NONINVASIVE GIANT PANDA PATERNITY EXCLUSION

Giant panda hair samples obtained by noninvasive methods served as a source of DNA for amplification of seven giant panda microsatellite loci utilizing the polymerase chain reaction. Thirteen giant pandas held in Chinese zoos were tested for identification of paternity. Some males listed as sires have been excluded as the biological father of captive-born giant pandas. Because of the death of some potential sires, paternity is still not assigned for some giant pandas, although there is a high likelihood that paternity assignment could be made if postmortem samples are available for genetic analysis. The DNA microsatellite variation assayed by the test we have developed provides a rapid, highly informative, and noninvasive method for paternity identification in giant pandas. (C) 1994 Wiley-Liss, Inc.

Sequence variation and genetic diversity in the giant panda

About 336-444 bp mitochondrial D-loop region and tRNA gene were sequenced for 40 individuals of the giant panda which were collected from Mabian, Meigu, Yuexi, Baoxing, Pingwu, Qingchuan, Nanping and Baishuijiang, respectively. 9 haplotypes were found in 21 founders. The results showed that the giant panda has low genetic variations, and that there is no notable genetic isolation among geographical populations. The ancestor of the living giant panda population perhaps appeared in the late Pleistocene, and unfortunately, might have suffered bottle-neck attacks. Afterwards, its genetic diversity seemed to recover to same extent.

Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Sox and Zfx genes in giant panda

By using PCR cloning techniques, the DNA sequences of the HMG box regions of six Sox genes (pSox) and the zinc finger domains of two Zfx genes (pZfx) in the giant panda were identified. The giant panda Sox genes fell into two subfamilies, SOX-S1 and SOX-S2. The pSox and pZfx genes of the giant panda were highly homologous to the corresponding genes in mammals and revealed close substitution rates to those in the primates.

The giant panda (Ailurapoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the Lest, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastocyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the interspecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not species-specific; (ii) there is compatibility between interspecies somatic nucleus and ooplasm during early development of the reconstructed egg.

Microsatellite DNA analysis proves nucleus of interspecies reconstructed blastocyst coming from that of donor giant panda

A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g(010) was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.

Sequencing, annotation and comparative analysis of nine BACs of giant panda (Ailuropoda melanoleuca)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.

The sequence and de novo assembly of the giant panda genome

Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.

Impacts of the Wenchuan Earthquake on the Giant Panda Nature Reserves in China

Ministry of Science and Technology of China [2008BAK47B02, 2008BAC44B04, 2008BAK50B06, 2008BAC43B01, 2006BAC08B06]

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the Giant Panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E. coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

濒危物种保护、生境破碎化及其恢复

保护区是否真的能起保护作用？保护区究竟如何布局？这是自然保护区的建设一直争论不休的课题。就大熊猫保护而言，目前面临的最紧迫问题可能不是保护区数量多少的问题，而是这些多个保护区组成的保护区群的配置或格局是否合理。因此，本研究以岷山大熊猫保护区群为研究对象，分析现有大熊猫及栖息地及其保护现状，指出目前保护区存在的问题，并对岷山大熊猫自然保护区群合理布局提出建议性设计，以期为大熊猫保护及自然保护区建设的理论提供借鉴。 到目前，为了保护大熊猫，在岷山地区已经建立了1 7个自然保护区，这些保护区的建立对大熊猫的保护起到了非常重要的重要。但是，大熊猫栖息地仍然持续下降。就其原因有两方面，即岷山地区人为活动的干扰，如旅游业的盲目发展。但，同时大熊猫自然保护区本身的布局存在一定的不合理性。最后，从景观尺度上提出岷山大熊猫保护区群合理建设的布局，即在分析现有保护布局基础上，找到优先保护的敏感地带建立廊道，将所有保护区连成布局合理的保护区群。 大熊猫受威胁的关键因子并不是竹子，而是森林．竹子复合系统的破碎化或质量的退化，故保护大熊猫应从保护森林、恢复森林．竹子复合生态系统的质量入手。但是，具体实践中究竞采取什么模式来恢复退化的大熊猫栖息地？选取不同模式的科学依据是什么？如何评判不同恢复模式的成本和效益？这些方面的工作到目前缺少系统研究。本研究在分析了岷山地区自然、社会与经济条件的基础上，从荒山荒地造林、天然林保护与低效林地改造主要植被恢复措施着手，全面分析各类植被恢复措施所形成植被的生态效益（保持水土、涵养水源、保持肥力等）、经济收益（木材、果品和林副特产品）、社会效益（种植结构、劳动力再分配等），总结出植被恢复成本和效益的构成要素，计算主要植被恢复模式的成本和效益，为选择适宜的植被恢复措施、典型植被恢复模式和合理确定植被恢复技术提供科学依据，从而推动岷山植被快速健康地恢复，为大熊猫提供适宜的生存空间。 三峡大坝的建设使其固有水域的人为改造导致水位上升，使原本连续的山地生态系统被分割成大小不等的岛屿。水位变化导致的这种生境岛屿化为栖息地破碎化和岛屿生物地理学提供了十分难得的实证研究的机会。然而，同时，这种栖息地的片段化无疑对三峡库区生物多样性保护和生态系统的完整性造成威胁。到目前，为了监测库区建设对生态环境的影响，已经建立了22个定位观测站，但没有一个是生境破碎化方面的，为此建议尽快建立生境破碎化方面的定位研究站。三峡库区生物多样性丰富，但保护区面积仅仅占库区面积的6.9%，而同期我国已经建立了1 999个自然保护区，占国土面积的15%，因此，非常迫切建立更多的保护区（至少达到全国平均水平）。为了从宏观尺度上加强对库区的综合保护，建议从景观生态学的角度出发，将库区作为一个自然、社会和经济复合生态系绕对待。

新基因的从头起源与几种重要物种的进化基因组学研究

自然界丰富的生物多样性不断地激发着包括达尔文在内所有生物学家的研 究热情。自从达尔文进化论提出以来，进化生物学所要回答的一个基本问题就 是生物是如何从一个共同祖先进化到如此丰富多样的。随着分子生物学中心法 则的发现和基因组时代的到来，比较不同物种的基因组（即通过进化基因组学 研究）找出进化过程中发生的遗传变异成为求解这一基本进化生物学问题的重 要方法。 通过比较不同物种的基因组可以发现新基因的诞生是在进化过程中普遍存 在的基本过程，对生物的进化发挥着重要的作用。大量前人的研究认为新基因 主要通过老基因的重复产生，新基因的从头起源很少发生或根本不存在。直至 最近在果蝇中发现了新基因从头起源事件才改变了人们的这种看法，然而这些 研究缺少功能的证据。我们通过比较酿酒酵母近缘种的基因组序列发现了酿酒 酵母中进化出的一个从头起源的新基因BSC4，并且提供了群体遗传学、转录 组、蛋白质组学和表型水平的证据支持这个基因的生物学功能和蛋白编码能 力。同时我们在其近缘种中发现其直系同源的非编码序列拥有RNA 水平的表达 活性，由此我们提出了一个蛋白基因从头起源的两步模型。我们认为一个非编 码DNA 序列进化为蛋白编码基因需要经历两个步骤：第一，DNA 序列先进化出 顺式元件来招募转录机器变成有RNA 转录活性的序列；第二，转录的序列通过 突变获得开放读码框并加入到翻译机器中。 进一步的分析提示BSC4 可能在酿 酒酵母转换到营养贫瘠的环境中并进入生长停滞期时对酿酒酵母的适应性作出 了贡献。酿酒酵母是一种对人类生活十分重要的微生物，它进行发酵的能力在 工业生产中具有重要应用价值。生长停滞期是酿酒酵母实际生产应用中频繁经 ii 历的过程，对这一阶段的适应性进化也对其工业应用有重要意义。 大熊猫是我国的国宝。它是一种具有独特特性的熊科动物，进化上属于食 肉目类群，食性确以竹子为主。为了适应其食性，其前掌的籽骨还发育出了著 名的“伪拇指”来帮助其进食。然而这些性状是如何进化出来的确一直是个未 解之谜。进化基因组学为解决这些问题提供了一个重要的思路和方法。我们通 过应用第二代测序技术对大熊猫基因组进行了从头测序和组装，通过和其它基 因组比较分析发现了大熊猫基因组中不存在编码降解纤维素酶的基因，提示了 大熊猫特殊食性的进化机制很可能是通过其肠道微生物的改变而发生的。同 时，我们也发现了大熊猫鲜味受体的退化，这很可能是一个伴随其食性进化而 发生的变异。 长雄野生稻是栽培稻的近缘种，它和栽培稻同属于AA 基因组。由于它具有 以发达的地下茎为生理表型的多年生特性和自交不亲和性，研究这些特性背后 的遗传机制对改良栽培稻一年生为多年生和构建自交不亲和的新杂交稻育种体 系有重要意义。我们从头测序并组装了长雄野生稻的基因组，通过和栽培稻基 因组的比较分析，在前人工作的基础上找出了决定上述两个重要性状的可能的 基因组区域，为进一步的实验验证提供了候选的基因。同时我们的序列提供了 对其它野生稻特性研究的重要基础。