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.

小麦胚乳蛋白组分和Glu-B3位点基因核心编码区差异与小麦品质关系的研究

小麦加工品质改良已成为我国小麦育种的主要目标之一。特别是我国加入WTO以后，对小麦产品的质量提出了更高的要求，小麦品质改良的任务将更加艰巨和重要，小麦胚乳蛋白是影响小麦加工品质性状的重要因素。因此，深入了解小麦胚乳蛋白对加工品质性状的影响及其分子基础，为品质改良提供理论依据和科学指导，对加速我国小麦品质育种和优质小麦生产具有重要意义。本研究选用在麦谷蛋白5个基因位点(Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3)上均含不同等位基因的小麦品种99G45和京771及Pm97034和京771杂交F9代共164个麦谷蛋白纯合系，及228个中国推广普通小麦品种和高代育成品系为试材，研究了麦谷蛋白Glu-1和Glu-3位点基因等位变异对籽粒蛋白、湿面筋含量、Zeleny沉降值和SDS沉降值间的关系；本研究还利用小麦A、B和D基因组中低分子量麦谷蛋白亚基（LMW-GS）基因特异引物，通过PCR方法克隆了1个Glu-A3位点和3个Glu-B3位点LMW-GS基因片段，在此基础上分析了不同等位基因对品质造成差异的分子基础；另外，本研究对中国近年推广的部分品种和育成的高代品系资源的多样性进行了分析。现将主要研究结果简述如下： 1. 对来自三个麦区的148份材料的醇溶蛋白组成进行了分析，结果表明，各麦区醇溶蛋白模式具有较大差异。在ω区,A7、B、E、F、G、J、P、Q、S和U仅存在于西南秋播麦区；A3、M、N、R、W和X仅存在于黄淮特种麦区；K仅存在于北方冬麦区；A6是北方冬麦区出现频率最高的带型模式，而西南秋播麦区中D出现的频率最高。ω-区的E、H和M几种模式是以前国内外未曾报道的。且初步确定，这些模式对品质性状具有正效应。至于γ区,A、B、D、E和F在各区均有出现，其中B和E在各区出现的频率都很高，在26.1-39.6％之间。相反，H 仅出现在黄淮特种麦区，J仅限于西南秋播麦区。对于β-区醇溶蛋白，B型模式在所有区中都相当高，而模式A仅存在于第三区.对于α-区，模式A在Ⅲ区而模式D在Ⅱ区出现的频率很高。1BL.1RS易位系在中国小麦品种中出现频率高达41.2％，在I, II和Ⅲ麦区的出现频率分别为 45.5、43.5和35.2%。各生态区模式的差异可能是品种适应不同生态条件和人为选择的结果，但这有待进一步证明。由于醇溶蛋白位点（Gli-1）与LMW-GS位点（Glu-3）紧密连锁，本结果可为下面确定普通小麦LMW-GS等位基因变异所用。 2. 利用Gli-1与Glu-3的紧密连锁，以228个小麦品种/系为材料，首次对中国小麦品种麦谷蛋白亚基的6个位点进行综合分析，研究小麦籽粒蛋白与品质性状间的关系，结果表明6个高分子量（HMW）和低分子量（LMW）麦谷蛋白位点对蛋白质含量的效应大小为，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3；对GMP含量的效应大小为, Glu-A3>Glu-B3>Glu-D1> Glu-B1>Glu-A1>Glu-D3；对湿面筋含量的效应大小为, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1；对Zeleny沉降值的效应大小为, Glu-A1> Glu-B3>Glu-D3>Glu-D1>Glu-B1>Glu-A3；对SDS沉降值的效应大小为, Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1。对蛋白含量而言，各位点的最佳组合方式为1、17+18、5+10、Glu-A3e、Glu-B3g、Glu-D3b；对湿面筋含量而言，各位点的最佳组合方式为1、6+8、5+10、Glu-A3d、Glu-B3c、Glu-D3b；对Zeleny沉降值而言，各位点的最佳组合方式为N、17+18、5+10、Glu-A3d、Glu-B3d、Glu-D3b；对SDS沉降值而言，各位点的最佳组合方式为1、7+8、2.2+12、Glu-A3b、Glu-B3g、Glu-D3b。另外，分析了稀有亚基对5+12与2.2+12与品质性状的关系，认为5+12对品质有负效应，2.2+12对品质有正效应。在品质育种时，应对优异组合或优异亚基加以利用。 3. 首次利用重组自交系（RILs）为材料，研究麦谷蛋白亚基表达量与品质性状的关系，通过对重组自交系中各HMW-GS表达量的分析，认为，就单个亚基的表达量而言，7亚基最高；其次为2亚基、5亚基、12亚基和10亚基；亚基9和1的表达量最小；N亚基不表达。对成对出现的亚基对而言，x型和y型亚基的总表达量2+12>5+10>7+9>17+18。就单个亚基与品质性状的关系而言，仅有10亚基的表达量与蛋白含量的相关性达5%的显著水平，2亚基的表达量与湿面筋含量呈负相关，显著水平也达5%，其余单个亚基对品质性状均无显著影响；就x型/y型亚基的比例来看，2/12和5/10对湿面筋含量都有显著的负效应；对某一位点等位基因控制的亚基表达总量来看，2+12对SDS沉降值有显著负效应。另外，本研究得出：2＋12的亚基对的负效应主要体现在2亚基上，且在同一位点上，x型亚基的表达量大于y型。所以推导稀有亚基组合2+10很可能也是劣质亚基。 4. 以 Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3作为5个因素对99G45/京771和Pm97034/京771杂交后代的蛋白质含量和SDS沉降值进行多因素方差分析。结果表明，Glu-A1和Glu-D3对蛋白含量的加性效应达5％显著水平；Glu-D1 * Glu-D3对蛋白质含量的互作效应也达5%显著水平；其余位点的加性和互作效应对蛋白质含量的影响均不显著。对SDS 沉降值而言，Glu-D1的加性效应最大,贡献率为4.2 % ,达1 %显著水平,其次是Glu-B1位点,贡献率为3.3% ,达5%显著水平。其余位点对SDS 沉降值的加性和互作效应均未达5%显著水平。总体而言, 各位点对蛋白含量的效应大小为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3；对SDS沉降值的效应大小为Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。Glu-D1和Glu-D3位点上等位基因变异对蛋白含量有显著或极显著影响，含Glu-D1d和Glu-D3 GD、Glu-D3 JD基因的株系分别比含Glu-D1a和Glu-D3 PD基因的株系有较高的蛋白含量；在该遗传背景下，麦谷蛋白各基因位点对蛋白含量的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9>17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。对SDS沉降值的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9=17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。所以，对蛋白含量和SDS沉降值均较好的组合为1，7+9，5+10，GB，GD。 5. 因为GB和PB对品质的效应有显著差异，选取LMW-GS位点特异扩增引物对京771、99G45和Pm97034的Glu-B3位点进行扩增，结果得到三个不一样的扩增片段（Genebank号为DQ539657－DQ539659），得到的基因片段与Genebank中已报道的同类序列高度同源。通过克隆片段组成的分析，发现对Pm97034的序列较京771和99G45段少一个7氨基酸的重复单元，这可能是它较另外两个片段对面筋强度影响小的主要原因；另外，在99G45的序列中，124位处出现L（亮氨酸）代替P（脯氨酸），158位处出现了T（苏氨酸）代换M(蛋氨酸)，这可能是99G45Glu-B3位点序列对SDS沉降值的效应显著优于Pm97034的原因。 6．通过对RILs各位点同普通小麦品种（系）各位点与品质关系的比较，发现对SDS沉降值的效应，各位点在不同研究材料中是不同的，普通小麦中：Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1，RILs中：Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。利用重组自交系材料（完全排除了1BL/1RS易位干扰）所得到的结果与Gupta and MacRitchie (1994)所得结论一致。进一步证实了1BL/1RS易位对小麦品质的重要影响。对蛋白含量而言，普通小麦品种（系）中，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3，RILs中，Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3，和对SDS沉降值的效应一样，推断在非1BL/1RS易位的情况下，各位点对其效应应为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3。 对同一位点的等位基因而言，普通小麦和重组自交系中Glu-A1和Glu-D1上的等位基因对品质性状的贡献是一致的，但Glu-B1上的等位基因对SDS沉降值的贡献发生了变化，普通小麦中17+18>7+9，RILs中7+9>17+18，这可能也是1BL/1RS造成的。 Baking quality improved is one of the main object of wheat bread in China. The overall objective of the present studies was to increase the understanding about protein quality in wheat, i.e. to make it possible to improve the production of wheat with desired quality for different end-uses. With the analysis of gluten protein in RILs, 99G45/Jing 771 and Pm97034/Jing, and 228 wheat cultivars or lines in China, the correlations between glutenin compositions and protein content, glutenin macropolymer(GMP), wet gluten content, Zeleny sedimentation value and SDS sedimentation value contentand breadmaking quality were studied. Also a rapid and efficient detection method of geneticpolymorphism at Glu-B3 loci in wheat was established using polymerase chain reaction(PCR).The results obtained were as follows: 1. Cultivated Chinese wheat germplasm has been a valuable genetic resource in international plant breeding. Patterns of gliadin among cultivated Chinese accessions are unknown, despite the proven value and potential novelty. The objective of this work was to analyse the diversity within improved Chinese wheat germplasm. The electrophoretic banding patterns of gliadin in common wheat cultivars and advanced lines were determined by acid-polyacrylamide gel electrophoresis. For 148 leading commercial cultivars and promising advanced lines used in our study, 48 patterns were identified, 29 corresponding to ω-gliadin, 9 to γ-gliadin, 5 to β-gliadin and 5 to α-gliadin. The most frequent patterns were A6 in ω; B in γ; B in β and A in the region of α. 116 band types appeared in the148 samples: 94 accessions had unique gliadin types, and 22 gliadin types while not unique were found in 54 accessions. The gliadin patterns of Chinese wheat cultivars and lines greatly differed from the patterns of wheat lines from other countries. Three patterns, E, J, H, M, N and O in the ω-zone had not previously been reported. Three wheat zones，the Northern Winter Wheat Region, the Yellow and Huai Valley River valleys Winter Wheat Region and the Southwestern Winter Wheat Region，in China showed different frequencies in their gliadin patterns. This information can be used to monitor genetic diversity with Chinese wheat germplasm. 2. To analyse the relationship between the loci and characteristics quality, we utilized the 228 cultivars/lines. The results showed that : For protein content, Glu-D1 >Glu-B3>Glu-A1=Glu-B1>Glu-A3=Glu-D3. For GMP content, Glu-A3>Glu-B3 >Glu-D1>Glu-B1>Glu-A1>Glu-D3. For wet gluten content, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1. For Zeleny sedimentation value, Glu-A1>Glu-B3> Glu-D3>Glu-D1>Glu-B1>Glu-A3, For SDS sedimentation value, Glu-B3>Glu-A1= Glu-D1= lu-A3>Glu-D3>Glu-B1。For protein content, the best combination of 6 loci is (1,17+18,5+10,Glu-A3e, Glu-B3g,Glu-D3b). For wet gluten content, the best combination of 6 loci is (1,6+8,5+10,Glu-A3d,Glu-B3c,Glu-D3b). For Zeleny sedimentation value, the best combination of 6 loci is (N,17+18,5+10,Glu-A3d, Glu-B3d, Glu-D3b). For SDS sedimentation value, the best combination of 6 loci is(7+8,2.2+12,Glu-A3b, Glu-B3g,Glu-D3b)。Additional, we analysed the relationship between the subunits 5+12 and 2.2+12, think that 5+12 was negative for quality, 2.2+12 is postive for quality. It should be effective utilized. 3. It’s the first time to utilize RILs to study the relationship between subunits expression quantity and characteristics quality. The results showed that: For single subunit, the expression quantity of 7 is the highest. Then the 2, 5, 12 and 10. The expression of subunit 9 and 1 is the lowest. Subunit N is not expressed. For subunits, the expression quantity of x type and y type are 2+12>5+10>7+9>17+18. The significant relation of 5% only showed between the expression quantity of subunit 10 and protein content. The relationship between expression quantity of others and characteristic quality was not significant. For x type/ytype, 2/12 and 5/10 is negative relation insignificant level. For the subunit(s) in a loci, Only 2+12 effect SDS sedimentation value negative in significant level. 4. With RILs 99G45/Jing 771 and Pm97034/Jing 771, we found that: The effective of Glu-A1, Glu-D3 and Glu-D1 * Glu-D3 for protein content is significant at 5% level. The effect of other loci for protein wre not significant. For SDS sedimentation value, the effect of Glu-D1is the highest, which contribution is 4.2 % .Then the Glu-B1, contribution is 3.3%. The effect of other loci for SDS sedimentationvalue were not significant. In total, for protein content: Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3; for SDS sedimentationvalue: Glu-D1>Glu-B1> Glu-D3>Glu-A1>Glu-B3. The effect of alleles in Glu-D1 and Glu-D3 loci are significant at 1% or 5%. In Glu-A1, 1>N; Glu-B1, 7+9>17+18>14+15; Glu-D, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. For SDS sedimentation, Glu-A1, 1>N; Glu-B1, 7+9=17+18>14+15; Glu-D1, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. The best combinations for SDS sedimentation value is 1，7+9，5+10，GB，GD. 5. Because of the difference of GB and PB for SDS sedimentation value, we selected the specific primer for LMW-GS loci to amplified the Glu-B3 of Jing771, 99G45and Pm97034. We got 3 amplify fragment (Gene Bank accession number are DQ539657－DQ539659）. We found that the fragment of Pm97034 were deleted a repetitive 7 amino acid domain, which is perhaps the reason effect the gluten strength. Furthermore, in the position 124 of sequence 99G45, L has been replaced with P. Position 158, T replaced M, which may be the reason why the Glu-B3 locus of 99G45 is prefer to Pm97034 when refer to SDS sedimentation value. 6. Comparing the results of RILs and common wheat, we found that perhaps just the1BL/1RS made the difference of loci in different accession.

Spatial and temporal flows of China's forest resources: Development of a framework for evaluating resource efficiency

Science & Technology Basic Work Program of China: Scientific Survey of the Middle-lower Reaches of Lantsang River and the Great Shangri-La Region [2008FY110300]; National Basic Research Program of China (973 Program): Ecosystem Services and Ecological Safety of the Major Terrestrial Ecosystems of China [2009CB421106]; National Natural Science Foundation of China [30670374]; EU ; European Commission, DG Research [003874]

Synthesis and Stabilization of Novel Aliphatic Polycarbonate from Renewable Resource

A novel aliphatic polycarbonate from renewable resource was prepared by copolymerization of furfuryl glycidyl ether and CO2 using rare earth ternary catalyst; its number-average molecular weight (M-n) reached 13.3 x 10(4) g/mol. The furfuryl glycidyl ether and CO2 copolymer (PFGEC) was easy to become yellowish at ambient atmosphere due to post polymerization cross-linking reaction oil the furan ring; the gel content was 17.2 wt % after 24 h exposure to air at room temperature. PFGEC could be stabilized by addition of antioxidant 1010 (tetrakis[methylene (3.5-di(tert-butyl)-4-hydroxhydrocinnamate)]methane) in 0.5-3 wt % after copolymerization. The Diels-Alder (DA) reaction between N-phenylmaleimide and the pendant furan ring was also effective for the stabilization of PFGEC by reducing the amount of furan ring and introducing bulky groups into PFGEC. The cyclization degree could reach 72.1% when the molar ratio of N-phenylmaleimide to furan ring was 3: 1, and no gel was observed after 24 h exposure to air. The glass transition temperature (T-g) of PFGEC was 6.8 degrees C, and it increased to 40.3 degrees C after DA reaction (molar ratio of N-phenylmaleimide to furan ring was 3: 1).

Development of SSR primers from EST sequences and their application in germplasm identification of Porphyra lines (Rhodophyta)

This paper reports the development of SSR markers from EST data and their utilization in germplasm identification of Porphyra. The publicly available EST (expressed sequence tag) sequences of Porphyra were searched from the Internet (www.kazura.or.jp/en/plant/porphyra/EST/). From a total of 20,779 obtained EST sequences, 391 SSRs (simple sequence repeats) were analysed with SSRIT software (www.gramene.org/db/searches/ssrtool). From those, 48 SSR primer-pairs were designed and tested by commonly used SSR reaction conditions using 22 Porphyra DNA samples as templates. Results showed that 41 SSR primer-pairs gave good amplification patterns. These were used to conduct SSR analyses of genetic diversity and variety identification of the 22 Porphyra lines. A dendrogram and the DNA fingerprints of the Porphyra lines were developed based on the obtained SSR data.

Identification of Porphyra lines (Rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique AFLP,fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germplasm identification-AFLP) was designed for identification of the 27 Porphyra lines. In addition, 21 specific AFLP markers from 15 Porphyra lines were identified; 6 AFLP markers from 4 Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of the Porphyra lines.

Identification of 27 Porphyra lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.

Genetic transformation in Kappaphycus alvarezii using micro-particle bombardment: a potential strategy for germplasm improvement

This study investigated the delivery of a SV40 promoter driving lacZ gene into cells of Kappaphycus alvarezii using particle bombardment. Thallus pieces 0.5-0.8 mm in diameter and 1 cm in length were prepared as gene recipients. Bombardment parameters of 450 psi (rupture pressures) x 6 cm (particle travel distances), 650 psi x 6 cm, 1,100 psi x 6 cm and 1,100 psi x 9 cm were used. A significant increase in transformation efficiency from about 33% under the rupture pressure of 450 psi to 87% at 650 psi was observed in transformed thalli. Most of the positive cells appeared in epidermal cells bombarded at 450 psi, whereas positive signals were seen in both epidermal and medullary cells at 650 psi. No positive transient expression was detected at a bombardment of 1,100 psi, or in negative or blank controls. For the conditions tested, the best parameter was obtained at 650 psi at a distance of 6 cm. Thus, the strategy of taking vegetative thalli as recipients, using particle bombardment, and combining this with micro-propagation, together with developing an in vivo selectable marker, is a viable way to produce stable transformants, to eliminate chimeric expression, and to achieve transgenic breeding in K. alvarezii.

The genetic analysis and germplasm identification of the gametophytes of Undaria pinnatifida (Phaeophyceae) with RAPD method

Eleven pairs of Undaria pinnatifida (Harv.) Suringar gametophytes were identified with random amplified polymorphic DNA (RAPD) technique. After screening 100 primers, 20 ten-base primers were determined for the RAPD analysis. A total of 312 polymorphic loci were obtained, of which 97.7% were polymorphic. The primer S198 was found to distinguish all the selected Undaria pinnatifida gametophytes. The genetic distances between each two of the twenty-two U. pinnatifida gametophytes ranged from 0.080 to 0.428, while the distances to the Laminaria was 0.497 on average. After reexamination, two sequences characterized amplification region (SCAR) markers were successfully converted, which could be applied to U. pinnatifida germplasm identification. All these results demonstrated the feasibility of applying RAPD markers to germplasm characterization and identification of U. pinnatifida gametophytes, and to provide a molecular basis for Undaria breeding.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (Triticum aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Defoliation, nitrogen, and competition: effects on plant growth and resource allocation of Cleistogenes squarrosa and Artemisia frigida

Two species, Artemisia frigida Willd. (C-3, semishrub, and dominant on overgrazed sites) and Cleistogenes squarrosa (Trin.) Keng (C-4, perennial bunchgrass, and dominant or codominant on moderately grazed sites) were studied to determine the effects of defoliation, nitrogen (N) availability, competition, and their interactions on growth, biomass, and N allocation in a greenhouse experiment. The main treatments were: two nitrogen levels (NO = 0 mg N pot(-1), N1 = 60 mg N pot(-1)), two defoliation intensities (removing 60% of total aboveground biomass and no defoliation), and three competitive replacement series (monocultures of each species and mixtures at 0.5:0.5). Our results were inconsistent with our hypothesis on the adaptive mechanisms of A. frigida regarding the interactive effects of herbivory, N, and competition in determining its dominant position on overgrazed sites. Cleistogenes squarrosa will be replaced by A. frigida on over-grazed sites, although C. squarrosa had higher tolerance to defoliation than did A. frigida. Total biomass and N yield and N-15 recovery of C. squarrosa in mixed culture were consistently lower than in monocultures, whereas those of A. frigida grown in mixtures were consistently higher than in monocultures, suggesting higher competitive ability of A. frigida. Our results suggest that interspecific competitive ability may be of equal or greater importance than herbivory tolerance in determining herbivore-induced species replacement in semi-arid Inner Mongolian steppe. In addition, the dominance of A. frigida on overgrazed sites has been attributed to its ability to shift plant-plant interactions through (lap colonization, root niche differentiation, and higher resistance to water stress.