芸薹属B基因组异附加系材料高效鉴定体系的创建

芸薹属B基因组包含许多在育种中有用的性状和基因,然而它的研究却非常有限。为了创建全套甘蓝-黑芥单体异附加系来解析B基因组,在本研究中我们针对芸薹属B基因组染色体创建了一套以SSR分子标记和FISH技术为核心的高效、快速、准确地鉴定和筛选体系。利用该体系,我们在以异源三倍体杂种(2n=26,CC.B)作为母本,用亲本甘蓝(2n=18,CC)作轮回亲本的回交后代中进行异附加系的鉴定和筛选。结果表明,采用该体系能够准确的鉴定和筛选出在C基因组上附加的每一条B基因组染色体的单体异附加系(2n=19,CC+1B1-8);并且效率很高,仅回交3代我们就获得了7个不同的单体异附加系。创建的甘蓝-黑芥单体异附加系对于黑芥基因组结构和进化的研究以及优良性状的应用都具有重要价值。     

大白菜-结球甘蓝2号单体、双体异附加系的获得与鉴定

以大白菜-结球甘蓝异源三倍体(AAC,2n=3x=29)与二倍体大白菜(AA,2n=2x=20)回交一代BC1F1及其自交后代BC1F2为试验材料,经过细胞学观察和核型分析,于BC1F1中鉴定出了附加甘蓝2号染色体的大白菜-结球甘蓝单体异附加系,于BC1F2中鉴定出了附加甘蓝2号染色体的大白菜-结球甘蓝双体异附加系,并对其进行了减数分裂与植株形态学观察.该异附加系的获得为分析大白菜和甘蓝的亲缘关系,进一步获得大白菜和结球甘蓝易位系、代换系提供了基础材料.     

普通小麦与Elymus rectisetus异附加系的分子细胞遗传学鉴定

Elymus rectisetus (Nees in Lehm) A. Löve et Connor是目前小麦族中发现的唯一的无融合生殖种,为了鉴定和标记从普通小麦与E. rectisetus BC₂F₂衍生后代中选育的2n=44株系1026A1、1057A1和1035A2的外源染色体,应用细胞学、基因组原位杂交和RAPD方法进行了研究。经细胞学鉴定,3个株系花粉母细胞减数分裂中期Ⅰ（PMC MⅠ）染色体构型均为2n=22Ⅱ,与普通小麦Fukuhokomugi杂交F₁的PMC MⅠ染色体构型均为2n =21Ⅱ+1Ⅰ,两两杂交F1的PMC MⅠ染色体构型均为2n=21Ⅱ+2Ⅰ,表明它们是分别附加了1对互不相同外源染色体的普通小麦-E. rectisetus二体异附加系。标记E. rectisetus品系1050的基因组DNA为探针DNA,对3个异附加系进行原位杂交,分别鉴定出附加的1对E. rectisetus染色体。应用13个引物对2个亲本和3个异附加系进行RAPD分析,获得了可分别用于检测1026A1和1057A1中所附加的E. rectisetus染色体遗传物质的分子标记OPB-14_900bp、OPE-09_750bp和OPB-14_1000bp。     

异附加系数量性状的遗传分析方法

本文根据异附加系的细胞遗传特点结合数量遗传学原理，提出了异附加系数量怀状遗传分析方法。要点如下：（１）以异附加系为被测系、正常品种（系）作测验系，组配成各世代群体；（２）将异附加系分成两类，第一类是杂种分离世代中可区分亚世代，第 是其杂种分离世代中不可区分亚世代；（３）给出了第一类异附加系的各世代（亚世代）的遗传组成，均数分量和变异分量；（４）给出了第二异附加的遗传组成和均数分量。     

普通小麦辉县红-荆州黑麦异染色体系的选育及其梭条花叶病抗性鉴定

为发掘和利用荆州黑麦所携抗梭条花叶病基因，综合利用分子细胞遗传学与分子标记技术结合多年抗性鉴定，从高感梭条花叶病小麦地方品种辉县红与荆州黑麦杂交后代（F₇~F₉）中选育出二体异附加系5个(分别添加1R、2R、R3、5R和R7)、5RS端二体异附加系1个和多重异附加代换系2个（染色体组成分别为20’’+2R(2D)’’+4R’’和19’’+1R（1B）’’+2R（2B）’’+4R’’）。鉴定表明，双二倍体荆辉1号高抗梭条花叶病，表明黑麦抗性基因可在小麦背景中稳定表达，2R、R7二体异附加系及2个含2R的多重异附加代换系均表现高抗，推测2R和R7上可能携带抗病基因。这些材料是研究荆州黑麦抗性基因遗传及小麦抗病育种的新种质。     

携带抗黄矮病基因染色体的分离

抗黄矮病小麦一中间堰麦草异附加系Z2 (2n=44)携带一对完整的中间僵麦草染色体,用Z2作母本与普通小麦品种中8601杂交,获得杂种F1(2n=43=21Ⅱ+1I)。利用激光显微切割技术将F1花粉母细胞减数分裂中期I及后期I的呈单价体的中间僵麦草染色体分离出来,经去蛋白、Sau3AI酶切后,进行PCR体外扩增。结果表明利用激光显微切割可分离     

基于SSR分子标记的Nicotiana tobacum–N.plumbaginifolia异源染色体植株的鉴定与筛选

以烟草(Nicotiana tabacum)品种云烟87的八倍体(2n=8x=96)和野生烟草N. plumbaginifolia (2n=2x=20)的基因组DNA为模板,对340对烟草SSR引物进行筛选以获得能扩增多态性条带的引物。利用多态性引物对种间杂交后代及190株回交后代的基因组DNA进行扩增,并对N.plumbaginifolia中的SSR标记的连锁情况进行简要分析。经筛选获得了多态性引物29对。结果显示,在190株后代中, 159株的基因组DNA能扩增出N. plumbaginifolia的特异SSR位点,可以判定该159株为N. tabacum的N. plumbaginifolia异源染色体植株,其余31株植株可能不含有N. plumbaginifolia的染色体。经UPGMA聚类分析,本群体中植株的遗传多样性较为丰富,部分分子标记在后代中的出现具有完全相关性。29个标记中14个可确定来源于5条不同染色体,N.plumbaginifolia的29个位点在回交后代中的扩增效率并不相同,且效率均较低(低于31.00%),说明该杂种中N. plumbaginifolia基因组的垂直传递效率较低。利用SSR分子标记可以判定云烟87八倍体与N.plumbaginifolia杂交获得的后代为真杂种,且自该远缘杂种回交后代中筛选获得大量异源染色体植株。这些结果和筛选获得异源染色体植株为进一步创制N.tabacum-N.plumbaginifolia抗黑胫病单体附加系以及易位系奠定了基础。     

抗白粉病八倍体小偃麦和双体异附加系的鉴定

对从中间偃麦草(Elytrigia intermedium)与普通小麦品种烟农15杂交后代中选育的双体异附加系山农Line15和八倍体小偃麦山农TE263的形态学、细胞学观察结果表明,它们的主要形态性状介于双亲之间,根尖细胞染色体数目分别为2n=44和2n=56,PMC MⅠ染色体构型分别为2n=22Ⅱ和2n=28Ⅱ;以中间偃麦草总基因组DNA为探针的荧光原位杂     

普通小麦-Elymus rectisetus衍生系的细胞学鉴定及其特征特性研究

利用细胞学方法对2个普通小麦-Elymus rectisetus(Nees in Lehm)A.Lve et Connor衍生系1059A1和1063A1进行了鉴定,并对它们的细胞学稳定性、白粉病抗性、高分子量麦谷蛋白亚基组成和植物学性状进行了研究。2个衍生系在形态学和细胞学上已经基本稳定,根尖体细胞含有44条染色体,花粉母细胞减数分裂中期I(PMC MI)染色体构型为2n=22Ⅱ,与普通小麦亲本Fukuhokomugi杂交F1PMC MI染色体构型为2n=20Ⅱ 3Ⅰ。结果表明,它们均为普通小麦-E.rectisetus异代换-附加系。白粉病抗性鉴定结果表明,2种异代换-附加系高感白粉病。2种异代换-附加系及亲本的SDS-PAGE分析表明,它们的高分子量麦谷蛋白亚基组成与Fukuhokomugi相同。     

甘蓝型油菜-埃塞俄比亚芥二体附加系植株形态及细胞学鉴定

从甘蓝型油菜品种“３－６３－４－５－１”与埃塞俄比亚芥品种“Ｄｏｄｏｌｌａ”杂种Ｆ１植株开放受粉获得的Ｆ２群体中筛选出一株半不育、矮杆、甘蓝型油菜类型植株，经连续４个世代自交、分离鉴定出一个二体附加系“９２Ｉ１０９６”。细胞学观察结果，其根尖细胞染色体数２ｎ＝４０，比其母本甘蓝型油菜（２ｎ＝３８）多两条额外染色体。花粉母细胞（ＰＭＣｓ）减数分裂中期（ＭＩ）染色体构型平均为０．４７Ｉ＋１９．７７Ⅱ，     

Stabilization of new types of diploids (2n=22, 24) through selfing of aneuploids (2n=21, 22) derived from crossing of sesquidiploids (2n=29, AAC) and Brassica campestris (2n=20 AA)

Summary Aneuploids with 2n=21 and 2n=22 derived from crossing of sesquidiploids (2n=29, AAC) and Brassica campestris (2n=20, AA) were selfed successively in order to follow the changes in chromosome number of the progenies for three consecutive generations. Progenies with 2n=22, 23 and 24 obtained after selfing of S₀ generation and the succeeding S₁, S₂ and S₃ generations were analyzed in terms of pollen stainability, % seed set as well as cytogenetically based on meiotic behaviour with the aim of determining the possibility of addition of one or more alien chromosomes into n=10 species which may lead to differentiation of single or plural disomic addition lines. The generation of aneuploids with 2n=21 progressed in such a way that most plants seem to revert to the 2n=20 chromosome number of B. campestris after selfing. From 2n=22 aneuploids, however, the succeeding progenies showed high frequency of plants with two additional chromosomes which accounted for 50.6% and 52.9% of total S₃ progenies via 2n=22 and 2n=24 S₂ generations, respectively. The meiotic behaviour of these progenies indicated evidence for a rule governing the frequency distribution of chromosome number among these addition lines and high possibility to breed such disomic plants with 2n=22. A method of selecting stable aneuploids was suggested in addition to the possible role of pollination biology at various processes of such breeding program.     

利用人工合成甘蓝型油菜建立白菜—甘蓝附加系

采用白菜子房培养和甘蓝胚培养方法得到了带有标记性状的白菜和甘蓝种间杂种，并将这些人工合成甘蓝型油菜回交于白菜，得到３１个白菜－甘蓝单体附加系（２ｎ＝２１），和１８个双体附加系（２ｎ＝２２），为进一步对附加染色体上的所载基因定位奠定了基础。此外，还研究了附加甘蓝染色体对雄笥和雌性育性的影响，结果表明，白菜中附加甘蓝染色体可明显降低育性，特别是可显著降低附加系的自交和杂交结实率。     

秋水仙素对辣椒生长的影响及多倍体诱导效应研究

以秋水仙素为诱变剂,比较了不同浓度和不同处理时长组合下辣椒(Capsicum annuum L.)的生长差异和多倍体诱导效应.结果表明:和对照植株相比,所有处理变异植株在形态上表现为叶片宽大,叶色较深,茎变粗且节间距长,气孔增大;相关指标都表现出显著或极显著的差异,而且随着处理强度的增加差异更为明显.秋水仙素诱导产生的变异植株经染色体倍性鉴定植株表征为嵌合体(2 n=24或2 n=48).相同处理时长下,不同浓度秋水仙素溶液对辣椒多倍体诱导效应差异显著,而对相同浓度下不同处理时长对植株叶片数和气孔面积影响差异不显著(P>0.05),其它指标差异极显著(P<0.01).秋水仙素浓度与处理时长之间存在交互作用,不同处理之间除气孔面积差异不显著之外,其它指标间差异显著或极显著.结合加倍率和植株死亡率综合分析后认为,用0.05%的秋水仙素溶液处理6天诱导效果最佳,诱导诱导加倍率可达26.1%.     

Chromosome pairing in haploid plants of radish derived from alien monosomic addition lines

Six haploid plants of radish were obtained via alien monosomic addition lines (2n = 19). One plant was derived from anther culture of an Raphanus sativus‐Brassica oleracea addition line, one plant from the selected smaller seed of an R. sativus‐Sinapis arvensis addition line and four plants from an R. sativus‐B. rapa addition line. During metaphase I of pollen mother cells, two plants exhibited the chromosome pairing of (0‐3)II+ (3‐9)I, three showed (0‐1)III + (0‐3)II + (3‐9)I and the remaining plant (0‐2)II + (5‐9)I. Trivalents seemed to be formed by the pairing between two larger chromosomes and the smaller one with somewhat loose pairing. All haploid plants were inferior to the radish cv. ‘Shogoin’ (2n = 18) with respect to vegetative growth. Their flowers were smaller, with sterile pollen grains, but a few normal flowers with fertile pollen sporadically developed and then produced a few seeds. It is suggested that the radish genome (R, n = 9) might comprise three pairs of homoeologous chromosomes, with the remaining three chromosomes carrying the homologous region(s) that results in a trivalent formation.     

Intergeneric crosses with Fragaria and Potentilla. I. Crosses between Fragaria moschata and Potentilla fruticosa

Summary Crosses between Fragaria moschata (2n=42) and Potentilla fruticosa (2n=14) produced many seeds; about half of these germinated, but only nine plants were obtained from 554 seedlings. Five plants died without flowering, and four were vigorous but sterile. Four plants had the expected chromosome number (2n=28), one plant had 21 chromosomes, and four were aneuploid with 23, 24, 25 and 27 chromosomes respectively. Response to growth substances giving elongated stem internodes and swollen but not succulent receptacles provided evidence that two of the plant were hybrids. It is suggested that all nine plants originated from a normal fertilisation which was followed in some cases by chromosome elimination at an early stage of embryo development.     

Resistance to barley yellow-dwarf-virus disease in derivatives of crosses between hexaploid wheat and species of Lophopyrum (Triticeae; Poaceae)

Among the wheatgrasses that are possible sources of genetic resistance for wheat to barley yellow-dwarf-virus disease (BYD) are those that have been commonly subsumed under the name Agropyron elongatum (Host) P. Beauv. Two of these wheatgrass species are the diploid Lophopymm elongatum (Host) Á. Löve (2n = 2x = 14) and the decaploid L. ponticum (Podp.) Á. Löve (2n = 10x = 70). These two species, the addition and substitution lines of L. elongatum chromosomes in hexaploid wheat (Triticum aestivum L.), and derivatives of hybrids between hexaploid wheat and L. ponticum, were screened for resistance to BYD, as defined by visual symptoms in field-grown plants. The two species, an amphiploid derived from L. elongatumבChinese Spring’ wheat, and the derivatives involving L. ponticum chromosomes were all highly resistant. The substitution and addition lines of L. elongatum chromosomes in ‘Chinese Spring’ revealed that the genetic control of resistance in L. elongatum must be complex, with more than one critical locus involved. Chromosomes 2E and 5E are involved and there are lesser contributions to resistance from the remaining wheatgrass chromosomes. One highly resistant derivative was determined to have only three pairs of L. ponticum chromosomes. It has a wheat-like morphology and shows promise for further characterization.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

Improving Fiber Quality Traits of Xinluzao 45 Using Gossypium barbadense Chromosome Segment Substitution Lines

Gossypium barbadense chromosome segment substitution lines could be used as an important germplasm resource for improving fiber quality traits of upland cotton. In this study, we used TM-1 background substitution lines CSSL-122, the Xinjiang upland cotton Xinluzao 45 and hybrid, backcross progeny BC₂F₁ populations comprising 120 individual plants as test materials. Nineteen SSR markers located in Gossypium barbadense chromosomes land inked with fiber length and strength were used to screen out obvious polymorphic primers between the parents. We then traced and detected chromosome segments of Gossypium barbadense in the BC₂F₁ populations. Simultaneously, we compared fiber quality traits of positive plants that contained Gossypium barbadense chromosome segments with non-positive plants in the BC₂F₁ populations. The results showed that two markers, NAU2987 and BNL3145, which were linked with fiber length, could accurately identify the positive plants. In addition, compared with non-positive plants, the increased fiber length and strength of the positive plants were very significant (P < 0.01). Our research suggested that alien Gossypium barbadense chromosome segments significantly improved the fiber quality traits of Xinluzao 45. Thus, the Gossypium barbadense chromosome segment substitution lines will provide a vital theoretical basis and practical reference for improving Upland Cotton fiber quality traits.     

The effect of colchicine treatment on Solanum acaule and S. bulbocastanum; A complete analysis of ploidy chimeras in S. bulbocastanum

Summary Seeds of tetraploid Solanum acaule (2n=48) and diploid S. bulbocastanum (2n=24) were germinated in petri-dishes on filter paper soaked in 0.3% colchicine. An additional treatment with 0.3% colchicine was applied one month after sowing at four successive days in the axils of the cotyledons of the seedlings. S. acaule appeared much more sensitive to colchicine (14 surviving seedlings from 500 seeds) than S. bulbocastanum (109 surviving seedlings from 450 seeds). Six S. acaule plants with 2n=96 chromosomes were obtained against 38 S. bulbocastanum plants with 2n=48 chromosomes.The ploidy level in each of the three germ layers L₁, L₂, L₃ was determined in 113 plants of S. bulbocastanum and the following results were obtained. Four of the eight possible ploidy types were detected, viz 2x-2x-2x (72 plants), 4x-2x-2x (3 plants), 2x-4x-4x (9 plants) and 4x-4x-4x (29 plants). Doubling the number of chromosomes resulted in a highly significant increase of the number of chloroplasts in the guard cells of stomata and a greatly significant decrease in the proportion of trimerous pollen, male fertility and leaf index. The variability for all characters studied, except for leaf index, was clearly lowest in the 2x-2x-2x group. All plants with a 2x-L₂ were highly male fertile and self-incompatible, also in the three bud stages tested. Male fertility of the plants with 4x-L₂ varied greatly: 12 plants had more than 90% stainability, 5 plants must be considered male sterile. All non-sterile plants with 4x-L₂ were found to be self-compatible, pointing to a gametophytic system of incompatibility in S. bulbocastanum.     

Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.