10.12302/j.issn.1000-2006.202103013

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (5): 109-118.doi: 10.12302/j.issn.1000-2006.202103013

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Growth and genetic diversity analysis of clones screened by phenotypical resistant to pine wilt disease in Pinus massoniana

GAO Jingbin¹^,²(), XU Liuyi²^,³, YE Jianren⁴^,^*()

1. Anhui Vocational & Technical College of Forestry, Hefei 230031, China
2. State Key Laboratory of the National Forestry and Grassland Administration for Pine Wood Nematode Disease Prevention and Control Technology, Hefei 230031, China
3. Anhui Academy of Forestry, Hefei 230001, China
4. Nanjing Forestry University, Nanjing 210037, China

Received:2021-03-04 Accepted:2021-08-28 Online:2021-09-30 Published:2021-09-30
Contact: YE Jianren E-mail:jingbingao@163.com;jrye@njfu.edu.cn

Abstract

Abstract:

【Objective】 Pine wilt disease caused by pine wood nematode is one of the most deadly forest quarantine diseases in the world. Screening and innovation of resistance genetic resources is an essential basis for breeding for pine wood nematode disease resistance of Pinus massoniana. The remained genetic resources in the severely disease-affected areas in natural distribution of masson pine, may be an important and potential genetic basis for resistant breeding, which is worth further exploration, systematic evaluation, exploring and utilization. The aim of this study was to comprehensively evaluate the genetic resources of masson pine with phenotypical resistance to pine nematode and its potential value in resistance breeding by analyzing the genetic diversity, growth traits and surveil rate of the 110 selected clones.【Method】 All 1 201 primary-selected clones of masson pine were screened by inoculated pine wood nematode, and 110 re-selected clones with pine wood nematode phenotypic resistance were involved in the evaluation of genetic diversity [by random amplified polymorphic DNA markers (RAPD)], and growth performance. From 14 pairs of SCAR markers, two pairs of primers with strong versatility and high polymorphic loci were selected for MuPS (Multiplex PCR of SCAR Markers) amplification and genetic polymorphic loci detection. The potential value of these masson’s pine genetic resources in resistance breeding was evaluated based on the genetic diversity parameters (estimated by Popgene 32 software) of the 79 clones with unique MuPS type, combined with the traits of single-tree volume and survival rate. 【Result】 There were 110 clones (from 81 families out of primary ) survived among the 1 201 primary selected clones (selected from the 251 primary families), after the inoculation with pine wood nematode. And 92 MuPS types were amplified from the 110 cloned by two pairs of primers, and 79 of the 110 clones were accurately identified by a unique MuPS type (71.81%). Based on the each unique MuPS type’s information of the 79 clones, the average number of effective alleles (N_e) at all loci was 1.508 1, the Nei’s gene diversity (H) was 0.302 3, and the Shannon’s diversity index (I) was 0.459 1. The N_e, H, I for the re-selected population was variated from 1.012 8 to 1.998 1, 0.012 6 to 0.499 5, and 0.038 5 to 0.692 7, respectively. There were 83 clones well survived out of the 110 re-selected clones after pine wood nematode inoculation. There were 83 clones well survived out of the 110 re-selected clones,after the pine wood nematode inoculation. The tree height, DBH and tree volume growth was ranged from 4.6 to 10.7 m, 6.7 to 21.7 cm, and 0.012 3 to 0.189 4 m ³, respectively. And the tree volume of the best performed clone (clone Xiu 3-3, 0.189 4 m³) was more than 15-fold over the lowest clone (clone Guang 27-2, 0.012 3 m³). 【Conclusion】 There were 110 clone among the 1 201 primary clones of masson’s pine, showed significant resistance to pine wood nematode disease after manual inoculation in field, which was around 9% of the primary selected clones. A significant variation/differentiation on tree volume growth was also found among the 110 re-selected clones with phenotypic pine resistance to wilt disease, which may indicated that it was possible to improve tree volume growth based on the disease resistance improvement background. It was worth to notice that, a higher genetic diversity was still existed in the reserved populations in this study, even though ninety percentage of the primary selected clones were eliminated through the disease resistance screening in this study. In conclusion, we believe that these precious genetic resources through resistance screening, have obvious potential value in P. massoniana growth and nematode disease resistance breeding.

Key words: Pinus massoniana, MuPS type, pine wilt disease resistance, genetic diversity, genetic improvement

CLC Number:

GAO Jingbin, XU Liuyi, YE Jianren. Growth and genetic diversity analysis of clones screened by phenotypical resistant to pine wilt disease in Pinus massoniana[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(5): 109-118.

Figures/Tables 7

Table 1

Fig.1

Table 2

The MuPS patterns of 110 clones of P. massoniana"

无性系 clone	MuPS型 MuPS type	无性系 clone	MuPS型 MuPS type	无性系 clone	MuPS型 MuPS type
广1-3	11001110100001	广51-1	00110110000101	休32-4	10011010110001
广2-5	10101011000001	广51-4	10001110000100	休33-2	10001010000001
广3-2	10001010100001	广52-1	10001110100101	休35-2	01101010000000
广3-4	10100010100101	广52-2	00001010100001	和1-2-5	10001011100101
广4-3	00000111000101	广54-1	00101010010001	和2-2-3	11101111000001
广5-4	10001111000101	广54-2	00000010000001	和4-1	00001011000100
广5-5	00001111000101	广55-2	11001010000001	和4-3	10101010010101
广6-5	10001010000100	广56-3	00001010100001	和5-4	11001111010101
广11-3	01101010010101	广57-1	10001011100001	和9-5	11000110000011
广12-1	10001001000001	广57-4	10011110100101	和10-3	10000010000001
广15-3	01110111010101	广59-3	00001111000001	和12-1	00001110000000
广16-2	10101110000001	广61-3	10001011000001	和12-4	10001111000001
广17-3	10001100000111	广61-4	10001010000101	和12-5	10000111101000
广18-2	00001110000001	广62-2	10001010100001	和13-1	10001111000101
广18-4	10011010000001	广62-5	10001011000001	和14-1	10001100100000
广19-1	11101111010000	广63-1	00011010000101	和14-2	10100010100100
广19-5	00110010000001	广63-2	10001010000101	和14-3	00001111100100
广20-1	10101000000001	广65-4	10001011100101	和15-1	00001010000101
广20-3	01001111000000	休1-4	00001010100000	和15-2	01001110000000
广23-3	10001010000000	休1-5	10010110100101	黄13-1	10000001100101
广24-4	10001000000001	休2-3	10000110100101	黄13-4	10000010000001
广26-3	00000010000101	休2-4	00001010000001	黄20-3	10000110100001
广26-5	10001010000100	休3-3	10001111000101	黄26-2	00101010100001
广27-2	10000010000101	休8-4	10001010100001	黄30-5	10101010100101
广27-4	10100010000000	休11-1	00000010000101	滁5-4	00001011100100
广31-4	10001010000100	休12-1	11001011000100	滁6-3	11001100100001
广33-2	11100011000101	休12-2	00001110000101	滁7-1	10000111100101
广33-4	10101010000001	休14-2	10101011000101	滁7-2	01000010100101
广33-5	10101011100101	休15-5	00001011100001	滁7-4	00001011100001
广34-5	10001111000101	休18-5	10000100000001	滁9-3	10001111100001
广35-2	10011011000100	休19-5	10101011100101	滁10-1	10001010010100
广37-4	10011010100001	休20-1	10010011000001	白14-1	10111000000001
广39-2	01011110000000	休20-5	10011111000001	小1-5	10011110000011
广39-4	01000111000000	休22-4	10011010000001	小3-1	10011011100101
广40-5	00100011000001	休28-2	10111100100101	小3-5	00011111100001
广42-1	10001010110001	休31-5	11001110110101	广西1-5	00101001000100
广49-3	10000010000101	休32-1	10001010100001	-	-

Table 2

Table 3

Table 4

Table 5

Tree height, DBH and single tree volume growth of 110 clones of P. massoniana with phenotypic resistant to pine wilt disease"

序号 No.	无性系 clone	树高/m tree height	胸径/cm DBH	材积/m³ volume	序号 No.	无性系 clone	树高/m tree height	胸径/cm DBH	材积/m³ volume
1	广1-3	6.9	17.5	0.095 3	56	休1-4	8.1	12.4	0.053 6
2	广2-5	8.7	17.0	0.106 2	57	休1-5	枯死
3	广3-2	7.3	11.4	0.042 1	58	休2-3	枯死
4	广3-4	7.7	12.7	0.054 2	59	休2-4	10.5	18.5	0.145 2
5	广4-3	6.8	13.5	0.056 1	60	休3-3	9.8	21.7	0.189 4
6	广5-4	枯死			61	休8-4	7.65	12.6	0.053 1
7	广5-5	6.9	15.0	0.070 0	62	休11-1	8.9	14.7	0.080 8
8	广6-5	6.7	13.8	0.058 0	63	休12-1	枯死
9	广11-3	7.0	9.0	0.025 5	64	休12-2	7.0	11.5	0.041 5
10	广12-1	6.9	16.1	0.080 6	65	休14-2	8.7	16.2	0.096 5
11	广15-3	7.5	12.7	0.053 2	66	休15-5	枯死
12	广16-2	4.6	12.4	0.036 7	67	休18-5	枯死
13	广17-3	9.8	19.1	0.146 7	68	广19-5	7.4	9.0	0.026 5
14	广18-2	8.8	20.1	0.149 8	69	休20-1	7.6	11.8	0.046 4
15	广18-4	枯死			70	休20-5	9.8	20.5	0.169 0
16	广19-1	枯死			71	休22-4	8.5	14.5	0.076 0
17	广19-5	8.9	17.6	0.115 8	72	休28-2	6.8	12.2	0.045 8
18	广20-1	8.1	13.7	0.065 5	73	休31-5	7.4	14.0	0.064 0
19	广20-3	9.0	18.3	0.126 3	74	休32-1	8.8	18.0	0.120 1
20	广23-3	枯死			75	休32-4	8.4	15.6	0.087 2
21	广24-4	7.9	16.9	0.097 8	76	休33-2	7.8	12.8	0.055 6
22	广26-3	5.9	11.1	0.034 4	77	休35-2	枯死
23	广26-5	8.5	15.1	0.082 4	78	和1-2-5	8.9	16.0	0.095 7
24	广27-2	5.7	6.7	0.012 3	79	和2-2-3	8.2	11.0	0.042 5
25	广27-4	枯死			80	和4-1	枯死
26	广31-4	8.0	12.3	0.052 3	81	和4-3	枯死
27	广33-2	7.8	11.8	0.047 2	82	和5-4	枯死
28	广33-4	8.5	12.9	0.060 1	83	和9-5	10.7	19.5	0.163 7
29	广33-5	9.0	17.9	0.120 8	84	和10-3	9.5	16.0	0.100 5
30	广34-5	10.5	19.2	0.156 3	85	和12-1	8.8	17.8	0.117 5
31	广35-2	枯死			86	和12-4	8.6	16.9	0.104 1
32	广37-4	7.5	13.1	0.056 6	87	和12-5	10.0	20.2	0.166 6
33	广39-2	枯死			88	和13-1	7.1	14.1	0.063 1
34	广39-4	8.3	12.3	0.053 7	89	和14-1	5.0	10.5	0.027 7
35	广40-5	8.1	12.2	0.051 9	90	和14-2	9.5	16.0	0.100 5
36	广42-1	6.8	7.3	0.016 4	91	和14-3	9.6	13.2	0.069 0
37	广49-3	枯死			92	和15-1	枯死
38	广51-1	8.2	10.1	0.035 9	93	和15-2	7.4	11.8	0.045 5
39	广51-4	7.6	14.9	0.073 9	94	黄13-1	8.3	12.9	0.059 1
40	广52-1	9.2	14.7	0.082 8	95	黄13-4	7.9	13.9	0.066 2
41	广52-2	枯死			96	黄20-3	枯死
42	广54-1	7.2	6.9	0.015 3	97	黄26-2	8.5	12.4	0.055 6
43	广54-2	9.6	19.0	0.142 9	98	黄30-5	8.9	13.8	0.071 2
44	广55-2	7.5	14.5	0.069 4	99	滁5-4	枯死
45	广56-3	8.0	12.1	0.050 6	100	滁6-3	枯死
46	广57-1	9.3	13.4	0.069 4	101	滁7-1	8.0	12.1	0.050 6
47	广57-4	枯死			102	滁7-2	8.4	17.4	0.108 4
48	广59-3	枯死			103	滁7-4	7.0	10.7	0.036 0
49	广61-3	枯死			104	滁9-3	9.2	16.0	0.098 1
50	广61-4	枯死			105	滁10-1	10.5	15.0	0.095 4
51	广62-2	7.9	10.8	0.039 9	106	白14-1	9.6	15.7	0.097 6
52	广62-5	8.2	13.2	0.061 3	107	小1-5	6.0	11.6	0.038 0
53	广63-1	8.8	17.5	0.113 5	108	小3-1	9.6	15.6	0.096 3
54	广63-2	枯死			109	小3-5	6.7	9.4	0.026 9
55	广65-4	7.0	15.0	0.070 7	110	广西1-5	6.9	13.7	0.058 4

Table 5

Table 6

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MuPS组	引物名称 primer		序列期望长度/bp expected length		引物序列(5'→3') sequence		引物设计^* primer design
Pmscar-M1	Pmscar157		887		F:CCTTGACGCATAAGAGAGAGTTAT		RAPD+14碱基
	Pmscar157		887		R:CCTTGACGCAGAGATGGGTGCATA		RAPD+14碱基
	Pmscar522		700		F:CCTTGACGCAGTATTTACATTGAA		RAPD+14碱基
	Pmscar522		700		R:CCTTGACGCACTTTATAGGTGTAG		RAPD+14碱基
	Pmscar361		620		F:TGCGCCCTTCCATGGATTTCTACA		RAPD+14碱基
	Pmscar361		620		R:TGCGCCCTTCACTTACTACTTCCC		RAPD+14碱基
	Pmscar305		507		F:CTTCCCCAAGCAACATATTTTTGG		RAPD+14碱基
	Pmscar305		507		R:CTTCCCCAAGAGTTCTTGATTTTC		RAPD+14碱基
	Pmscar007		425		F:CAGGCCCTTCTTTGAAATTAAACA		RAPD+14碱基
	Pmscar007		425		R:CTGACGAAGGGATGTAAAGAGTGA		内部
	Pmscar083		334		F:TAGATGAAGGGTTTATGGATTGATTTG		内部
	Pmscar083		334		R:GACCGCTTGTGAATATTATTGGCT		RAPD+14碱基
	Pmscar053		206		F:TGGAAACGCTGAGTATCGAAGTTTTG		内部
	Pmscar053		206		R:TCGGCGATAGTTAATATAATTATTAAAGG		RAPD+19碱基
Pmscar-M2		Pmscar626		825		F:TGATGGCGTCGAAAATTGTCCATA	RAPD+14碱基
		Pmscar626		825		R:TGATGGCGTCATAGGTACGATGAG	RAPD+14碱基
		Pmscar561		700		F:TTCCCCCGCATGACAAATAGA	RAPD+11碱基
		Pmscar561		700		R:TTCCCCCGCAATTTCCATGA	RAPD+10碱基
		Pmscar082		606		F:GACCGCTTGTACTTTGGAACTATG	RAPD+14碱基
		Pmscar082		606		R:GACCGCTTGTAAGGGAGGTTCTAA	RAPD+14碱基
		Pmscar039		481		F:CAATCGCCGTAAGAAATTTT	RAPD+10碱基
		Pmscar039		481		R:AAGAAGTCCAAGAATCCATGAAGG	内部
		Pmscar465		404		F:CCATCGTTCAAGCTAGTTTCTTTCC	内部
		Pmscar465		404		R:CAGGCCCTTCTTTGACTGCAT	RAPD+11碱基
		Pmscar499		298		F:CCTTGACGCAGGGTTTATTCGAAA	RAPD+14碱基
		Pmscar499		298		R:CCCTTTGTAAAATTGTAAATCCCAATG	内部
		Pmscar025		237		F:GCTAGGTGGTTTTTATTATTATGCCACT	内部
		Pmscar025		237		R:TCCTCAGTCATTGGAATCTTGGTA	内部

引物名称 primer	识别能力 recognition ability	多态位点出现率 polymorphic site occurrence rate
Pmscar 157	68.18	12.60
Pmscar 522	16.36	3.02
Pmscar 361	23.63	4.36
Pmscar 305	17.27	3.19
Pmscar 007	75.45	13.94
Pmscar 083	38.18	7.05
Pmscar 053	90.00	16.63
Pmscar 626	38.18	7.05
Pmscar 561	36.36	6.72
Pmscar 082	9.09	1.68
Pmscar 039	0.90	0.16
Pmscar 465	46.36	8.57
Pmscar 499	2.72	0.50
Pmscar 025	78.18	14.45

扩增位点 amplification site	N_e	H	I
Pmscar 157	1.914 2	0.477 6	0.670 6
Pmscar 522	1.270 9	0.213 1	0.369 5
Pmscar 361	1.381 9	0.276 4	0.448 9
Pmscar 305	1.253 4	0.202 2	0.355 0
Pmscar 007	1.998 1	0.499 5	0.692 7
Pmscar 083	1.674 6	0.402 8	0.592 6
Pmscar 053	1.879 1	0.467 8	0.660 6
Pmscar 626	1.502 9	0.334 6	0.517 2
Pmscar 561	1.461 6	0.315 8	0.495 5
Pmscar 082	1.139 3	0.122 3	0.241 7
Pmscar 039	1.012 8	0.012 6	0.038 5
Pmscar 465	1.587 9	0.370 2	0.557 1
Pmscar 499	1.039 1	0.037 6	0.094 8
Pmscar 025	1.998 1	0.499 5	0.692 7
平均值mean	1.508 1	0.302 3	0.459 1
标准差SD	0.346 1	0.165 7	0.215 3

生长性状 growth index	平方和 SS	自由度 df	均方 MS
树高tree height	1 506.614	82	13.696
胸径DBH	838.956	82	10.231
材积volume	0.128	82	0.002

Growth and genetic diversity analysis of clones screened by phenotypical resistant to pine wilt disease in Pinus massoniana

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