摘要
遗传连锁图谱是系统进行基因组学和数量性状位点研究的基础。莲集观赏、食用及药用功能于一身,具有极高的经济价值,但在莲的分子遗传连锁图谱研究方面,至今未见相关研究成果发表。基于以上研究背景,本文利用SSR分子标记对来源于中国、泰国和美国的莲野生居群进行了遗传多样性及进化关系分析；根据莲居群遗传多样性分析的结果,选择具有较高DNA序列多态性且遗传背景差异较大的中国莲种质‘中国古代莲’和美洲黄莲种质‘AL1’作为亲本,并利用RAD-seq技术快速检测亲本及其F1分离群体的SNP基因分型,首次构建了莲的高密度遗传连锁图谱。
1莲野生居群遗传多样性评价
1.1莲属植物物种水平上的遗传多样性较高(I=0.95、He=0.50和PIC=0.46),可能与该物种具有的独特进化历史有关。但其居群水平上的遗传多样性却相对较低(I=0.26、He=0.20和PIC=-0.14),其原因可能是：一、适生生境遭到破坏,导致莲野生居群的分布范围及种群数量大大缩减乃至部分消失；二、由于人为干扰导致了莲生存生境的片段化和居群分布的间断化。同时生境片断化后居群间地理距离的增加会进一步阻断居群间的基因流；三、莲本身繁育系统的特殊性也是造成其居群水平上遗传多样性较低的主要原因。
1.2来源于美国的2个美洲黄莲居群、黑龙江省的7中国莲居群和泰国的2个泰国莲居群野生种质间的遗传关系为：泰国莲与美洲黄莲居群间的亲缘关系最远,中国莲与美洲黄莲居群间的亲缘关系居中,而泰国莲与中国莲居群之间的亲缘关系最近。并且美洲黄莲与亚洲莲居群在遗传相似系数(GS)为0.14处以100%靴带支持率分化为两支,表明两者间的遗传差异较大。
1.3支持莲属2个物种的独立分类地位。虽然来自于化石的证据、叶绿体基因rbcL和核糖体DNA ITS区序列的高同源性、染色体及血清学反应的同源性等显示,美洲黄莲和亚洲莲的亲缘关系较近。但两者被太平洋隔离的地理分布格局、一定的杂交生殖障碍、居群间较远的遗传距离及显著的遗传分化变异,再加上植株形态特征的显著不同及其F1植株的自交不结实现象,本文支持莲属2个物种的独立分类地位。
1.4对莲野生居群的保护建议采取下列措施：一、对居群间遗传分化较大的7个中国莲和2个美洲黄莲居群,及遗传多样性相对较高的泰国莲居群THL1实施就地保护；二、在对野生莲进行就地保护的同时,应尽量避免引入栽培莲品种；三、迁地保护。建议采集种子以达到保育尽可能多的居群遗传多样性的目的。
2莲高密度遗传连锁图谱的构建
2.1构建了莲的遗传连锁框架图谱。母本‘中国古代莲’的图谱包括8个连锁群,共32个标记,覆盖基因组长度362.52cM,标记间平均图距为11.33cM,连锁群平均长度为45.315cM；父本美洲黄莲‘AL1’的图谱包括11个连锁群,共137个标记,覆盖基因组长度495.37cM,标记间平均图距3.62cM,连锁群平均长度为45.03cM。
2.2RAD-seq技术是快速获得大量SNP标记的一条有效途径。本研究以‘中国古代莲’的全基因组序列为参考,利用RAD-seq技术分析亲本与分离群体不同单株的SNP标记基因型,共获得了适合图谱构建的6622个SNP多态性标记。
2.3构建了美洲黄莲‘AL1’的高密度遗传连锁图谱。以SSR遗传连锁框架图谱为基础,利用SNP标记构建了一张由1515个标记组成的10个连锁群的高密度遗传图谱。该图谱覆盖基因组长度563.6cM,平均图距为0.38cM。每个连锁群上的标记数介于95-194之间,长度为39.9-83.8cM。
2.4RAD标记是加密图谱和构建高密度遗传连锁图谱的优势分子标记。本文利用RAD标记构建的遗传图谱与莲SSR标记连锁框架图谱相比,总容量增加了1378个标记,总图距增加了68.23cM,标记间平均图距缩小了3.24cM。
2.5本文中不同连锁群上偏分离标记的分布是不均匀的,LG5、LG7和LG9上无偏分离标记,LG3、LG8和LG10上仅有2-3个偏分离标记,而LG1(21)、LG2(19)、LG4(80)和LG6(127)上分布较多,并且LG4和LG6上存在偏分离标记聚集现象。其原因可能是：一、为了增加群体的多态性,采用亲缘关系相对较远的‘中国古代莲’和美洲黄莲‘AL1’作为亲本。亲本间较远的遗传背景差异导致杂种染色体的配对和重组行为受到不同程度的影响,从而使连锁位点间的重组率降低而最终导致偏分离；二、作图群体亲本之一‘中国古代莲’杂合性较低；三、作图群体较小(80个F1单株)也有可能加重偏分离情况。
Researches on genetic diversity of Nelumbo are mainly focused on Chinese lotus with abundant variety resources. About the evaluation of genetic diversity among natural populations of American lotus, Thai lotus and Chinese lotus, few reports was involved. Construction of a genetic linkage map is a powerful research tool for studies on plant genetics and analysis of quantitative trait loci (QTL) underlying traits of economic importance, however, little progress on the genomic constitution of Nelumbo species has been reported. In this studies, genetic diversity and the evolutionary relationship among wild lotus populations in its main distribution area are analyzed. And based on an F1population derived from a cross between Nelumbo nucifera'China Antique' and Nelumbo lutea'AL1', a high-density genetic linkage map of lotus has been constructed using SSR and SNP markers which are discovered by restriction site associated DNA sequencing (RAD-Seq). The main results are as follows:
1Assessment of genetic diversity among wild lotus populations
1.1Wild lotus had a high genetic diversity at species level (I=0.95, He=50and PIC=0.46), and a unique evolutionary history of lotus was likely to be the reason for this. However, a low genetic diversity was revealed at population level. And three possible factors might contribute to the low genetic diversity within populations. Firstly, habitat destruction resulted in the reducing or loss of distribution area and population numbers of wild lotus. Secondly, the natural habitats of wild lotus had become fragmented and isolated, which increased the geographical distance and blocked gene flow between populations. Finally, the specificity of lotus' breeding system was the main reason for its low genetic diversity.
1.2The populations from N. lutea were separated from those from N. nucifera at a similarity coefficient of0.14with the bootstrap of100%, which confirmed that N. lutea and N. nucifera were genetically distinct. And the ranks of genetic distance between lotus populations are AmL&ThL> AmL&ChL> ThL&ChL. The population AmL was related more closely with the population ChL than with the population ThL.
1.3The paper supported that N. nucifera and N. lutea are the two independent species of Nelumbo. According to the fossil evidence, very high similarity of rbcL gene and nuclear DNA ITS sequence, high homology in serological reaction and chromosome analysis, N. nucifera and N. lutea were considered as one species. But on the basis of the facts that N. nucifera and N. lutea were separated by Pacific, and had a certain barrier of reproductive isolation, greater genetic distance, significant genetic variation, divergence in plant traits and the self-imcompatibility of F1plants, the author supported that N. nucifera and N. lutea are two independent species of Nelumbo.
1.4Some actions about the protection of lotus population are suggested as follows. Firstly, try to protect the natural habitats of wild lotus from damage. Second, it is imperative to preserve in situ all the populations surveyed to conserve genetic diversity and evolutionary processes. Finally, ex situ conservation could also be applied because of the low genetic diversity within populations. And seeds and rhizomes could be harvested for ex situ conservation.
2Construction of a high-density genetic linkage map of lotus
2.1A framework map of lotus was constructed. The map for female parent N. nucifera 'China Antique' was362.52cM with32markers distributed in eight linkage groups. The mean genetic distance between markers was11.33cM and the mean length of linkage group was45.315cM. The map for male parent N. lutea 'AL1' was495.37cM with137markers distributed in11linkage groups. The mean genetic distance between markers was3.62cM and the average length of linkage group was45.03cM.
2.2RAD-seq technology was an effective way to discover rapidly thousands of SNP markers. In line with the reference genomes of N. nucifera'China Antique', we identified6622potential SNP markers for lotus map construction using high-throughput genotyping of F1population of RAD system.
2.3A high-density genetic linkage map of American lotus 'AL1' was constructed on the basis of a SSR framework map. And the map mainly consisting of RAD markers was563.6cM with1515markers distributed in ten linkage groups. The average distance between markers was0.38cM. There were95to194markers per linkage group, and the genetic distance of each linkage group varied from39.9cM to83.8cM.
2.4RAD was a powerful molecular marker of increasing marker density and extending linkage groups. In this project, compared with the framework map in common with SSR markers, the high-density map mainly consisting of RAD markers contained1378more markers and increased genetic distance of68.23cM in length, and the average distance between markers was decreased by3.24cM.
2.5We found that marker segregation distortion was unevenly distributed on linkage groups. There were no markers deviated significantly from Mendelian segregation ratios on LG5, LG7and LG9. Two and three markers of segregation distortion were observed on LG3, LG8and LG10. Many markers on LG1(21), LG2(19), LG4(80) and LG6(127) showed moderate segregation distortion. And obvious clusters of deviated marker loci were investigated on LG4and LG6, which might be explained by the following factors:1) in order to improve the polymorphism of F1population, Nelumbo nucifera 'China Antique'and Nelumbo lutea 'AL1', diverging strongly in their geographical distributions and important morphological traits, were chosen as parents for hybridization.2) the high level of homozygosity of the female parent Nelumbo nucifera 'China Antique', which was unexpected, mainly contributed to the considerable segregation distortion in the F1population.3) the number of individuals in F1population was not very large.
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