60Co-γ射线对2种狼尾草属牧草的诱变效应

doi:10.3864/j.issn.0578-1752.2019.03.003

摘要/Abstract

摘要：

目的探究 ⁶⁰Co-γ射线对“热研4号”杂交狼尾草及“华南”象草的诱变效应,创制新种质,以推动狼尾草的育种工作。方法在前期剂量筛选基础之上,以2种狼尾草品种（“热研4号”杂交狼尾草和“华南”象草）种茎为材料,利用 ⁶⁰Co-γ射线进行辐射,剂量为30 Gy,剂量率为1 Gy·min ^-1。将辐射后的种茎与未辐射的种茎栽植于大田中,常规田间管理,1个月后统计成活率。待成活的植株生长成熟后,在2个诱变系群体中分别随机挑选30株进行形态指标的测定,共测定7个指标。随后利用分子标记探究辐射对遗传变异的影响。结果 2个品种的辐射材料,分别随机选取30株进行形态指标测定,与对照相比,发现“热研4号”诱变系中有28株的株高,21株的茎节数及17株的叶宽有显著或极显著差异。“华南”诱变系中有20株的株高有显著或极显著差异,其他形态指标差异不大。2种牧草的突变体大都表现出株高的降低,因此,生物量也随之减少。还发现一个辐射材料RY4-17,由于分蘖数多于对照导致生物量增加。SSR分子标记研究结果表明,在“热研4号”诱变系中,20对引物的平均多态信息含量为0.245,共扩增出83条带,其中多态性条带52条,诱变系与对照之间的遗传相似系数为0.67—0.89,平均值为0.81,差异位点数为9—27,平均为15.9个,UPGMA聚类结果显示突变体RY4-9和RY4-23与对照遗传距离最远,变异程度最大。在“华南”诱变系中,20对引物的平均多态信息含量为0.394,共扩增出81条带,其中多态性条带65条,诱变系与对照之间的遗传相似系数为0.54—0.86,平均值为0.77,差异位点数为10—38,平均为18.8个,UPGMA聚类结果显示突变体HN-24与对照遗传距离最远,变异程度最大。结论适宜剂量的 ⁶⁰Go-γ射线辐射可以使2种牧草种茎成活率显著降低,同时可以有效诱导2种牧草在形态学和遗传学水平上发生变异。 ⁶⁰Go-γ射线辐射诱变是解决狼尾草因无性繁殖为主而导致遗传资源多样性匮乏的有效途径。

关键词: 狼尾草, ⁶⁰Co-γ射线, 表型变异, 遗传变异, SSR分子标记

Abstract:

【Objective】 Exploring the mutagenic effects of ⁶⁰Co-γ-rays on P. purpureum × P. americanurn. cv. Reyan No.4 and P. purpureum Schum. cv. Huanan and creating new germplasm, which aims to promote the process of Pennisetum breeding. 【Method】 Based on the pre-dose screening, using stems of the two species of Pennisetum as materials and irradiated by ⁶⁰Co-γ-rays and the dose was 30 Gy, the dose rate was 1 Gy·min ^-1. Then, the irradiated stems were planted in the field and conventional field cultivation management was performed. The survivor rate was counted one month later. After the plants to be survived mature, we randomly selected 30 individual plants from the two species Pennisetum and 7 morphological indicators of them were measured. Then, molecular markers were used to explore the effects of radiation on genetic variation. 【Result】 30 individual plants of the two species Pennisetum were selected randomly to measure the morphological indicators, respectively. The heights of 28 mutants, internodes of 21 mutants and leaf breadth of 17 mutants of Reyan No.4 have significant differences at 0.05 or 0.01 levels with the control. The heights of 20 mutants of Huanan have significant differences at 0.05 or 0.01 levels with the control, but there were no significant differences in other morphological indicators with the control. Most of the two species of Pennisetum mutants showed a decrease in plant height, and the biomass also decreased. However, we found that a mutant RY4-17 whose biomass is more than control because it has more tillers. The results of SSR molecular markers indicated that the average PIC of the 20 primers was 0.245 in the Reyan No.4 mutants, and a total of 83 bands were obtained, of which 52 bands were polymorphic. The GS between the mutants and the control was 0.67-0.89, the average was 0.81, the number of different sites was 9-27, with an average of 15.9. The UPGMA cluster showed that the distance between RY4-9, RY4-23 and control was the longest and indicated the mutation degree was greatest. In the Huanan mutants, the average PIC of the 20 primers was 0.394, a total of 81 bands were obtained, of which 65 bands were polymorphic. And the GS between the mutants and the control was 0.54-0.86, the average was 0.77, the number of different sites was 10-38, with an average of 18.8. The UPGMA cluster showed that the distance between HN-24 and control was the longest and indicated the mutation degree was greatest. 【Conclusion】 The appropriate dose of ⁶⁰Co-γ-rays radiation can significantly reduce the survival rate of the two species Pennisetum, and can effectively induce the mutations in the morphological and genetic. The method is an effective way to solve the lack of genetic resources caused by asexual reproduction of Pennisetum.

Key words: Pennisetum, ⁶⁰Co-γ-rays, phenotypic variation, genetic variation, SSR molecular markers

武炳超, 童磊, 杜昭昌, 胡家菱, 张欢, 陈燚, 刘伟, 张新全, 黄琳凯. ⁶⁰Co-γ射线对2种狼尾草属牧草的诱变效应[J]. 中国农业科学, 2019, 52(3): 414-427.

WU BingChao, TONG Lei, DU ZhaoChang, HU JiaLing, ZHANG Huan, CHEN Yi, LIU Wei, ZHANG XinQuan, HUANG LinKai. Mutagenic Effects of ⁶⁰Co-γ-rays on Two Species of Pennisetum Genus Forages[J]. Scientia Agricultura Sinica, 2019, 52(3): 414-427.

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https://www.chinaagrisci.com/CN/Y2019/V52/I3/414

图/表 8

表1

“热研4号”杂交狼尾草突变体形态指标"

材料 Material	分蘖数 Tiller	株高 Plant height (cm)	茎节数 Internode	茎粗 Stem thick (mm)	叶长 Leaf length (cm)	叶宽 Leaf breadth (mm)	生物量 Yield (kg)
RY4-1	18	469.1±11.3	18.7±4.0	21.8±4.2	123.5±13.0	55.9±4.2**	19.1
RY4-2	24	382.4±15.7**	16.8±3.3	16.4±2.9*	114.2±11.9	56.5±4.0**	13.4
RY4-3	21	444.9±21.2**	14.4±5.1**	20.4±2.3	120.5±6.3	55.7±6.7**	15.3
RY4-4	14	256.2±18.0**	6.3±3.5**	15.2±2.3**	96.9±10.7**	53.0±1.0	2.3
RY4-5	24	385.7±11.5**	14.3±4.1**	18.0±2.0	121.8±13.4	50.1±4.3	10.7
RY4-6	15	375.9±23.3**	14.3±5.2**	20.3±1.9	127.8±8.8	56.5±5.8**	10.8
RY4-7	28	415.9±18.3**	18.2±1.9	18.5±3.5	120.9±16.7	56.1±2.2**	18.3
RY4-8	22	360.6±22.0**	15.8±1.6	20.2±4.3	123.1±13.8	58.8±3.2**	18.9
RY4-9	17	337.4±27.2**	9.5±5.0**	18.7±2.2	105.9±19.1	61.1±4.7**	7.1
RY4-10	19	304.7±21.3**	11.0±3.6**	17.0±1.9	106.1±16.3	51.1±3.8	13.0
RY4-11	19	444.5±30.0**	13.7±6.6**	18.3±1.9	119.3±12.8	54.5±7.0**	17.1
RY4-12	18	414.4±31.0**	15.2±5.3*	16.4±1.7*	102.1±31.6*	52.2±5.7*	8.8
RY4-13	8	370.1±18.5**	13.3±6.6**	17.2±2.9	126.2±12.3	53.4±4.0*	9.9
RY4-14	13	359.2±10.8**	13.2±2.8**	17.1±3.7	106.6±6.1	40.2±2.7*	12.0
RY4-15	26	393.7±16.1**	14.3±4.3**	20.1±2.1	126.7±39.9	52.9±5.8*	16.9
RY4-16	14	362.3±22.4**	12.6±4.0**	17.2±2.7	91.8±7.3**	55.8±4.8**	4.8
RY4-17	31	441.9±11.3**	13.0±5.1**	19.0±3.5	115.4±8.4	40.5±6.2**	19.9
RY4-18	12	214.8±30.4**	7.6±5.9**	15.2±3.9**	94.5±23.5**	48.3±6.0	2.3
RY4-19	17	417.2±10.4**	15.2±3.2*	21.3±3.4	121.0±7.1	54.8±6.1**	15.3
RY4-20	15	299.9±9.6**	9.2±3.1**	15.2±2.3**	116.5±14.6	48.7±7.2	5.2
RY4-21	21	376.5±16.7**	14.1±5.8**	19.9±3.6	132.4±4.1	51.8±2.5	9.6
RY4-22	20	349.6±12.2**	10.7±3.3**	17.4±3.5	111.3±12.6	56.3±3.9**	8.6
RY4-23	21	383.8±10.6**	12.4±0.5**	19.3±0.6	121.8±9.1	50.2±5.0	19.0
RY4-24	17	374.2±22.8**	15.4±2.3	17.8±1.7	120.0±5.8	48.2±6.2	14.0
RY4-25	3	352.0±32.5**	14.7±2.5	13.5±1.5**	105.3±17.2	46.0±8.5	1.2
RY4-26	13	352.1±50.6**	12.0±4.5**	16.6±1.5*	108.3±18.0	43.9±6.6	4.1
RY4-27	11	411.1±52.3**	18.4±6.3	18.8±3.7	119.6±8.0	48.7±9.2	7.6
RY4-28	22	471.9±33.5	18.5±3.7	18.5±1.4	127.6±10.6	55.9±2.8**	18.0
RY4-29	10	400.3±41.6**	17.8±2.2	15.1±1.9**	131.6±17.2	49.6±5.4	17.8
RY4-30	19	422.4±43.8**	15.0±5.0*	15.2±2.6**	123.3±11.3	42.9±4.5	12.4
CK	19.7±1.5	476.3±42.8	20.1±4.0	19.5±2.3	120.0±23.4	47.4±4.1	17.4±0.7

表1

表2

“华南”象草诱变系形态指标"

材料 Material	分蘖数 Tiller	株高 Plant height (cm)	茎节数 Internode	茎粗 Stem thick (mm)	叶长 Leaf length (cm)	叶宽 Leaf breadth (mm)	生物量 Yield (kg)
HN-1	29	435.5±16.1	20.6±2.9*	17.7±2.4	88.6±6.4	42.0±6.3*	18.4
HN-2	18	399.5±34.8**	11.4±5.5	19.0±2.4	92.4±14.3	47.0±4.4	13.7
HN-3	23	386.2±45.2**	16.1±7.1	15.9±1.8	81.0±5.6*	45.5±5.0	13
HN-4	36	422.1±12.0**	15.2±4.8	17.3±2.5	88.4±11.1	44.6±3.9	18.9
HN-5	19	426.9±17.8**	15.2±5.6	17.1±1.6	93.3±16.4	43.5±5.1	12.3
HN-6	18	408.4±15.1**	14.8±4.9	17.6±2.7	93.1±11.0	40.7±4.0**	10.8
HN-7	13	261.2±31.1**	12.7±2.8	11.6±1.6**	86.5±10.4	36.6±4.3**	3.2
HN-8	25	449.2±16.9	15.3±6.1	18.0±3.0	82.4±14.0*	45.4±4.2	15.8
HN-9	25	429.2±29.6*	14.8±5.0	16.4±2.0	101.4±9.6	44.0±5.1	14.1
HN-10	26	410.8±17.5**	17.5±5.1	16.2±2.0	95.0±12.6	40.6±4.1**	13.7
HN-11	14	421.8±29.0**	17.1±3.9	18.4±1.8	94.1±8.1	43.4±3.6	8.9
HN-12	43	469.6±15.9	18.3±5.2	18.1±2.2	106.8±13.1**	47.7±3.6	24.2
HN-13	43	493.0±25.9**	17.6±3.4	18.2±2.4	93.2±10.0	42.1±1.7	28.6
HN-14	26	489.6±17.2**	17.1±4.8	19.0±2.1	89.3±9.2	42.3±3.8*	14.5
HN-15	32	402.6±18.3**	19.7±4.5	18.6±2.2	82.9±12.5	52.6±4.0*	20.9
HN-16	22	340.5±32.1**	15.8±3.4	18.2±2.4	84.1±6.7	51.4±6.4	9.9
HN-17	21	428.6±12.5*	17.1±6.2	19.5±3.5*	88.0±28.4	45.2±5.1	10.8
HN-18	35	441.7±13.2	22.1±5.5**	16.6±4.2	78.8±6.1**	44.9±2.8	20.4
HN-19	23	432.3±12.7*	16.1±7.5	19.1±2.1	96.1±10.4	40.8±5.3**	10.6
HN-20	23	454.0±20.3	17.6±4.9	17.3±1.1	82.7±5.5	45.1±2.8	13.1
HN-21	19	366.7±34.4**	16.9±5.5	16.1±1.7	83.9±8.5	45.4±4.4	10.8
HN-22	13	376.6±17.6**	15.1±5.5	16.4±2.0	97.1±8.7	44.1±4.7	7.9
HN-23	47	471.0±15.7	16.8±1.3	19.7±4.3*	105.2±6.3*	43.9±4.2	22.6
HN-24	26	432.0±28.6	17.0±1.6	15.5±1.8	95.2±8.6	43.9±3.0	10
HN-25	21	409.3±25.9**	17.7±7.8	19.3±3.3*	87.0±13.2	42.3±5.7*	21.1
HN-26	25	461.3±18.6	15.5±7.2	19.4±3.1*	73.4±5.2**	52.6±5.6*	21.4
HN-27	17	318.6±27.5**	14.1±4.0	16.7±2.4	80.9±10.1*	46.2±4.8	9.4
HN-28	18	434.6±27.0	17.1±5.4	23.3±3.0**	83.4±12.1	62.0±14.9**	15.7
HN-29	19	387.9±19.3**	13.6±3.5	15.7±2.6	92.0±11.5	44.2±4.7	8.4
HN-30	36	451.2±12.7	18.1±5.6	20.8±2.3**	80.0±12.4*	40.4±5.3**	18.9
CK	26.3±2.1	454.5±21.9	15.2±7.0	17.0±2.7	92.7±7.9	47.8±2.8	18.8±0.9

表2

图1

表3

表4

表5

表6

图2

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引物名称	上游引物	下游引物	退火温度	扩增条带数	多态性条带数	多态性条带比率	多态性信息量
Primer name	Forward primer	Reverse primer	Annealing	Total number of amplified bands	The number of polymorphic bands	Percentage of polymorphic bands (%)	PIC
	(5′-3′)	(5′-3′)	(℃)				(%)
SSR8	TCCCTCTAATCACAAATGAGTCCA	TTTGGACTGTTTCCCTTTCAAATAA	62	3	3	100	28.5
SSR14	GAGAAGATCAGAGGAGGGTAGCAG	CGATGAATGCAATGCAAGCTA	62	3	2	66.67	17.5
SSR16	ACTAGAACTACAGGAGGTCGGGG	ATATGGGATGTGTTGAGGTGTGG	62	3	3	100	47.7
SSR19	CTCTCTTTCTCTCTCTGTTGCGTG	CACCACACTGCTCCACCACT	62	6	6	100	29.9
SSR24	ACCACCATTCCTTCAATTCGTACT	GAGAAGTGAGTGCTACAGGTGCAT	62	2	0	0	0
SSR33	AGGCTCAAGCTCTACGTGGC	GACAAACGGCAAACATTTTCAA	58	6	6	100	18.6
SSR39	GACCTGGGGTAGGTCTGATCC	CGCTTTGTTTTGGTGTAGATTGAG	62	7	6	85.71	43
SSR42	TTTAGAGAACTCCTACGGTACGGC	ATCCACCTAGACACCGTACGAAAT	62	2	1	50	23.2
SSR43	GTTTCTTGCAAGTTACTCGCTTCA	CGTTAGTTCCTCCATTCTCTTTTGA	62	3	3	100	75.5
SSR46	CGCAACCAGCATCTCTTCAG	CACTGTCGTAGCTCAAGAAGTCGT	62	6	4	66.67	24
SSR47	CAATGAAACTATGCACTTAAGCCG	AGTCTGTTTTGAGTCGAGGGAATC	62	4	2	50	18.4
SSR50	GCATCAGGAACCATATATGTTGGA	AACACAATCGCTAGCTGATCCATA	62	5	2	40	21.9
SSR51	GTTTGCTTCAACATAACGCAAAAG	GATCTGGTGGCTCCTGACTTG	58	3	1	33.33	18
SSR52	AAAGAGCAAAGGCTTTATTTGCAC	CGACACAGAGGGAGTGTTATATTTTG	62	4	1	25	50.8
SSR53	AAATGAAAGTGAAAGGGGAAAACA	TTTTTCTTCTGTTATTATTCTCGTGTCC	62	3	0	0	0
SSR54	GAAAACTCACCGGAAGGTCAAC	GAGGAGAAGACGAACGGTGACT	62	4	3	75	9.3
SSR55	AAGTCCACATCATCCCGGTC	GTGGCTTACCTGATCCGAGC	62	8	2	25	7.9
SSR59	CTCTAATCTCTCTCCTCCCGCTC	CTGCTCACCATCAGCAGTGAG	60	5	3	60	26.7
SSR65	GCCTGCCTATTGCCTAGACG	AAGGAAGATGTGACTCACGGATTT	62	3	3	100	29.2
SSR70	AGCTGATCTTATTTGGCTACTGCC	TGTAAAAGCCTACAAGGATGCGTA	62	3	0	0	0
总计Total				83	52
平均Average				4.2	2.6		24.5

引物名称	上游引物	下游引物	退火温度	扩增条带数	多态性条带数	多态性条带比率	多态性信息量
Primer name	Forward primer	Reverse primer	Annealing	Total number of amplified bands	The number of polymorphic bands	Percentage of polymorphic bands (%)	PIC
	(5′-3′)	(5′-3′)	(℃)				(%)
SSR1	AGCATCGATAGCTTTTTCCTGC	GAGGGGAGGAGAGGATGCTT	62	4	3	75	58
SSR3	GTCGCACTGCAATCCAACATA	TCCATTCATTCATTCTTCAGTGATTT	62	3	3	100	55.3
SSR9	CGTACCTTCTCGCAATAAGGAGAT	GAAGAAGGTTCACCTCTTTCGGTA	62	3	2	66.67	47.7
SSR11	AGAGGAGACAAAGGACCTCCG	GTCCATGGAGGACGAGCTTC	62	8	7	87.5	28.6
SSR12	GTCGAGGAAGTCCTTGGTGTG	GACGAGGGGTTCATCCACAT	62	4	4	100	58.5
SSR19	CTCTCTTTCTCTCTCTGTTGCGTG	CACCACACTGCTCCACCACT	62	6	2	33.33	13.3
SSR23	AGGTTCTGCTTCTGGAGGTGAC	TAGATTTGTTCATGATGATGCGTG	62	3	2	66.67	56.1
SSR24	ACCACCATTCCTTCAATTCGTACT	GAGAAGTGAGTGCTACAGGTGCAT	62	4	2	50	18.7
SSR25	ATTTCTCTGGTTTTGTTCAGCTCC	ACAGAGGTGGTATGATCGACTTGA	62	4	4	100	68.5
SSR29	GCTTTCAAGTGATACAACGACACC	CCACTCGTCCTTCAATCTCTACCT	62	4	3	75	10.8
SSR35	ATGTGTTCGTTGCCATCTGTAGTT	GCACGGCAAACAAACAAAAA	62	2	2	100	65.9
SSR38	TCTTTAGATCTCTGCCATGTAGCG	GCTAGCACTACCCCTGCATTTTAT	62	3	1	33.33	18
SSR43	GTTTCTTGCAAGTTACTCGCTTCA	CGTTAGTTCCTCCATTCTCTTTTGA	62	7	7	100	49.1
SSR47	CAATGAAACTATGCACTTAAGCCG	AGTCTGTTTTGAGTCGAGGGAATC	62	5	5	100	61.5
SSR50	GCATCAGGAACCATATATGTTGGA	AACACAATCGCTAGCTGATCCATA	62	4	3	75	35.3
SSR53	AAATGAAAGTGAAAGGGGAAAACA	TTTTTCTTCTGTTATTATTCTCGTGTCC	62	2	2	100	76.5
SSR54	GAAAACTCACCGGAAGGTCAAC	GAGGAGAAGACGAACGGTGACT	62	4	3	75	27.4
SSR55	AAGTCCACATCATCCCGGTC	GTGGCTTACCTGATCCGAGC	62	4	4	100	27
SSR59	CTCTAATCTCTCTCCTCCCGCTC	CTGCTCACCATCAGCAGTGAG	60	4	4	100	7.8
SSR66	ATAATGGAAAGTGAGTTTTGCCGT	GGAGCCTTCAGCTTTAAAAGACAA	62	3	2	66.67	4.2
总计Total				81	65
平均Average				4.1	3.3		39.4

⁶⁰Co-γ射线对2种狼尾草属牧草的诱变效应

Mutagenic Effects of ⁶⁰Co-γ-rays on Two Species of Pennisetum Genus Forages

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诱变系名称 Mutants	遗传相似系数 GSC	诱变系名称 Mutants	遗传相似系数 GSC	诱变系名称 Mutants	遗传相似系数 GSC
RY4-1	0.81	RY4-21	0.77	HN-11	0.65
RY4-2	0.83	RY4-22	0.78	HN-12	0.77
RY4-3	0.77	RY4-23	0.72	HN-13	0.79
RY4-4	0.87	RY4-24	0.81	HN-14	0.69
RY4-5	0.80	RY4-25	0.76	HN-15	0.74
RY4-6	0.87	RY4-26	0.88	HN-16	0.72
RY4-7	0.78	RY4-27	0.84	HN-17	0.78
RY4-8	0.84	RY4-28	0.83	HN-18	0.81
RY4-9	0.67	RY4-29	0.86	HN-19	0.80
RY4-10	0.83	RY4-30	0.88	HN-20	0.83
RY4-11	0.77	HN-1	0.79	HN-21	0.83
RY4-12	0.80	HN-2	0.78	HN-22	0.80
RY4-13	0.82	HN-3	0.72	HN-23	0.75
RY4-14	0.81	HN-4	0.78	HN-24	0.54
RY4-15	0.86	HN-5	0.74	HN-25	0.85
RY4-16	0.77	HN-6	0.75	HN-26	0.83
RY4-17	0.72	HN-7	0.77	HN-27	0.86
RY4-18	0.76-	HN-8	0.72	HN-28	0.80
RY4-19	0.83	HN-9	0.67	HN-29	0.84
RY4-20	0.89	HN-10	0.78	HN-30	0.85

诱变系名称 Mutants	差异位点数（个） Different sites	差异位点百分率 Percentage of different sites (%)	诱变系名称 Mutants	差异位点数（个） Different sites	差异位点百分率 Percentage of different sites (%)
RY4-1	16	19.3	HN-1	17	21.0
RY4-2	13	15.7	HN-2	18	22.2
RY4-3	19	22.9	HN-3	23	28.4
RY4-4	11	13.3	HN-4	18	22.2
RY4-5	17	20.5	HN-5	21	25.9
RY4-6	11	13.3	HN-6	20	24.7
RY4-7	18	21.7	HN-7	19	23.5
RY4-8	13	15.7	HN-8	23	28.4
RY4-9	27	32.5	HN-9	27	33.3
RY4-10	14	16.9	HN-10	18	22.2
RY4-11	19	22.9	HN-11	28	34.6
RY4-12	17	20.5	HN-12	19	23.5
RY4-13	15	18.1	HN-13	17	21.0
RY4-14	16	19.3	HN-14	24	29.6
RY4-15	12	14.5	HN-15	21	25.9
RY4-16	19	22.9	HN-16	23	28.4
RY4-17	23	27.7	HN-17	18	22.2
RY4-18	20	24.1	HN-18	15	18.5
RY4-19	14	16.9	HN-19	16	19.8
RY4-20	9	10.8	HN-20	14	17.3
RY4-21	19	22.9	HN-21	14	17.3
RY4-22	18	21.7	HN-22	16	19.8
RY4-23	23	27.7	HN-23	20	24.7
RY4-24	16	19.3	HN-24	38	46.9
RY4-25	20	24.1	HN-25	12	14.8
RY4-26	10	12.0	HN-26	14	17.3
RY4-27	13	15.7	HN-27	10	12.3
RY4-28	14	16.9	HN-28	16	19.8
RY4-29	12	14.5	HN-29	13	16.0
RY4-30	10	12.0	HN-30	12	14.8

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