药用野生稻(Oryza officinalis Wall)和转bar基因水稻(Oryza sativa L.)花粉杂交的基因漂移

采用生殖生物学方法研究了药用野生稻和转bar基因水稻花粉杂交的基因漂移。结果表明 ,供试水稻花粉在药用野生稻柱头上的萌发生长与药用野生稻自花授粉花粉的萌发生长有一定差异 ,表现在穿过柱头的花粉粒百分率及内容物释放和正在凝缩、释放的花粉粒百分率较少。虽然转基因水稻花粉能在药用野生稻柱头上正常萌发生长 ,并能释放内容物 ,但杂交后结实率为 0 ,表明转基因水稻和药用野生稻杂交不亲和。他们的不亲和性不是在花粉的萌发和穿过柱头这一阶段 ,具体原因有待进一步研究。在本实验条件下转基因水稻和药用野生稻没有发生成功的基因交流。

用荧光显微镜技术观察药用野生稻(Oryza officinalis Wall)和转基因水稻的不亲和性

采用荧光显微镜技术观察转基因水稻花粉在药用野生稻柱头上的萌发及在花柱内的生长过程,以明确两者之间不亲和性发生的阶段,为判断其能否发生基因漂流提供依据。结果表明,两种转基因栽培稻(Y003和99t)的花粉在药用野生稻柱头上的萌发率均比药用野生稻自花授粉的低,花粉管在花柱中的生长速度较慢,且分别在到达花柱中部(Y003)或花柱基部(99t)时停止生长,顶端异常膨大,杂交子房逐渐萎缩,结实率为0。药用野生稻与栽培稻杂交不亲和的原因是花粉管在花柱中停止生长、不能进入胚囊完成受精,在自然条件下转基因栽培稻中的外源基因向药用野生稻漂流的可能性较小。

稻属种间杂种(Oryza sativa×O.officinalis)回交世代的形态学研究

两个组合（中８６－４４×ＹＤ１７８５、中１５６×３－６６）的种间杂种（Ｏ．ｓａｔｉｖａ×Ｏ．ｏｆｆｉｃｉｎａｌｉｓ）Ｆ１植株，完全不育。以栽培稻为轮回亲本进行回交，结合胚拯救技术，获得两组合回交一代（ＢＣ１Ｆ１）植株。两组合回交一代（ＢＣ１Ｆ１）群体中分别出现６个和７个类型的植株，完全不育。通过继续回交获得的组合中８６－４４×ＹＤ１７８５回交二代（ＢＣ２Ｆ１）植株中，出现了４个类型的植株，株型似栽培稻，大部分植株可育。经自交获得株型好，结实正常，抗性强的中间材料。结果表明：（１）栽野杂种回交世代出现疯狂分离，药用野生稻遗传上存在较大异质性；（２）通过连续两次回交，结合胚拯救技术，可改变杂种野生性状，获得具有药用野生稻优良基因的中间材料，如７５－２、７９－１、７９－１１三个株系；（３）证实胚拯救技术在种间杂交转移药用野生稻有利基因中是一条行之有效的途径。

Comparative Physical Mapping of Bph3 with BAC-FISH in Oryza officinalis and O. sativa

A FISH procedure was adopted to physical mapping rice RFLP marker RZ69 and the BAC clone 38J9 screened by RZ69 which is linked to gene Bph3 in Oryza saliva L. and O. officinalis Well ex: Watt. The FISH results showed that both 38J9 and RZ69 were located in the middle of 4S in O. officinalis and the centromere area of 4S in O. sativa. In O. officinalis the percentage distances from the centromere to the hybridization sites were 20.00 +/- 5.40 and 22.12 +/- 3.44, the detection rates were 50.00% and 6.14%, but in O. sativa they were 0 and 0, 56.10% and 6.29% correspondingly. The results obtained from the BAC and RFLP clone were almost the same in the cultivated rice and O. officinalis. It was suggested that die marker RZ69 of the cultivated rice and its homologous sequence in O. officinalis were in the same BAC clone, the homologous sequence of Bph3 in O. officinalis should be located at the sites hybridized by both RZ69 and 38J9. Many signals on different chromosomes of O. officinalis were observed under no blocking with Cot-1 DNA, showing that the repetitive sequences were also homologous between O. sativa and O. officinalis. The identification of chromosome 4 of O. officinalis is based on comparative map with RG214 and BAC clone screened by RG214. The feasibility of comparative physical mapping performed between O. sativa and O. officinalis with rice BAC clones was discussed.

Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis

Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid （AA, 2n=2x=24） and autotetraploid （AAAA, 2n=4x=48） cultivated rice were crossed as maternal parents with O. officinalis （CC, 2n=2x=24）, none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations （DAP）. By rescuing hybrid embryos, in vitro F1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globularor pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes.

Comparative Embryological Studies on Infertility of Interspecific Hybridizations Between Oryza sativa with Different Ploidy Levels and O. officinalis

As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.

Taxonomic Relationship Between Oryza minuta and O． officinalis Based on AP－PCR Analysis

The arbitrarily primed polymerase chain reaction(AP-PCR)technique was used to detect polymorphisms in genomic DNA from Oryza minuta, O. officinalis, and related rice species. The results show that the genetic distance between O.minuta and O.officinalis is much higher than that between two known species,O.sativa and O. rufipogon, showing that O.minuto and O.officinalis should be classified as two different spedes in genus Oryza,and that the AP-PCR technique can be used as a powerful tool for studying taxonomic relationships among related spedes, and even subspecies.

Isozyme analysis on progenes of somatic hybrids of Oryza sativa L.and O.officinalis

Some wild species of Oryza possess agronomi-cally useful traits such as disease/insect resis-tance and salt tolerance.However,the use ofwild Oryza species for rice improvement hasbeen hampered by the sexual incompatibility orsterility of hybrids obtained by conventionalmethods.We have used protoplast fusion as amethod to incorporate useful trait of wild O-ryza species into cultivated rice and~ obtainedsomatic hybrid plants between O.sativa and

Genetic Analysis of Monosomic Alien Addition Line MAAL8 of O. officinalis(CC)-O. sativa(AA)

[Objective] By carrying out the anther culture on the monosomic alien addition line MAAL8 of O.officinalis-O.sativa and the back crossing with O.sativa H1493,the genetic characteristics of monosomic alien addition line were studied.[Method] The phenotype analysis was used to study the separation proportion of progeny.Moreover,SSR and the methylation analysis were used to study the transmission behavior of nonhomologous chromosome.[Result] 78 plants of 145 backcross progenies preserved the rolled leaf mark trait of MAAL8.In 32 anther culture plants,five plants had the normal rolled leaves,and two plants had the extremely rolled leaves.The rest had the flat leaves.14 couples of SSR markers were used to analyze,and it indicated that all the rolled-leaf plants could obtain the characteristic band type of O.officinalis,but the flat-leaf plants showed none of them.11 polymorphic RFLP markers were used to carry out Methylation-Sensitive Southern analysis.It showed that the methylation variation manners of eight markers between AA and CC genomes were different.[Conclusion] The nonhomologous chromosome of MAAL8 could pass to the progenies independently and integrally via the meiosis,and the phenotype characteristics didn＇t change.Moreover,the methylation manner of O.officinalis could inherit stably in the hybrid progeny as the addition of single chromosome.The stability of methylation might have the certain effect on the relatively independent inheritance of nonhomologous chromosome in the genome environment of O.sativa.

广西药用野生稻重要农艺性状遗传多样性分析

药用野生稻具有抗病虫、抗逆境、高生物产量、高光效等优良特性,蕴藏有许多优良基因。本研究用广西所有药用野生稻分布点收集的代表性资源199份,对抽穗期、花药长度、粒宽、粒长与粒重5个重要农艺性状进行遗传多样性、主成分等分析。结果表明:广西药用野生稻具有丰富的遗传多样性,抽穗期、花药长度、粒宽、粒长和粒重的变异系数分别为9.12%、11.90%、3.39%、4.64%和6.24%,多样性指数分别为1.4878、2.0453、1.7727、1.1567和2.0379。主成分分析表明:第1、第2主成分主要与产量性状相关,累计贡献率达63.759%,第3、第4主成分特征值相近,贡献率分别为16.306%和15.542%,抽穗期与花药长度是影响的决定因子,主要反映植株的生长特征。7个地理居群按遗传多样性大小排序为:梧州市(1.6731)>玉林市(1.5784)>贵港市(1.5070)>南宁市(1.3325)>来宾市(1.2131)>贺州市(0.9629)>钦州市(0.3819),梧州市的多样性指数最高,表明梧州市是广西药用野生稻表型遗传多样性的中心。

药用野生稻基因组DNA提取方法比较

以药用野生稻为试验材料,分别选用传统的CTAB提取法、CTAB改良方法、碱裂解法、磁珠法、吸附柱法、尿素提取法及酶消化法7种方法提取水稻叶片组织DNA,7种方法分别标记为A~G。通过比较这些方法提取出DNA的浓度和纯度,分析这些方法的优劣势。7种方法均能获得较好的基因组,按获得DNA浓度表现为A>B>F>C>G>E>D,纯度表现为D>E>C>G>B>F>A。综合考量DNA的纯度、浓度、提取时间、成本、安全无毒等方面因素,若对基因组的质量要求不高,只是做简单的检测且考虑成本问题,可以选择CTAB提取法、CTAB改良方法和尿素提取法;若需要高纯度基因组,可以选择磁珠法、吸附柱法、碱裂解法;若考虑安全无毒、快速提取及成本可控,可以选择磁珠法、吸附柱法;若样品稀有,可以选用CTAB提取法和CTAB改良法。

筛选和转化药用野生稻TAC克隆获得耐旱水稻

【目的】利用分子生物学技术建立一种可有效转移药用野生稻有利基因的方法,为进一步大量开展利用药用野生稻有利基因提供技术基础。【方法】以植物抗性相关转录因子AP2/EREBP和bZIP家族基因保守序列为探针,采用同位素α-32P对探针进行标记,通过Southern杂交对药用野生稻TAC文库进行第一轮筛选;所获得阳性克隆用地高辛标记的探针进行第二轮筛选;最后利用PCR方法对第二轮筛选所获得的阳性克隆进行验证。通过农杆菌介导法将阳性克隆转入栽培稻品种日本晴,利用TAC载体插入片段两端的抗性标记Hpt和SacB,通过PCR扩增检测和和Southern杂交对转化植株进行鉴定。采用PEG胁迫法和生理指标变化对转基因T2植株的芽期、苗期耐旱性进行鉴定。【结果】第一轮TAC文库筛选共获得1 073个阳性克隆,其中,以AP2/EREBP的保守序列为探针筛选出606个、bZIP为探针筛选出467个。对这些阳性克隆进行第二轮筛选,从中共获得147个阳性克隆,其中,AP2/EREBP为探针筛选出95个、bZIP为探针筛选出52个。PCR验证有103个阳性克隆能够扩增出目的片段,分别是利用AP2/EREBP设计的引物筛选出63个克隆和bZIP的40个克隆,阳性克隆得率分别为66.32%和76.92%。通过农杆菌转化,以编号为52D-M16和22D-P2(与AP2/EREBP相关)以及55R-A17、49R-O14和8-A24(与bZIP相关)的克隆转化水稻日本晴均获得转化植株,转化试验结果发现外源插入片段越大则克隆转化的成苗率越低。抗性鉴定、PCR和Southern杂交结果均证明外源片段已转入受体水稻品种日本晴中。芽期抗旱性鉴定表明R12-23、R15-41和R1-8 3份材料的耐旱性优于受体日本晴,苗期鉴定显示M63-9和R12-23的耐旱性较强。综合认为,R12-23具有较好的耐旱性。【结论】通过构建药用野生稻TAC文库并结合特异克隆筛选和遗传转化方法,可以进行有利基因的转移,结合进一步的育种,有望实现药用野生稻在育种上的利用。

广西野生稻资源抗稻瘟病材料的鉴定与评价

经过3代自交纯化与稻瘟病抗性鉴定,在1500份普通野生稻（Oryza rufipogon Griff.）中发现38份抗病材料,在113份药用野生稻（O.officinalis Wall.ex Watt.）中发现18份抗病材料。普通野生稻和药用野生稻相同材料在不同年份的田间病区诱发鉴定中,各级抗性植株分布差异不显著,鉴定结果表现一致。与普通野生稻相比,广西药用野生稻抗性基因发生的频率显著较高、抗性稳定,是一种特殊抗性类型,从中获得具有重要利用价值的新抗性基因的可能性较大。研究还表明,自交提纯能够明显提高这两种野生稻的平均抗性水平。在田间病区诱发条件下,广西普通野生稻抗性材料出现的频率与地区居群的遗传多样性没有显著相关性。

野生稻细菌性条斑病抗性资源筛选及遗传分析

对1655份普通野生稻(Oryza rufipogon Griff.)和31份药用野生稻(O.officinalis Wall.ex Watt)进行了细菌性条斑病(Xanthomonas oryzae pv.oryzicola,简称细条病)抗性鉴定,结果发现在普通野生稻中有57份抗病材料,其中3级抗性有31份,占总数的1.87%;5级中抗有26份,占总数1.57%。在药用野生稻资源中,有15份抗病材料,占总数的48.4%。选取了8份普通野生稻抗性资源(分别命名为DP1、DP3、DP5、DP9、DP15、DP16、DP17和DP20)与9311杂交,再自交或与9311回交后,分别获得BC1、F1和F2后代。在接种鉴定中发现这些抗性资源的BC1或F1所有植株均对细条病表现感病,说明这8份材料的抗性属于隐性遗传。在DP3与9311杂交的F2群体中,抗感植株的分离比符合1∶15的比例,说明DP3的抗性由2对隐性重叠作用基因控制。研究结果表明,在野生稻中可以获得一批具有较大利用价值的细条病抗性资源,其中药用野生稻资源中抗性材料所占的比例较大。

药用野生稻转育后代一个抗白叶枯病新基因的定位

从药用野生稻渗入后代选育的水稻株系B5表现为高抗褐飞虱、白背飞虱和白叶枯病。对B5与籼稻品种明恢 6 3杂交组合的 187个重组自交系 (RILs)进行了抗白叶枯病接种鉴定 ,采用分离集团分析法 (BulkedSegregantAnalysis,BSA) ,在第 1染色体上筛选到与水稻抗白叶枯病基因相连锁RFLP分子标记。利用RILs抗病性表现型鉴定资料和构建的分子标记连锁图谱 ,将抗白叶枯病基因定位在第 1染色体短臂的C90 4和R5 96之间 ,这两个分子标记间遗传距离为 1 3cM。该基因对RILs群体抗病性变异的贡献率为 5 2 96 % ,是一效应值较大的主效基因。这一抗白叶枯病基因不同于已报道的抗白叶枯病基因的位点 ,因此将其命名为Xa2 9(t)。

栽培稻-药用野生稻杂种F_1及回交后代的基因组原位杂交鉴定

以ｄｉｇｏｘｉｇｅｎｉｎ标记的药用野生稻（Ｏｒｙｚａ ｏｆｆｉｃｉｎaｌｉｓ）总ＤＮＡ作探针，未标记的栽培稻（Ｏｒｙｚａ ｓａｔｉｖａ）总ＤＮＡ封阻（１：１６），对栽培稻一药用野生稻Ｆｉ及回交后代材料根尖体细胞染色体制片进行基因组原位杂交，鉴定出３个双单体异源附加系和４个单体异源附加系。当标记探针用ａｎｔｉ－ｄｉｇｏｘｉｇｅｎｉｎ－ＰＯＤ和ＤＡＢ检测时，药用野生稻染色体显棕色，栽培稻染色体则因Ｇｉｅｍｓａ对染而显浅蓝色；标记探针用ａｎｔｉ－ｄｉｇｏｘｉｇｅｎｉｎ－ＦＩＴＣ检测时，药用野生稻染色体发出黄绿色荧光，而栽培稻染色体则因ＰＩ对染而发出红色荧光。在异源附加系的有丝分裂间期细胞中，药用野生稻染色质所占区域也很明显。由此看来，有可能利用ＧＩＳＨ技术来区分Ａ与稻属的其他基因组。

珍稀濒危植物——云南药用野生稻自然生态群的新发现及其特性

野生稻包括云南药用野生稻 (OryzaofficinalisWall .exWatt.,染色体组CC ,染色体数2n =2 4 )是宝贵的基因资源库 ,自然分布急剧减少。 2 0 0 1年 ,在云南耿马孟定遮甸新发现了1个药用野生稻分布点 ,该地药用野生稻植株高度可塑性大 ,可以根据密度和荫蔽情况自动大幅度调节株高 ;比其它生态群的结实率高 ;而其自然发病和人工接种鉴定发现抗稻瘟病能力中等偏上 ,抗白叶枯病害能力强 ,抗螟虫和稻飞虱能力也很强。品质分析结果表明该居群药用野生稻蛋白质含量比其它野生稻的高 ,而直链淀粉含量偏低 ,说明品质较好。本发现对采取有效措施进行药用野生稻原生地保护 。

药用野生稻抗源对褐稻虱的抗性遗传及利用研究

利用广谱高抗褐稻虱的药用野生稻(CC染色体组)与感虫栽培稻(AA染色体组)品种进行远缘杂交,通过幼胚培养获得绿苗,经过4代回交和4代自交,成功地将抗性基因转移到栽培稻中,获得了高世代B4F4株系。对其亲本和后代的鉴定结果表明,药用野生稻抗性受1对显性主效基因控制;其抗性基因与药用野生稻主要农艺性状没有连锁遗传关系。

药用野生稻抗稻褐飞虱鉴定与利用技术研究

从 198份药用野生稻资源中筛选出一批广谱高抗褐飞虱抗源 (其中 3份为免疫级 )并对这些抗源进行了抗性遗传研究。研究结果表明其抗性是受一对显性基因控制的。通过离体幼胚培养获得绿苗 ,经过多代回交和自交 ,成功地将抗性基因转移到栽培稻中 ,获得了高世代 (B4 F5)株系。同时利用外源DNA花粉管导入法对药用野生稻广谱高抗褐飞虱基因导入栽培稻进行了研究 ,获得了具有某些性状的后代。

广西野生稻耐冷性鉴定与遗传纯合研究

【目的】筛选耐冷性强的野生稻种质,研究野生稻耐冷性遗传的稳定性,为水稻耐冷性育种和研究提供抗源。【方法】利用人工气候箱对收集的广西野生稻资源进行苗期耐冷性鉴定,在桂北高寒山区自然低温条件下进行宿根期耐冷性评价;选择耐冷性较强的材料通过连续套袋自交或花药培养进行耐冷性遗传纯合研究。【结果】筛选出苗期1级耐冷材料广西普通野生稻20份,广西药用野生稻1份,普通野生稻花培株系92份;宿根期3级耐冷材料广西普通野生稻43份,药用野生稻2份,普通野生稻花培株系5份;耐冷性较强的直立或半直立型种质8份;广西普通野生稻苗期和宿根期耐冷性呈极显著正相关(r=0.26),广西药用野生稻苗期和宿根期耐冷性相关性不显著(r=-0.11);广西普通野生稻在苗期耐冷性与宿根期耐冷性上,平均耐冷等级均表现为匍匐型耐冷性最强,倾斜型次之,半直立型与直立型间无差异且最弱;来自桂北的野生稻苗期耐冷性最强;来自桂南的野生稻宿根期耐冷性表现最强。【结论】鉴定筛选出的材料可用于耐冷育种及抗逆机理研究。筛选出的材料可用于水稻耐冷性研究和育种,同时可以通过套袋自交与花药培养加快获得耐冷性更强的纯合株系。