Morphological variability between wild populations and inbred stocks of a Chinese minnow, Gobiocypris rarus

Gobiocypris rarus, a small, native cyprinid fish, is currently widely used in research on fish pathology, genetics, toxicology, embryology, and physiology in China. To develop this species as a model laboratory animal, inbred strains have been successfully created. In this study, to explore a method to discriminate inbred strains and evaluate inbreeding effects, morphological variation among three wild populations and three inbred stocks of G. rarus was investigated by the multivariate analysis of eight meristic and 30 morphometric characters. Tiny intraspecific variations in meristic characters were found, but these were not effective for population distinction. Stepwise discriminant analysis and cluster analysis of conventional measures and truss network data showed considerabe divergence among populations, especially between wild populations and inbred stocks. The average discriminant accuracy for all populations was 82.1% based on conventional measures and 86.4% based on truss data, whereas the discriminant accuracy for inbred strains was much higher. These results suggested that multivariate analyses of morphometric characters are an effective method for discriminating inbred strains of G. rarus. Morphological differences between wild populations and inbred strains appear to result from both genetic differences and environmental factors. Thirteen characters, extracted from stepwise discriminant analysis, played important roles in morphological differentiation. These characters were mainly measures related to body depth and head size.

High variability of the gvpA-gvpC region in Microcystis

Gas vesicles provide buoyancy to Microcystis and other common cyanobacterial bloom-forming species. gvpA and gvpC are structural genes encoding gas vesicle proteins. Phylogenetic analyses of 10 Microcystis strains/uncultured samples showed that gvpC and each intergenic segment of the gvpA-gvpC region can be divided into two types. The combination of different types of gvpC and intergenic segments is an important factor that diversifies this genomic region. Some Microcystis strains isolated in China possess a 172 to 176 bp sequence tag in the intergenic segment between gvpA and gvpC. The gvpA-gvpC region in Microcystis can be divided into at least 4 classes and more numbers of subclasses. Compared to rbcLX and other regions, the high variability of the gvpA-gvpC region should be more useful in identifying geographical isolates or ecotypes of Microcystis.

Microsatellites assessment of Chinese sturgeon (Acipenser sinensis Gray) genetic variability

Four microsatellites were used to examine the genetic variability of the spawning stocks of Chinese sturgeon, Acipenser sinensis, from the Yangtze River sampled over a 3-year period (1999-2001). Within 60 individuals, a total of 28 alleles were detected over four polymorphic microsatellite loci. The number of alleles per locus ranged from 4 to 15, with an average allele number of 7. The number of genotypes per locus ranged from 6 to 41. The genetic diversity of four microsatellite loci varied from 0.34 to 0.67, with an average value of 0.54. For the four microsatellite loci, the deviation from the Hardy-Weinberg equilibrium was mainly due to null alleles. The mean number of alleles per locus and the mean heterozygosity were lower than the average values known for anadromous fishes. Fish were clustered according to their microsatellite characteristics using an unsupervised 'Artificial Neural Networks' method entitled 'Self-organizing Map'. The results revealed no significant genetic differentiation considering genetic distance among samples collected during different years. Lack of heterogeneity among different annual groups of spawning stocks was explained by the complex age structure (from 8 to 27 years for males and 12 to 35 years for females) of Chinese sturgeon, leading to formulate an hypothesis about the maintenance of genetic diversity and stability in long-lived animals.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

水稻同源四倍体高代自交系农艺性状分析及遗传差异研究

课题组在不断地创制新的同源四倍体材料的同时，连续多年以提高结实率为目的培育、筛选自交系材料，已获得自交繁殖十二年的高代自交系材料。相对于诱导创制初期，材料表现出的结实率低，同种系单株间的差异较大；高代材料已表现出较显著的结实率提升和较一致的农艺性状表型。 本实验选取课题组多年培育的同源四倍体水稻高代自交系材料，通过形态学、农艺性状和细胞遗传学比较，研究水稻同源四倍体与二倍体之间的异同。结果显示，所有同源四倍体材料的染色体组成均为2N=4X=48，花粉母细胞(PMC)减数分裂行为较正常，99%以上的染色体都能在减数分裂中期I（MI）发生联会、配对，形成四价体和二价体，这与理论染色体组构成相符。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。统计分析表明，二价体和四价体的比例对结实率没有显著影响，但是三价体的数目对结实率有一定影响。这一结果表明了结实率和细胞减数分裂行为可能存在相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了依据。 然后，对同源四倍体水稻高代自交系材料进行农艺性状和品质性状的统计与分析。主要针对结实率、每穗实粒数、有效分蘖和穗长等主要农艺性状，以及直链淀粉含量这一重要的品质性状进行统计。将统计结果与1996年诱导加倍的初代材料的数据相对比分析，结果显示所有材料经过多代选育培养，其农艺性状已经有了较显著的提高，同时同源四倍体材料的农艺性状稳定性也有了较显著的提升。如结实率的提高幅度较大，所有材料的平均结实率均显著高于加倍初代，而同种材料不同单株间的结实率差异也显著地减少，变异系数（CV）的平均值由1996年的41.15%减少到了2008年的28.81%。其他重要农艺性状也有不同程度的改良，种内变异系数也相应地降低。此外，实验测量了同源四倍体材料和来源二倍体材料的直链淀粉含量。分析结果显示，部分材料的直链淀粉含量与二倍体亲本产生了较显著的差异，这可能是诱导加倍过程中的遗传变异造成的；同源四倍体材料的种内变异系数（CV）平均值由1996年的6%下降到了2008年的3.88%，显示出在品质性状方面，同源四倍体材料的遗传稳定性也有较显著的增加。同源四倍体材料农艺性状经过多年的选育，表现出一定的提升，同时，经过多年自交纯化，所有材料种系内的性状差异逐渐缩小，说明同源四倍体水稻的遗传稳定性随着自交纯化而增强，这为同源四倍体水稻的进一步选育打下了良好的基础。 最后，通过测量连续两年的自交系材料的遗传多态性，分析材料间遗传差异和种群遗传结构，深入研究连续两代材料间的遗传差异，研究同源四倍体水稻与二倍体材料遗传稳定性之间的差异。实验采用18对SSR微卫星标记对连续两代15个材料，共94份样本进行差异分析。通过扩增条带长度多态性分析，计算不同材料以及同种材料不同世代间的遗传距离，构建同源四倍体和二倍体水稻的分子指纹库，并绘制聚类图。结果显示，同源四倍体和二倍体不同材料间的遗传差异比较大，遗传距离处于0.4757至0.2816之间；而相同品种不同世代材料间的遗传差异较小，但也表现出一定的遗传差异。同种同源四倍体材料不同世代间的遗传差异比二倍体材料更大，两代四倍体材料间遗传距离处于0.1359至0.0485之间；而两代二倍体材料间的遗传距离处于均小于0.0388。结果表明，同源四倍体水稻高代材料具有一定的遗传稳定性，但与来源二倍体材料相比，其世代间的遗传变异性仍然较强。这种结果说明，经过多代的自交纯化培育，同源四倍体水稻材料能够建立起相对稳定的遗传结构，同时，其强于二倍体亲本的变异性有能够为新品种的选育，农艺性状、品质性状的改良提供一定的遗传基础。此外，分析结果表明通过分子标记辅助检验，水稻材料间的遗传多态性能够有效地区分不同的品种，这为水稻品种的分子鉴定提供了一定的依据。 本研究从细胞学鉴定，农艺性状统计分析以及分子标记辅助聚类分析多方面地对同源四倍体水稻高代系进行了研究，对探究同源四倍体水稻的遗传规律，进一步揭示其遗传特性、农艺性状的遗传构成，为进一步选育优质的多倍体水稻提供了一定的理论依据。 This group insists on creating new Autotetraploid Rice (Oryza sativa L.) materials, while improving the seed-setting of them for many years, cultivated and selected the inbred line materials, has obtained the high generation inbred lines after twelve years cultivation. Compared to the early induced materials, which shown the low seed setting, and the large difference between the different plants in the same germ-line; the high generation materials have shown significant improvement in seed setting and more uniform phenotype agronomic traits. The autotetraploid rice high generation inbred lines material, which has been cultivated for more than 12 years, was chose in this experiment. The similarities and differences between autotetraploid and diploid rice was studied through morphological, agronomic and cytogenetic ways. The results showed that all the chromosome of autotetraploid materials are composed of 2N=4X=48, the pollen mother cells (PMC) meiosis behavior is normal, more than 99% chromosomes in metaphase I(MI) were federated and paired to form tetravalents or bivalents, which constitutes a consistent theory of genome. In the meiosis process, the material with a higher seed setting showed less chromosome abnormal than the material whose seed setting is lower. However, statistical analysis showed that the bivalent and tetravalent rate had no significant impact on seed setting, but the number of trivalent had a certain impact on seed setting. The result shows that the seed setting may be related to the meiosis behavior, which provides a basis to cultivate new autotetraploid germ line with high seed setting through the meiotic behavior. Furthermore, the agronomic and quality traits of autotetraploid rice high generation inbred material were statistically analyzed. The statistically analysis was focused on major agronomic traits such as: seed setting, grains per panicle, effective tillers and panicle length, as well as the important quality trait amylose content. The statistic data was compared with the data in 1996, when the first induced generation of autotetraploid material, and the result shows that after a multi-generation breeding, the agronomic traits has been significantly improved in all the materials, while the stability of agronomic traits also significant upgraded. For instant, the seed setting increased significantly, the average seed setting of all materials was significantly higher than the first induced generation, and the differences between different plants in the same species also significantly reduced, the average of the coefficient of variation (CV) was reduced from 41.15% in 1996 to 28.81% in 2008. Other important agronomic traits had improved in different degrees; the coefficient of variation within species is also reduced accordingly. In addition, the amylose content of autotetraploid and diploid materials was measured in this experiment. The results shows that the amylose content of some of the material differed from diploid parents significantly, it may caused by the genetic change during the inducing, autotetraploid materials intra-specific coefficient of variation (CV) average reduced from 6% in 1996 to 3.88% in 2008, shows that this is a significant increase of quality traits stability in autotetraploid rice. Agronomic traits of autotetraploid material shows some improvement after years of breeding, at the same time, after years of purification, all material within the germ-line gradually narrow the differences in traits indicates that autotetraploid rice genetic stability was enhanced, which laid a good foundation for the further autotetraploid rice breeding. Finally, this experiment studied the genetic differences between materials of two generations and researched the difference of genetic stability between diploid and autotetraploid rice materials through investigating the genetic polymorphism, genetic differences between materials and population genetic structure of inbred line materials of two consecutive years.18 pairs of SSR microsatellite markers for 15 materials of two generations were used in this experiment, and the total of 94 samples were analyzed. Through the amplification length polymorphism analysis of different materials and materials in different generations, the genetic distance between materials and generations was analyzed, a diploid and autotetraploid rice molecular fingerprint database and map rendering cluster were constructed. The result shows that the genetic distance is between 0.4757 to 0.2816 among different autotetraploid and diploid materials; the genetic distance between different generations of same species was less, but also shows a certain degree of genetic differences. The inter-generational genetic differences of autotetraploid materials were greater than of the diploid materials, which are 0.1359 to 0.0485 as the genetic distance; comparing with the 0.0388 of diploid materials. The result shows that high generation inbred autotetraploid rice material has a certain genetic stability, but the genetic variation between generations is still strong comparing with the source diploid materials. It indicates that, after many generations of purification cultivation, autotetraploid rice materials established a relatively stable genetic structure, at the same time, stronger variability than its diploid parents are useful in the breeding of new varieties, provides a genetic foundation to the agronomic and quality traits improvement. In addition, the analysis result shows that the through the molecular marker-assisted testing, rice genetic polymorphism between materials can effectively distinguish different species, provides a certain basis for molecular identification of varieties of rice. A series of investigation such as cytological identification, statistical analysis of agronomic traits, molecular marker-assisted cluster analysis was applied in this experiment to research genetic pattern of autotetraploid rice high generation inbred lines, revealed the genetic characteristics and the genetic composition of agronomic traits, provides a theoretical basis for the further selection of high quality autotetraploid rice.

potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.