水稻是我国主要的粮食作物之一,其产量的提高主要依赖于杂种优势的利用。水稻光温敏核不育系的发现,使两系法利用水稻杂种优势成为可能。由于该不育系的育性转换受光周期和温度的调控,可以一系两用,不育期杂交制种,可育期自交繁种[1]。目前,生产上广泛使用的是长光高温不育而短光低温可育的光温敏核不育系,要求在长光高温条件下不育性稳定制种安全而在短日低温条件下育性易恢复繁种容易。对于这类光温敏核不育系,已报道定位的不育基因有pms1 [2-3]、pms2 [2,4]、pms3[5]、pms4[6]、OsPCD5[7]、tms1[8]、tms2[9-10]、tms3[11]、ms-h[12]、tms4[13]、TGMS[14]、tms5[15-17]、tms6(t) [18]、tms6[19]、tmsX[20]、ptgms2-1[21]、tms8[22]、p/tgms12-1[23]、tms9 [24]和tms9-1[25],其中pms1[26]、pms3[27]、p/tgms12-1[23]、OsPCD5[7]、ms-h[28-29]和tms5[30]已被克隆和功能分析。但在同一不育基因控制下的不同光温敏核不育系,由于受遗传背景的影响不育基因的表达仍然存在差异。对于异常低温影响光温敏核不育系制种安全的不育性不稳定问题,前人已从不育临界温度 [31-34]、低温耐受度[35-38]等方面进行了较多研究,选育不育临界温度低且耐受低温时间长的不育系以降低制种风险已是共识。然而,对于影响光温敏核不育系自交繁种的育性恢复难易问题,研究相对较少,陈雄辉等[39]认为除光温临界值外,育性的光温敏感度对繁种产量也有重要影响,对于光温反应类型及光温临界值极相近的2个不育系,敏感度高的不育系,一旦可育的光温条件满足,育性就迅速恢复并有较高的结实率,而敏感度低的不育系,可育的光温条件达到后育性恢复缓慢。随着分子生物学技术的发展,He等[40]以农垦58S来源的培矮64S和8902S为材料,通过不同光温处理定位了影响制种的光敏核不育性不稳定性QTL L2、L3a、L3b、L5、L6、L7和L10和影响繁种的育性可转换性QTL S2、S3a、S3b、S5、S8和S10。本研究将以冷繁结实率存在明显差异的温敏核不育水稻N38S和N727S为材料,定位影响繁种育性转换的低温敏感度QTL,可为实用型光温敏核不育水稻的分子标记辅助选育提供理论依据。
1 材料和方法
1.1 试验材料
以冷水灌溉30 d后自交结实率存在明显差异的水稻温敏核不育系N38S(来源于华68S,自交结实率实生苗4.3%,禾头6.7%)和N727S(来源于籼S,自交结实率实生苗90.0%,禾头73.3%)为材料,通过人工气候箱21.0 ℃低温处理获得可育材料与田间不育材料进行杂交获得F1,F1自交获得F2定位群体。试验材料种植于华南农业大学农场试验田。
1.2 育性转换的鉴定
将发育至幼穗分化第Ⅳ期(即雌雄蕊原基分化期)的亲本N38S、N727S及其F1、F2群体移入人工气候箱(加拿大Conviron PGV36),箱内温度设置为加权平均温22.17 ℃(表1),光照时间设置为11.5 h,光照度为1. 0 × 104 lx,RH≥75%。当低温处理至单株抽穗时,从抽穗单株上选取3穗的中上部颖花3～6朵,镜检时将每穗花药混合压片,用1% I2-KI溶液染色,将花粉圆形、深染色的计为正常可育花粉,其他皆计为败育花粉(包括无花粉、典败、圆败、染败),然后统计花粉可染率,单株的花粉育性为3穗花粉可染率的平均值。
表1 人工气候箱各时段温度设置
Tab.1 Temperature settings of different times in plant growth chamber
人工气候箱时段Different times of plant growthchamber温度设置/℃(平均温22.17 ℃)Temperature setting(The average temperature of 22.17 ℃ )6:00-8:0021.58:00-11:0022.511:00-16:0023.516:00-19:0022.519:00-6:0021.5
1.3 SSR标记分析
叶片DNA提取采用TPS简易抽提法,SSR引物由广州睿博兴科生物技术有限公司合成,PCR扩增参照Panaud等[41]的方法稍加修改进行,对PCR产物的检测采用6%聚丙烯酰胺凝胶电泳和银染的方法。
1.4 分子遗传连锁图谱的构建及QTL分析
运用Mapmaker 3.0及WinQTLCart 2.5软件对温敏核不育水稻进行育性转换的低温敏感度QTL分析的数据处理。Mapmaker 3.0用于构建遗传连锁图谱,得到各标记间的遗传连锁距离(cM)；WinQTLCart 2.5用于生成LOD曲线,确定QTL位点及在染色体上的分布。
2 结果与分析
2.1 F2定位群体的表型分析
经人工气候箱低温处理至抽穗,N38S的花粉育性为1.0%,N727S的花粉育性为65.0%,F1的花粉育性为67.5%,108株F2群体单株的花粉育性分布见图1。由图1可知,F2群体单株花粉育性呈连续分布,符合数量遗传的特点,可结合基因型鉴定结果对影响温敏核不育水稻育性转换的低温敏感度QTL进行检测分析。
2.2 遗传连锁图谱的构建
选取扩增效果较好且基本均匀分布于水稻12条染色体上的363对SSR标记对N38S和N727S进行亲本间的多态性分析。结果显示,有78对SSR标记在亲本间表现出多态性,多态率为21.5%。利用亲本间的多态标记对N38S×N727S的F2群体单株进行基因型分析,并运用Mapmaker 3.0软件对F2群体单株的表型鉴定数据和多态标记带型数据进行遗传连锁分析,构建基于SSR标记的分子遗传连锁图谱。结果显示,相互连锁的SSR标记构成8个连锁群,分别位于水稻的第1,4,7,8,9,10,11,12染色体上(图2)。
图1 N38S×N727S的108株F2群体单株的花粉育性分布
Fig.1 Distribution of pollen fertility of 108 F2 plants from the cross of N38S and N727S
图2 遗传连锁群在水稻染色体上的分布
Fig.2 Distribution of genetic linkage group on chromosomes in rice
2.3 育性转换的低温敏感度QTL定位
运用WinQTLCart 2.5软件对各连锁群上的QTL进行分析,一般当LOD>2.5时即认为存在QTL。通过复合区间作图法,检测到3个低温敏感度QTL位点,分别命名为QTL1、QTL2和QTL3,其中QTL1位于第1染色体的PSM12与RM583标记区间,QTL2位于第4染色体的PSM194与RM273标记区间,QTL3位于第4染色体的RM273与PSM103标记区间(图3)。
图3 影响温敏核不育水稻育性转换的低温敏感度QTL分析的LOD曲线
Fig.3 LOD curve of QTL analysis on low temperature sensitivity affecting fertility reversibility in TGMS rice
对定位的3个QTL进行遗传效应分析的结果见表2。由表2可知,3个QTL位点的加性效应值均为负值,分别为-12.14,-19.65,-10.40；显性效应值均为正值,分别为21.63,16.59,42.46；它们对育性转换的贡献率分别为7%,16%,3%。
表2 复合区间作图法检测到的低温敏感度QTL
Tab.2 QTLs of low temperature sensitivity detected by composite interval mapping method
染色体Chromosomes标记区间Interval QTLLOD值LOD value 加性效应Additive effect显性效应Dominance effect贡献率/%Percentagevariance explained1PSM12～RM583QTL14.0-12.1421.6374PSM194～RM273QTL23.8-19.6516.59164RM273～PSM103QTL32.9-10.4042.463
3 讨论与结论
一个优良的光温敏核不育系既要制种安全又要繁种容易。本研究利用SSR标记对影响温敏核不育水稻自交繁种的育性转换进行了低温敏感度QTL定位,获得3个QTL位点,其中QTL1位于第1染色体的标记PSM12(2 606 kb)与RM583(8 329 kb)之间,QTL2和QTL3分别位于第4染色体的PSM194(20 032 kb)与RM273(24 048 kb)之间和RM273(24 048 kb)与PSM103(32 060 kb)之间(http://www.ncbi.nlm.nih.gov)。这3个在低温条件下定位的QTL位点与He等[40]在短日条件下定位的6个影响繁种的育性可转换性QTL位点,即S2、S3a、S3b、S5、S8和S10位于不同的染色体上,因而彼此并不等位。而在已定位的光温敏核不育基因中,CSA位于第1染色体上[42],pms4位于第4染色体上[6],通过基于连锁标记的序列查询和Blast分析(http://blast.ncbi.nlm.nih.gov/Blast.cgi)可知,QTL1位于第1染色体标记RM583(8 329 kb)的上侧,与已报道的位于第1染色体标记RM292(9 566 kb)下侧的CSA不等位； QTL2和QTL3均位于第4染色体PSM194(20 032 kb)的下侧,与已报道的位于第4染色体RM6659(6 576 kb)下侧3.0 cM处的pms4相距较远。因此,本研究定位的3个影响温敏核不育水稻育性转换的低温敏感度QTL区间可能存在新的基因位点,并且这3个QTL位点的加性效应均为负值,说明N727S携带的等位基因增加花粉可育率和结实率,在利用分子标记辅助选择时,对QTL1、QTL2、QTL3应该选择N727S的标记类型,以提高自交繁种的产量。在3个QTL中,QTL2的低温敏感性最强,对于育性转换的贡献最大。本研究对3个低温敏感度QTLs的定位,将在利用分子标记辅助选择易于转育繁种的温敏核不育水稻方面发挥重要作用。
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