Screening and identification of rice salt-tolerant mutants at germination stage induced by space mutagenesis and heavy ion radiation
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摘要:目的
筛选萌发期耐盐突变体，为耐盐水稻新品种的创制与培育提供优异的种质资源。
方法对优质籼稻品种‘航聚香丝苗’干种子进行空间诱变和重离子诱变处理，并对诱变后代进行耐盐突变体的定向筛选。将野生型(WT)‘航聚香丝苗’种子用不同浓度NaCl溶液处理，探索萌发期耐盐突变体筛选的适宜浓度，以发芽率为指标对5 205份M2代诱变材料进行筛选，并在M3代进行验证。对WT和突变体进行耐盐相关生理指标的测定与分析，考查农艺性状；采用48个SSR标记对诱变后代与WT的遗传相似性进行评估。
结果萌发期耐盐突变体筛选的适宜NaCl溶液浓度为340 mmol/L，筛选得到12份萌发期耐盐突变体。生理指标测定结果显示，2个代表性突变体9-8和22-7的SOD、POD、CAT、APX活性均显著高于WT的，MDA、H2O2和O2−含量均显著低于WT的，耐盐突变体呈现出更强的抗氧化能力。突变体和野生型的农艺性状基本一致。SSR标记检测结果表明，WT ‘航聚香丝苗’与突变体19-49、20-15、20-49、20-62、15-6、16-65、22-7、20-19、20-38均无SSR差异位点，与突变体9-8、19-18、42-113的差异SSR位点均为1个。
结论筛选到12份萌发期耐盐突变体，且与WT ‘航聚香丝苗’的遗传背景高度一致，对水稻抗盐新品种的培育及抗盐性遗传机理的研究具有一定的理论和实际意义。
Abstract:ObjectiveTo screen salt-tolerant mutants during germination, provide excellent germplasm resources for creating and breeding new salt-tolerant rice varieties.
MethodThe dried seeds of the high quality indica rice variety ‘Hangjuxiangsimiao’, were treated with spatial mutagenesis and heavy ion mutagenesis, and the mutagenised progeny were subjected to targeted screening for salt-tolerant mutants. Wild type (WT) ‘Hangjuxiangsimiao’ seeds were treated with different concentrations of NaCl solution to explore the appropriate concentration for screening salt-tolerant mutants at the germination stage, and then 5 205 M2 mutant materials were screened using germination rate as an index and verified in the M3 generation. WT and mutants were measured and analysed for salt-tolerance-related physiological indices and agronomic traits. 48 SSR markers were used to assess the genetic similarity between the mutant progeny and WT.
ResultThe appropriate NaCl concentration for screening salt-tolerant mutants at the germination stage was 340 mmol/L, and 12 salt-tolerant mutants were screened at the germination stage. The results of physiological indices showed that the SOD, POD, CAT and APX activities of two representative mutants 9-8 and 22-7 were significantly higher than those of WT, and the MDA, H2O2 and O2− contents were significantly lower, the salt-tolerant mutants exhibited stronger antioxidant capacity. The results of SSR marker detection showed that there were no SSR differentiation locus between WT seedlings and mutants of 19-49, 20-15, 20-49, 20-62, 15-6, 16-65, 22-7, 20-19 and 20-38, and one differentiation SSR locus between them and mutants of 9-8, 19-18, 42-113.
ConclusionTwelve salt-tolerant mutants were screened at the germination stage, which were highly consistent with the genetic background of wild-type ‘Hangjuxiangsimiao’. It has certain theoretical and practical significance for breeding new salt-tolerant rice varieties and studing the genetic mechanism of salt tolerance.
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Keywords:
- Rice /
- Space mutation /
- Heavy ion radiation /
- SSR marker /
- Mutant screening /
- Germination stage /
- Salt tolerance
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图 1 不同浓度NaCl溶液处理下‘航聚香丝苗’干种子的萌发表型
Figure 1. The germination phenotypes of ‘Hangjuxiangsimiao’ dried seeds under different NaCl concentrations
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图 2 不同浓度NaCl溶液处理下‘航聚香丝苗’干种子的萌发情况
Figure 2. The germination state of ‘Hangjuxiangsimiao’ dried seeds under different NaCl concentrations
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图 4 M3代耐盐突变体盐处理7 d后的发芽率、发芽势和芽长对比
“**”表示突变体与野生型在0.01水平差异显著(t检验)；其中，红色柱子表示极耐盐突变体，蓝色柱子表示中度耐盐突变体。
Figure 4. Comparison of germination rate, germination potential and bud length of M3 generation salt-tolerant mutants after 7 days of salt treatment
“**” indicates that the mutants differed from the wild-type respectively at 0.01 levels of significant difference (t test); Red bars represent highly salt-tolerant mutants, while blue bars indicate mutants with moderate salt tolerance.
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图 5 盐胁迫对2个代表性耐盐突变体抗逆相关生理指标的影响
各小图中不同小写字母表示差异显著(P<0.05，Duncan’s 法)。
Figure 5. Effect of salt stress on stress-related physiological indexes of two representative salt-tolerant mutants
Different lowercase letters in each figure indicate significant differences (P<0.05, Duncan’s method).
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图 6 部分SSR引物对野生型材料及突变体的电泳图谱
Figure 6. Electrophoretic profile of partial SSR primers in the WT and the mutants
1~13：WT、9-8、20-62、42-113、20-38、15-6、16-65、19-49、20-15、20-19、20-49、22-7、19-18；红色箭头所指为差异位点。The red arrow indicates the differential loci.
表 1 空间诱变和重离子诱变的水稻突变体M3代株系的主要农艺性状1)
Table 1 The main agronomic characters of the rice mutants derived from space mutation and heavy ion mutation
株系
Line株高/cm
Plant
height剑叶长/cm
Flag-leaf
length剑叶宽/cm
Flag-leaf
width单株穗质量/g
Panicle weight
per plant穗长/cm
Panicle
length十粒长/cm
10-grain
length十粒宽/cm
10-grain
width千粒质量/g
1000-grain
weightWT 112.67±2.52b 34.60±2.04a 1.83±0.21a 22.30±2.82a 24.16±1.57a 11.13±0.16a 1.87±0.26a 21.16±1.50a 9-8 110.14±1.11b 33.75±2.02a 1.85±0.05a 22.24±0.15a 24.36±0.05a 10.59±0.02b 1.87±0.02a 18.52±0.43b 15-6 117.89±1.45a 34.05±0.32a 1.78±0.06a 21.81±1.26a 24.22±0.23a 11.08±0.07a 1.88±0.05a 21.27±0.32a 16-65 113.26±1.56b 33.71±2.18a 1.79±0.14a 22.72±1.31a 23.98±1.32a 11.12±0.09a 1.90±0.04a 20.89±1.03a 22-7 112.03±1.25b 34.19±1.65a 1.82±0.02a 22.99±1.06a 24.10±1.04a 11.13±0.06a 1.89±0.12a 21.25±0.14a 19-18 109.79±1.80b 34.04±1.93a 1.79±0.11a 22.57±1.47a 23.92±1.45a 10.71±0.12b 1.88±0.13a 21.03±0.36a 19-49 112.15±0.32b 33.92±1.21a 1.19±0.12a 22.40±2.03a 24.05±0.96a 11.09±0.11a 1.86±0.18a 20.84±1.21a 20-19 112.98±1.23b 33.92±2.08a 1.78±0.15a 22.16±1.65a 24.31±0.32a 11.14±0.03a 1.87±0.02a 20.96±1.07a 20-15 117.65±1.75a 35.24±1.15a 1.80±0.12a 22.90±1.54a 24.14±1.17a 11.12±0.12a 1.87±0.06a 21.08±0.35a 20-38 110.42±2.11b 34.13±1.33a 1.81±0.04a 22.62±1.63a 24.27±1.26a 11.10±0.11a 1.87±0.11a 21.23±0.22a 20-49 112.67±2.30b 33.68±1.82a 1.83±0.19a 23.14±1.24a 23.89±1.03a 11.03±0.21a 1.88±0.07a 21.35±0.53a 20-62 110.67±2.52b 34.15±1.63a 1.82±0.15a 22.09±2.12a 24.03±1.12a 11.19±0.03a 1.87±0.16a 21.57±0.28a 42-113 110.14±2.11b 30.50±1.29b 1.82±0.04a 22.36±1.36a 23.79±1.39a 11.01±0.22a 1.89±0.08a 21.24±0.55a 1) WT：航聚香丝苗；9-8为重离子诱变后代；其他株系为空间诱变后代；表中数据为平均值±标准误，n=5，同列数据后不同小写字母表示不同株系之间差异显著(P<0.05，Duncan’s法)。
1) WT: Hangjuxiangsimiao; 9-8 represents heavy ion-induced mutant; Other strains represent space-induced mutants; The data in the table are mean value ± standard error, n=5, different lowercase letters after the data in the same column indicate significant differences among different lines (P<0.05，Duncan’s method).
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