小麦Vp-1基因的表达特性和STS标记的开发与应用
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摘要
小麦成熟期穗发芽是一种世界性的小麦灾害,严重影响小麦品质和产量。目前我国北方的大部分白粒小麦品种对穗发芽高度敏感,收获期遇雨极易引起穗发芽而造成重大经济损失。如何控制小麦穗发芽,是小麦品种改良的关键问题。本试验利用不同穗发芽抗性材料,分离Vp-1及其等位基因;采用半定量RT-PCR的方法,分析Vp-1及其等位基因的表达特性;基于Vp-1及其等位基因序列差异,开发并验证Vp-1 STS标记,并就其有效性与已开发的标记作比较研究;利用Vp-1 STS标记,结合不同穗发芽测定方法,鉴定具有不同穗发芽抗性机制的小麦品种。主要结果如下:
1.在小麦3B染色体上发现了2个与穗发芽抗性相关的Vp-1的新型等位变异,分别命名为Vp-1Bb和Vp-1Bc,它们与Vp-1Ba(AJ400713)分别具有95.43%和97.89%的同源性;与Vp-1Ba相比,Vp-1Bb和Vp-1Bc在第3个内含子中分别具有193bp的插入和83bp的缺失,分别是由反转座子和转座子引起,在此基础上开发出与穗发芽抗性相关的Vp-1B STS标记,并命名为Vp1B3;经验证,Vp1B3标记可以有效地区分具有不同穗发芽抗性的小麦品种基因型,可用于种质资源的筛选和分子标记辅助育种。
2.对开花后不同发育时期的Vp-1A、Vp-1B和Vp-1D基因的表达分析显示:开花后25d、30d和35d的幼胚中,虽然Vp-1A、Vp-1B和Vp-1D均存在转录本的错误剪切现象,但只有Vp-1B的正常剪切的转录本丰度在抗、感穗发芽品种间存在差异,且Vp-1Bb和Vp-1Bc比Vp-1Ba具有更高的表达量;用30uM ABA浸泡2d后,3个Vp-1B等位基因在胚中均观测到转录本的表达,尤以Vp-1Bb转录本的表达量最高,但在30uM ABA浸泡4d后,均未观测到Vp-1Bb和Vp-1Bc转录本的表达,而Vp-1Ba转录本的表达量基本没有变化,说明这3个等位基因对ABA的敏感性是不同的,其原因是插入和缺失影响了胚对ABA的敏感性,最终导致了品种对穗发芽抗性的不同差异。
3.将开发的STS标记Vp1B3与已报道的小麦穗发芽抗性标记MST101、wmc104、Xgwm155的有效性比较可知,Vp1B3、Xgwm155标记与穗发芽抗性相关,而MST101、wmc104标记与穗发芽抗性无关,利用Vp1B3和Xgwm155标记来筛选小麦穗发芽抗性品种,将会提高选择效率和准确性。
4.利用Vp1B3标记结合整穗发芽法和发芽指数鉴定33份小麦新品系的穗发芽抗性结果表明:红粒抗穗发芽品系CA0489属于Vp1Bb基因型和红色种皮休眠型,CA0481属于Vp1Bc抗穗发芽基因型;白粒抗穗发芽品系CA0509和CA0459的抗性为非Vp1B3类型,其抗性可能与穗部性状和颖壳抑制物有关。
5.应用Vp1B3标记对中国的38份地方品种、94份历史品种和106份当代品种的Vp-1B基因多样性检测结果表明:Vp1-Ba、Vp1-Bb、Vp1-Bc和Vp-1Be基因型在地方品种中的分布频率分别为45%、18%、34%和3%,在历史品种中分别为30%、10%、60%和0%,在当代品种中分别为31%、1%、68%和0%;而在欧洲小麦材料中发现的Vp-1Bd基因型在本研究的238份实验材料中没有出现。
Pre-harvest sprouting (PHS) of wheat is a international balefulness which reduces the quality and economic value of the grains. Breeding for PHS tolerant cultivars is important in the Northern and Northeastern as well as in the Yangtze River valley region in China. So improvement of PHS tolerance is one of the most important objectives in wheat breeding. In this study, Vp-1 and Vp-1’s allelic varieties had been isolated from different PHS tolerance cultivars, and the expression characterization of the Vp-1 gene was studied by Semi-quantitative RT-PCR. According to the sequence and expression characterization of the Vp-1, a STS marker had been developed and validated, the efficiency was assessed with other three PHS markers and had been used in molecular assisted- selecting. The main details of the result as follows:
1. Two new Viviparous-1 allelic variants related to PHS tolerance were identified on chromosome 3B of bread wheat, and designated as Vp-1Bb and Vp-1Bc, respectively. Sequence analysis showed that Vp-1Bb and Vp-1Bc had an insertion of 193-bp and a deletion of 83-bp fragment, respectively, which were begot to retrotransposon and transposon. A co-dominant STS marker of Vp-1B gene covering the insertion and deletion region was developed and designated as Vp1B3. Statistical analysis indicated that Vp1B3 was strongly associated with PHS tolerance suggesting that Vp1B3 could be used as an efficient and reliable co-dominant marker in the evaluation of wheat germplasm for PHS tolerance and marker-assisted breeding for PHS tolerant cultivars.
2. Semi-quantitative RT-PCR analysis showed that alternatively spliced transcripts of the Vp-1A, Vp-1B and Vp-1D homologues were present and there were no differences in the splicing patterns or abundances of Vp-1A and Vp-1D from 25, 30 and 35 DAP embryos between PHS-tolerant and susceptible cultivars. The protein was expressed more highly in genotypes with Vp-1Bb and Vp-1Bc than in those with Vp-1Ba. Furthermore, genotypes with different levels of tolerance to PHS showed different responsiveness to ABA exposure and differences in transcript levels of Vp-1Ba, Vp-1Bb and Vp-1Bc were observed after ABA treatment. The results indicated that insertion or deletion in the third intron region may affect the expression of the Vp-1B gene and its sensitivity to ABA, and thus its resistance to PHS.
3. Another three PHS tolerance-associated markers: MST101, wmc104 and Xgwm155, as well as Vp1B3, were employed, aimed at assessing the efficiency of these markers in selecting genotypes with higher PHS tolerance. Variations at Vp1B3 and Xgwm155 were associated with PHS response, but not for STS marker MST101 and STMS marker wmc104, and the selection efficiency would be improved by use of both markers together.
4. Three methods, i.e., spike germination rate (SGR), germination index (GI), and molecular marker Vp1B3, were used to screen the PHS tolerance of 33 new wheat lines. The red grain PHS resistance line CA0489 possessed both Vp1Bb tolerance gene and red grain related dormancy gene; red grain PHS resistance line CA0481 had Vp1Bc tolerance gene. The two white grain PHS resistance lines CA0509 and CA0459 did not contain Vp1 tolerance gene, their PHS tolerance might be related to the characters of spike and the stayers excreted from glumes.
5. In addition, 38 landraces,94 historical cultivars and 106 modern cultivars were used to evaluate the diversity of the Vp-1B gene associated with PHS tolerance. Four alleles were found in the wheat varieties tested. Frequencies of Vp1Ba alleles, Vp1Bb, Vp1Bc and Vp1Be genotypes were 45%, 18%, 34% and 3%, respectively, in landraces; 30%, 10%, 60% and 0%, respectively, in historical wheat; while 31%, 1%, 68% and 0%, respectively, and no Vp-1Bd genotype in studied 238 cultivars, which presented in Europe wheat cultivars, was found in this study.
1.在小麦3B染色体上发现了2个与穗发芽抗性相关的Vp-1的新型等位变异,分别命名为Vp-1Bb和Vp-1Bc,它们与Vp-1Ba(AJ400713)分别具有95.43%和97.89%的同源性;与Vp-1Ba相比,Vp-1Bb和Vp-1Bc在第3个内含子中分别具有193bp的插入和83bp的缺失,分别是由反转座子和转座子引起,在此基础上开发出与穗发芽抗性相关的Vp-1B STS标记,并命名为Vp1B3;经验证,Vp1B3标记可以有效地区分具有不同穗发芽抗性的小麦品种基因型,可用于种质资源的筛选和分子标记辅助育种。
2.对开花后不同发育时期的Vp-1A、Vp-1B和Vp-1D基因的表达分析显示:开花后25d、30d和35d的幼胚中,虽然Vp-1A、Vp-1B和Vp-1D均存在转录本的错误剪切现象,但只有Vp-1B的正常剪切的转录本丰度在抗、感穗发芽品种间存在差异,且Vp-1Bb和Vp-1Bc比Vp-1Ba具有更高的表达量;用30uM ABA浸泡2d后,3个Vp-1B等位基因在胚中均观测到转录本的表达,尤以Vp-1Bb转录本的表达量最高,但在30uM ABA浸泡4d后,均未观测到Vp-1Bb和Vp-1Bc转录本的表达,而Vp-1Ba转录本的表达量基本没有变化,说明这3个等位基因对ABA的敏感性是不同的,其原因是插入和缺失影响了胚对ABA的敏感性,最终导致了品种对穗发芽抗性的不同差异。
3.将开发的STS标记Vp1B3与已报道的小麦穗发芽抗性标记MST101、wmc104、Xgwm155的有效性比较可知,Vp1B3、Xgwm155标记与穗发芽抗性相关,而MST101、wmc104标记与穗发芽抗性无关,利用Vp1B3和Xgwm155标记来筛选小麦穗发芽抗性品种,将会提高选择效率和准确性。
4.利用Vp1B3标记结合整穗发芽法和发芽指数鉴定33份小麦新品系的穗发芽抗性结果表明:红粒抗穗发芽品系CA0489属于Vp1Bb基因型和红色种皮休眠型,CA0481属于Vp1Bc抗穗发芽基因型;白粒抗穗发芽品系CA0509和CA0459的抗性为非Vp1B3类型,其抗性可能与穗部性状和颖壳抑制物有关。
5.应用Vp1B3标记对中国的38份地方品种、94份历史品种和106份当代品种的Vp-1B基因多样性检测结果表明:Vp1-Ba、Vp1-Bb、Vp1-Bc和Vp-1Be基因型在地方品种中的分布频率分别为45%、18%、34%和3%,在历史品种中分别为30%、10%、60%和0%,在当代品种中分别为31%、1%、68%和0%;而在欧洲小麦材料中发现的Vp-1Bd基因型在本研究的238份实验材料中没有出现。
Pre-harvest sprouting (PHS) of wheat is a international balefulness which reduces the quality and economic value of the grains. Breeding for PHS tolerant cultivars is important in the Northern and Northeastern as well as in the Yangtze River valley region in China. So improvement of PHS tolerance is one of the most important objectives in wheat breeding. In this study, Vp-1 and Vp-1’s allelic varieties had been isolated from different PHS tolerance cultivars, and the expression characterization of the Vp-1 gene was studied by Semi-quantitative RT-PCR. According to the sequence and expression characterization of the Vp-1, a STS marker had been developed and validated, the efficiency was assessed with other three PHS markers and had been used in molecular assisted- selecting. The main details of the result as follows:
1. Two new Viviparous-1 allelic variants related to PHS tolerance were identified on chromosome 3B of bread wheat, and designated as Vp-1Bb and Vp-1Bc, respectively. Sequence analysis showed that Vp-1Bb and Vp-1Bc had an insertion of 193-bp and a deletion of 83-bp fragment, respectively, which were begot to retrotransposon and transposon. A co-dominant STS marker of Vp-1B gene covering the insertion and deletion region was developed and designated as Vp1B3. Statistical analysis indicated that Vp1B3 was strongly associated with PHS tolerance suggesting that Vp1B3 could be used as an efficient and reliable co-dominant marker in the evaluation of wheat germplasm for PHS tolerance and marker-assisted breeding for PHS tolerant cultivars.
2. Semi-quantitative RT-PCR analysis showed that alternatively spliced transcripts of the Vp-1A, Vp-1B and Vp-1D homologues were present and there were no differences in the splicing patterns or abundances of Vp-1A and Vp-1D from 25, 30 and 35 DAP embryos between PHS-tolerant and susceptible cultivars. The protein was expressed more highly in genotypes with Vp-1Bb and Vp-1Bc than in those with Vp-1Ba. Furthermore, genotypes with different levels of tolerance to PHS showed different responsiveness to ABA exposure and differences in transcript levels of Vp-1Ba, Vp-1Bb and Vp-1Bc were observed after ABA treatment. The results indicated that insertion or deletion in the third intron region may affect the expression of the Vp-1B gene and its sensitivity to ABA, and thus its resistance to PHS.
3. Another three PHS tolerance-associated markers: MST101, wmc104 and Xgwm155, as well as Vp1B3, were employed, aimed at assessing the efficiency of these markers in selecting genotypes with higher PHS tolerance. Variations at Vp1B3 and Xgwm155 were associated with PHS response, but not for STS marker MST101 and STMS marker wmc104, and the selection efficiency would be improved by use of both markers together.
4. Three methods, i.e., spike germination rate (SGR), germination index (GI), and molecular marker Vp1B3, were used to screen the PHS tolerance of 33 new wheat lines. The red grain PHS resistance line CA0489 possessed both Vp1Bb tolerance gene and red grain related dormancy gene; red grain PHS resistance line CA0481 had Vp1Bc tolerance gene. The two white grain PHS resistance lines CA0509 and CA0459 did not contain Vp1 tolerance gene, their PHS tolerance might be related to the characters of spike and the stayers excreted from glumes.
5. In addition, 38 landraces,94 historical cultivars and 106 modern cultivars were used to evaluate the diversity of the Vp-1B gene associated with PHS tolerance. Four alleles were found in the wheat varieties tested. Frequencies of Vp1Ba alleles, Vp1Bb, Vp1Bc and Vp1Be genotypes were 45%, 18%, 34% and 3%, respectively, in landraces; 30%, 10%, 60% and 0%, respectively, in historical wheat; while 31%, 1%, 68% and 0%, respectively, and no Vp-1Bd genotype in studied 238 cultivars, which presented in Europe wheat cultivars, was found in this study.
引文
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