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作者简介:

邓颢珂(1997-),硕士研究生,研究方向为生态与进化生物学,(E-mail)19210700001@fudan.edu.cn。

通讯作者:

张文驹,博士,教授,研究方向为生态与进化生物学,(E-mail)wjzhang@fudan.edu.cn。

中图分类号:Q943

文献标识码:A

文章编号:1000-3142(2023)10-1838-11

DOI:10.11931/guihaia.gxzw202209007

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目录contents

    摘要

    基因组大小是物种基因组的重要特征,通常用DNA C值来衡量,能够用于快速判断基因组倍性,并为分类学与进化生物学提供重要依据。海三棱藨草(Scirpus mariqueter)是长江口和杭州湾具有重要生态意义的标志性物种,被认为是扁秆藨草(S. planiculmis)和藨草(S. triqueter)的杂交种,因染色体小而难以准确确定倍性。近年来,部分研究者指出该物种的分类和命名存在疑点。该研究通过基因组Survey分析检测海三棱藨草样本CJ1的基因组特征,测序深度约为120 ×,并以绿豆(Vigna radiata)为参考标准,利用流式细胞术测定了海三棱藨草及其同域近缘种扁秆藨草和藨草以及海三棱藨草和扁秆藨草的杂交F1共13个样本的DNA C值和相对倍性。结果表明:(1)基因组Survey分析测得CJ1的基因组大小为244.12 Mbp,杂合率为0.68%,重复序列比例为42.38%,GC含量为37.25%。(2)流式细胞术测得来自不同区域的海三棱藨草各样本的基因组倍性相同,1C值在234.87 ~ 242.5 Mbp之间,其中CJ1的基因组大小与基因组Survey检测结果高度一致。(3)扁秆藨草的1C值在251.77 ~ 264.13 Mbp之间,藨草1C值为537.33 Mbp。根据上述基因组大小,认为海三棱藨草不可能是这两者的杂交种。该研究补充了海三棱藨草及其近缘种的基因组特征,为后续全基因组测序奠定基础,同时也否定了海三棱藨草起源于扁杆藨草和藨草杂交的假说。

    Abstract

    Genome size is an important feature of a species’ genome and is usually measured by the DNA C-value, which can be used for quickly testing genome ploidy and provide an important basis for taxonomy and evolutionary biology. Scirpus mariqueter is a species with important ecological effects in the Yangtze River estuary and Hangzhou Bay, China. It is considered as a hybrid of S. planiculmis and S. triqueter, and it is difficult to accurately determine ploidy due to its small chromosomes. However, in recent years, some researchers, based on molecular markers, have raised doubts about the classification and nomenclature of S. mariqueter. Therefore, more experimental evidence on the taxonomic attributes, genomic characteristics and possible ploidy variation of S. mariqueter and its related species is needed. In this study, the genomic characteristics of S. mariqueter sample CJ1 were determined by genome survey analysis with a sequencing depth of approximately 120 ×. The DNA C-value and relative ploidy of 13 samples of S. mariqueter and its sympatric, related species (S. triqueter and S. planiculmis) were estimated by flow cytometry with Vigna radiata as a reference. The results were as follows: (1) Genome Survey analysis showed that the genome size of CJ1 was 244.12 Mbp, with a 0.68% heterozygosity rate, 42.38% sequence repeat, and 37.25% GC content. (2) The flow cytometry results showed that the ploidy of S. mariqueter samples from different regions was the same, with 1C values ranging from 234.87 Mbp to 242.5 Mbp, and the genome size of CJ1 was highly consistent with the genome Survey results. (3) The 1C value of S. planiculmis was between 251.77 Mbp and 264.13 Mbp, and the 1C value of S. triqueter was 537.33 Mbp. Because the genome size of hybrids is usually between or larger than those of their parents, it is unlikely that S. mariqueter is a hybrid of the two species based on the abovementioned genome size. This study provides genomic characteristics of S. mariqueter and its related species and lays a foundation for its whole-genome sequencing. At the same time, it also rejects the hypothesis that S. mariqueter originated from hybridization between S. planiculmis and S. triqueter.

  • 海三棱藨草(Scirpusmariqueter)是莎草科(Cyperaceae)藨草属(Scirpus)的多年生克隆植物,主要分布在长江口和杭州湾潮间带、冲击岛屿和河口泥滩,其独特的生物学性状使其在河口以及滨海生态系统中发挥着重要的生态学功能(欧善华和宋国元,1992),包括促进淤积(Yang,1998),其球茎和种子为白鹤等迁徙鸟类提供丰富的食物来源(Ma et al.,2003),为底栖动物和鱼类生长繁育提供重要栖息场所等(袁兴中等,2002;张衡等,2017)。尽管海三棱藨草生态意义重要,但其分类和命名依然存在争议,Tang和Wang(1961)在给其命名时认为它可能是藨草和扁秆藨草的杂交种(S. planiculmis × S. triqueter)。方永鑫(1992)对海三棱藨草及其假定亲本进行染色体数目分析,发现藨草2n=40,海三棱藨草2n=64,扁秆藨草2n=50,但仍无法确定海三棱藨草是否为杂交起源,Tatanov(2007)视海三棱藨草为一个属间杂种(× Bolboschoenoplectusmariqueter)。杨梅 (2010)用AFLP分子标记发现海三棱藨草和藨草之间的遗传距离约是海三棱藨草和扁秆藨草的4倍,同时ITS序列也显示海三棱藨草是一个独立的分类群。鉴于有些莎草科植物类群即使在种内也常常存在多种倍性(Nishikawa et al.,1984),我们在野外观察和同质园实验中也发现海三棱藨草长江口种群和杭州湾种群大部分表型都存在较大差异(李昕骥,2015),在对该物种进行全基因测序研究之前,对该物种分类属性、基因组特征及可能的倍性变异需要更多实验研究。

  • 基因组大小(genome size)能为研究物种间分类和进化提供关键信息(Dolezel et al.,2007),通常用细胞DNA C值(DNA C-value)来衡量,由于DNA C值这一概念存在多种定义(Greilhuber et al.,2005),为了避免术语歧义,本研究的“DNA C值”(简称C值)采用Bennett和Smith(1976)的定义,特指配子细胞核的DNA含量(1C),等于未经复制的体细胞的细胞核DNA含量(2C)的一半,这也是植物DNA C值数据库所采用的标准(Leitch et al.,2019)。基因组大小不仅可以用于判断倍性(郗连连等,2020),也是确定杂交的重要指标(周香艳,2009),更是对其进行全基因组高通量测序的基础(伍艳芳等,2014)。流式细胞术是目前被广泛应用的测定基因组大小的方法之一,通过记录待测样品和标准品的相对荧光强度计算待测品基因组大小,快速而简便(Dolezel &Bartos,2005; Hare &Johnston,2012; Jingade et al.,2021),这一方法需要精确而合适的参照种。近年来随着测序技术的提升和成本的下降,基因组Survey分析逐渐成为测定基因组大小的替代手段(Chen et al.,2015;霍恺森等,2019;Mgwatyu et al.,2020),这一方法不需要特定的对照样品,但其费用依然比流式细胞术高很多。因此,将上述两者结合起来测定基因组大小不失为较好的方法。

  • 植物 DNA C值数据库目前包含 12 273 个物种的C值(Leitch et al.,2019)。C值的常用单位为pg,但也可以通过1 pg=978 Mbp进行单位换算。其中莎草科基因组大小范围很大,1C值在196~9 657.9 Mbp之间(Nishikawa et al.,1984; Kaur et al.,2012)。测定结果主要集中于薹草属(Carex)的物种。在藨草属中,过去的研究只测定了水葱(S. tabernaemontani)、林生藨草(S. sylvaticus)、沼生水葱(S. lacustris)的基因组大小(Mowforth,1986; Leitch et al.,2019),为了更好地保护和利用基因资源,亟须加强对海三棱藨草及其同域分布的近缘种如藨草(S. triqueter)和扁秆藨草(S. planiculmis)的基因组大小的研究。

  • 本研究以长江口和杭州湾地区的海三棱藨草及其近缘种为研究对象,通过基因组Survey分析测定海三棱藨草的基因组特征信息,并以绿豆(Vigna radiata)为标准品,通过流式细胞仪测定海三棱藨草及其近缘种和杂交材料共13个样本的基因组大小,以探讨以下问题:(1)探究基因组Survey分析与流式细胞术测得藨草属物种基因组大小的准确性;(2)鉴定海三棱藨草是否起源于扁杆藨草和藨草杂交;(3)探讨海三棱藨草种内可能的倍性变化和基因组大小变化,以及基因组大小的进化意义。本研究旨在扩充藨草属植物基因组数据信息和鉴定海三棱藨草的物种起源,并为未来的全基因组测序工作提供重要参考信息。

  • 1 材料与方法

  • 1.1 植物材料

  • 实验材料见表1,包括目标种海三棱藨草,同域分布的近缘种扁秆藨草和藨草,以及海三棱藨草和扁秆藨草的杂交材料。上述材料均属于多年生草本克隆植物,具有有性繁殖和克隆繁殖的能力。在生长季节,植株长出花穗并主要通过风媒传粉,同时一些根茎会在植株的基部发育,最终长成多个克隆分株。在秋末,种子逐渐成熟,植株地上部分枯死,地下部分根尖膨大形成球茎。种子和球茎在次年春天发芽为新苗和克隆分株(Sun et al.,2001)。

  • 本实验中海三藨草的6个样本为采自长江口地区的CJ1、CJ2、CJ3和采自杭州湾地区的HZ1、HZ2、HZ3,涵盖该物种的整个分布范围和主要的生境类型。在其他研究工作中,这些样本在遗传上的差异通过RAD测序被确定。其中,CJ1和CJ3采自低盐围垦地,很少受到潮汐的影响,因此盐度逐渐降低至0‰~0.5‰;CJ2和HZ3采自中盐滩涂,夏季表层平均盐度约5‰;HZ1和HZ2采自高盐滩涂,受潮汐影响较大,夏季表层水体盐度均值约为10‰。扁秆藨草的3个样本采自浙江杭州,生境水体盐度接近淡水。海三棱藨草(♀)和扁秆藨草(♂)杂交材料来源于杨梅等人的杂交实验(Yang et al.,2013)。藨草的1个样本采自江苏启东(表1)。上述材料均种植在复旦大学江湾校区玻璃温室,相同的淡水环境下对所有样品取球茎或地下茎进行克隆繁殖,待幼苗长至约20 cm高,取样品进行后续实验。

  • 1.2 海三棱藨草基因组Survey分析

  • 1.2.1 DNA提取和检测

  • 提取海三棱藨草样本CJ1的基因组DNA,紫外可见分光光度计检测DNA浓度,1%琼脂糖凝胶电泳检验DNA完整性。将海三棱藨草样本CJ1的基因组DNA送到北京诺禾致源科技有限公司进行基因组Survey分析。

  • 1.2.2 建库测序和分析组装

  • 将质检后的DNA样品随机打断,经末端修复、加A尾、加测序接头、纯化、PCR扩增等(Rozen &Skaletsky,2000)构建文库,对文库进行质量检测,检测合格后,通过Illumina HiseqTM 2000平台进行Paired ends(PE)双端测序,对测序得到的数据进行过滤,评估GC分布、质量值Q20、Q30等。对过滤后的高质量数据进行K-mer分析,以K-mer深度为横坐标,K-mer数量频率或K-mer种类频率为纵坐标绘制频率分布图,通过公式(基因组大小= K-mer总数/K-mer期望深度)得到海三棱藨草样本 CJ1的基因组大小、杂合情况和重复序列比例等。

  • 通过Soapdenovo软件(Li et al.,2010)对基因组数据进行初步拼接。将DNA片段随机截断成更小的序列,利用序列间重叠关系构建de Bruijin图,对de Bruijin图进行化简,截断重复区域边界获得Contig序列,将Contig序列构建成Scaffold序列并对其Gap区域进行填补。对组装的Contig绘制GC含量深度图(Li et al.,2010)。

  • 表1 实验材料及来源

  • Table1 Information on materials used in this study

  • 1.3 流式细胞仪检测藨草属植物基因组大小

  • 1.3.1 酶和裂解液

  • 纤维素酶和果胶酶分别购买自麦克林试剂公司的生物技术级 C6339 纤维素酶、M6346 果胶酶,配置100 mL 2%的酶制剂:称取2 g纤维素酶和2 g果胶酶,加纯水定容至100 mL,4℃保存。选取Galbraith和WPB两种裂解液用于细胞核悬液制备,裂解液购买自青岛捷世康生物科技有限公司所配置好的100 mL成液,4℃保存。Galbraith裂解液和WPB裂解液的成分参照汪艳等(2015)对多种裂解液的总结。

  • 1.3.2 内标和外标

  • 采用绿豆作为内标,绿豆的1C值为1C= 0.53 pg(518.34 Mbp)(Bennett &Smith,1991)。测定藨草属各物种样本的相对倍性时,以同物种的一个样本(CJ1或SP1或MP1)作为外标参考样品。

  • 1.3.3 细胞悬液制备

  • 细胞核悬液的制备参照Dolezel等(2003)的实验方法并进行了部分改进。取新鲜的植物叶片,洗净并擦干,切成1 cm左右的小段。取1.5 mL预先配置的混合均匀的酶溶液置于2 mL EP管中;在使用内标测定时分别加入0.15 g待测植株叶片和0.15 g内标叶片,使用外标测定时加入0.3 g待测植株叶片或外标叶片,进行 37℃水浴30 min;转移至培养皿中,加入0.5 mL Galbraith解离液以及0.5 mL WPB解离液,使用刀片快速切碎并置于室温孵化15 min;通过尼龙膜过滤,进行500 r·min-1低速离心5 min,取上清液,再进行2 000 r·min-1高速离心5 min,弃去上清液,加入0.5 mL WPB解离液对细胞核进行重悬;使用含RNase A的碘化丙啶(PI)染液对细胞核进行避光染色15 min。每个样品设置3次生物学重复。

  • 1.3.4 流式细胞仪检测和分析

  • 使用流式细胞仪检测被染色的细胞核的荧光强度。上机前使用滤膜再次过滤,并使用Cell Quest软件的FL-2荧光通道获取荧光强度。上机时先调整电压强度,使得待测样品的G0/G1峰均值在50~100之间,调整后保持参数不变,收集最少10 000个细胞,每个样品进行3次技术重复。

  • 使用ModFit软件对收集的数据进行分析,生成各样品荧光分布直方图,根据公式[待测植物的基因组大小1C值(pg或Mbp)= 参考样品的基因组大小1C值 × 目标样品 G0/G1 峰荧光均值/参照样品 G0/G1 峰荧光均值]计算1C值(Dolezel et al.,2007),比较相同物种各样本G0/G1 峰荧光均值比例以确定物种内是否存在多种倍性。

  • 使用SPSS软件对数据进行统计分析,使用单因素方差分析比较藨草属植物基因组大小,Fisher LSD多重比较法(P<0.05,n=3)对藨草属植物基因组大小进行物种间两两比较。

  • 2 结果与分析

  • 2.1 海三棱藨草样本CJ1基因组Survey分析

  • 2.1.1 基因组数据统计和特征

  • 我们对海三棱藨草样本CJ1的基因组原始数据进行过滤处理,并获得了高质量数据共31.17 G。我们采用K-mer= 17进行分析,频率分布图见图1,在横坐标K-mer深度102有一个纯合峰,即K-mer期望深度为102(图1)。K-mer总数约23.66 G,由公式计算得到海三棱藨草样本CJ1基因组大小(1C)为249.07 Mbp,修正后为244.12 Mbp,杂合率为0.68%,重复序列比例为42.38%。

  • 2.1.2 基因组初步组装结果

  • 采用K-mer= 41对海三棱藨草样本CJ1的测序数据进行基因组初步组装,对大于100 bp的Scaffold及其内部的Contig进行统计,结果见表2,得到的Contig N50为9 096 bp,总长为188 364 877 bp,将Contig组装成Scaffold N50为17 287 bp,总长为189 819 219 bp(表2)。

  • 图1 海三棱藨草样本CJ1的17-mer分布曲线

  • Fig.1 17-mer distribution curve of Scirpusmariqueter sample CJ1

  • 统计基因组Contig分布和GC含量信息,结果见图2。海三棱藨草样本CJ1的Contig分布在79左右有最高峰(图2:A,C),基因组GC含量为37.25%,GC含量的深度信息显示GC图存在分离现象(图2:B,D,E),Contig和NCBI核酸库比对条数最多的物种均为植物,而非微生物或昆虫,表明没有受到外源污染或污染程度低,可以忽略。

  • 2.2 流式细胞仪测定藨草属植物DNA C值

  • 以绿豆为内标测定其他藨草属植物(共13个样本)基因组大小的流式细胞分析图见图3,换算出各样本的DNA C值见表3,为了与基因组Survey分析所测基因组大小比较,流式细胞术所测1C值统一换算为Mbp单位。由表3可知,标准品的DNA峰平均变异系数(CV)值大部分在5%以下,少数在6%左右,待测样品的DNA峰平均CV值在5.31%~6.97%之间。各藨草属植物1C值在234.87~537.33 Mbp之间:海三棱藨草1C值在234.87~242.50 Mbp之间,扁秆藨草1C值在251.77~264.13 Mbp之间,海三棱藨草和扁秆藨草的杂交材料1C值在254.08~263.44 Mbp。其中,最大的是藨草样本ST1[1C=(537.33 ± 22.75)Mbp],最小的是海三棱藨草样本HZ1[1C=(234.87 ± 4.53)Mbp](图3,表3)。

  • 表2 海三棱藨草样本CJ1的基因组组装结果统计

  • Table2 Statistics of the genome assembly results of Scirpusmariqueter sample CJ1

  • 图2 海三棱藨草样本CJ1的Contig分布及GC含量与测序深度关联分析

  • Fig.2 Contig distribution and correlation analysis between GC content and sequencing depth of Scirpusmariqueter sample CJ1

  • 测量的藨草属内不同物种1C均值从小到大排列为海三棱藨草<杂交材料≈扁秆藨草<藨草,杂交材料1C均值(257.25 Mbp)处于父本扁秆藨草(259.08 Mbp)和母本海三棱藨草(238.28 Mbp)之间。对藨草属内不同物种间基因组大小差异进行显著性分析,结果有显著差异(P<0.05,n=3)。对内标法测得结果进行物种间两两比较,海三棱藨草与扁秆藨草、杂交材料以及藨草之间有显著差异,扁秆藨草与藨草之间差异显著,而与杂交材料之间差异不显著(表3)。

  • 测量结果显示同一物种不同样本之间1C值呈现一定的波动,但变化较小(表3),同一物种各样本和同物种样本1(CJ1或SP1或MP1)的1C值比率在0.91~1.16之间,即同一物种的各样本拥有相同倍性。

  • 3 讨论与结论

  • 3.1 海三棱藨草及其近缘种基因组特征测定

  • 本研究首次通过基因组Survey分析对海三棱藨草基因组特征进行了评估,为流式细胞术测量藨草属植物基因组大小提供参考,同时Survey分析比流式细胞法能获得更全面的信息,为后续基因组De novo技术策略的制定提供重要依据。根据基因组Survey分析获得的海三棱藨草样本CJ1的基因组特征,判断该物种基因组为低重复、高杂合的一般复杂基因组,可能对组装造成一定影响(霍恺森等,2019);其GC含量所处范围可以降低测序和拼接的偏差(Aird et al.,2011)。综合以上指标,后续可以采用全基因组鸟枪法进行基因组组装(霍恺森等,2019)。本研究还首次利用流式细胞仪测定了海三棱藨草、扁秆藨草、藨草的基因组的C值,海三棱藨草种内基因组大小存在小范围波动,但变异程度较低(样本间变异系数为1%),其中流式细胞术测定的CJ1的C值与前述利用Survey技术测定的基因组大小值十分接近,表明本实验测定的C值较为准确和可靠。

  • 表3 利用内标法测定的海三棱藨草及近缘种植物DNA C值及其CV

  • Table3 DNA C-value and CV value of Scirpusmariqueter and its related species detected by the internal standard method

  • 注: *字母(a,b,c)不同的数值表示LSD检验下存在显著差异(P<0.05)。

  • Note: *The numerical values with different letters (a, b, c) indicate significant differences (P<0.05) according to LSD test.

  • 本研究同时采用了内标法和外标法来保证检测流式结果的可靠性,前者主要用于检测样品C值,后者主要用于检测藨草属物种内各样本的相对倍性。内标法大多数标准品DNA峰的CV值小于5%,但也有两个标准品CV值在5%~6.3%之间,同时待测品CV值在5.31%~6.97%之间。通常认为不可接受CV高于5%的测量结果,但也存在某些富含多酚、基因组很小的样品,无法实现低于5%的CV值(Dolezel et al.,2007)。然而,外标法测得样品DNA峰的CV值均低于5%,证实仪器校准、取材质量、核裂解液成分等和CV值相关的因素对结果的负面影响较小。有研究证实,标准样品的选择也会影响CV值(Dolezel &Bartos,2005;方其等,2011),我们猜测内标法测得DNA峰的CV值略高于5%的结果可能是标准品和待测品混合而致。需要注意的是,虽然外标法样品DNA峰的CV值较内标法低,但每次重复测量之间结果变化较大,导致标准差偏大,这可能是因为外标法制备细胞悬液时标准品和待测品为分开进行,处理时间、切碎手法可能有所差异,上机时测量标准品和待测品之间存在时间间隔也会导致峰值偏移,内标法更适于基因组大小测定(Vindelov et al.,1983),因此我们优先采用内标法的C值测量结果。

  • 图3 内标法测定的流式细胞分析图

  • Fig.3 Flow cytometry histograms determined by internal standard method

  • 3.2 基因组大小鉴定海三棱藨草并非扁秆藨草和藨草的杂交种

  • 海三棱藨草的发现者根据形态性状的相似性和分布特点,推测该种可能是藨草和扁秆藨草的杂交种(Tang &Wang,1961)。新形成的杂交种基因组大小往往介于父母本之间(Baack et al.,2005;周香艳,2009;弓娜,2011),自然环境中长期形成、成熟稳定的杂交种似乎表现出超过亲本基因组大小的普遍倾向(Baack et al.,2005;周香艳,2009;Cicek et al.,2015),杂交基因组大小比两个亲本都低的例子似乎较为罕见。该研究测得海三棱藨草基因组显著低于其两个假定的亲本(藨草和扁秆藨草),这从基因组大小层面否定了海三棱藨草是通过藨草与扁杆藨草杂交起源的假说,这与杨梅根据AFLP研究获得的结果一致(杨梅,2010)。尽管只检测了藨草的一个样本,但其他研究者检测到不同分布地区藨草染色体数目十分稳定,来自印度以及中国的多个样品的染色体数目都在40~42条之间(Bir &Sidhu,1991;方永鑫,1992;Bir et al.,1993;Hoshino,1993),因此上述结论应该可靠。此外,本研究测定了海三棱藨草与扁秆藨草人工杂交F1的C值,杂交材料1C均值处于父母本之间,略大于两者之和的一半,与通常研究结果一致,也支持上述结果。本研究人工杂交材料偏向于基因组更大的父本扁秆藨草,而杂交材料与海三棱藨草基因组大小显著的差异意味着更容易辨别海三棱藨草和杂交类型。

  • 3.3 海三棱藨草基因组大小与生境关联

  • 以往研究表明基因组大小与其地理位置、气候条件密切相关(Smith &Gregory,2009),这种差异甚至也存在于物种内(Cavallini &Natali,1994),特别是同一物种也可能存在不同倍性的类型。本实验所采集的海三棱藨草样品来自十分不同的生境且覆盖该种的主要分布区域,检测的海三棱藨草各样本拥有相同的倍性,表明该物种的基因组倍性较为稳定。但是,我们把海三棱藨草6个样本按基因组大小从小到大排列时,其基因大小似乎表现出和生境类型之间的联系:来自低盐围垦地的CJ1和CJ3基因组最大,来自中盐滩涂的CJ2和HZ3基因组次之,来自高盐滩涂的HZ1和HZ2基因组最小。我们猜测这种排序可能是由生境盐度而导致,较小基因组和较大生境压力的关联并不是个例(Price et al.,1981; Price,1988; Castrojimenez et al.,1989)。例如,Price等(1981)测定加州24个植物种群的基因组大小发现基因组较大的植物种群处于土壤发育良好的生境中。因此我们推测,海三棱藨草基因组大小的分布式样可能是受自然选择产生的适应性分化,盐度对DNA合成的限制可能是高盐环境下倾向于小基因组植物的内在原因(Lazarevićet al.,2015;Zahradníček et al.,2018)。但是,本研究每一类生境只测定了两个样本,这一物种的C值与盐度的关系还需测定更多样本才能确认。

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    • FANG Q, YIN LP, GUO SL, 2011. One method application of flow cytometry to determine plant DNA C-value [J]. Plant Quar, 25(2): 40-44. [方其, 印丽萍, 郭水良, 2011. 应用流式细胞术测定植物DNA C-值的实验方法研究 [J]. 植物检疫, 25(2): 40-44. ]

    • FANG YX, 1992. The chromosome numbers of three species of genus Scirpus [C]// OU SH. A collection of research papers on population characteristics of Scirpus mariqueter and its silt-promoting efficiency in the coastal tidal flats in Shanghai. Shanghai: J Shanghai Norm Univ (Nat Sci): 49-52. [方永鑫, 1992. 藨草属三种植物的染色体 [C]//欧善华. 上海市海岸带滩涂海三棱藨草种群特征及其促淤效能研究论文集. 上海: 上海师范大学学报(自然科学版): 49-52. ]

    • GONG N, 2011. Variations in the nuclear DNA content of the homoploid hybrid species in Senecio [D]. Lanzhou: Lanzhou University: 1-30. [弓娜, 2011. 千里光属同倍体杂交物种核DNA含量变异 [D]. 兰州: 兰州大学: 1-30. ]

    • GREILHUBER J, DOLEŽEL J, LYSÁK M A, et al. , 2005. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents [J]. Ann Bot, 95(1): 255-260.

    • HARE EE, JOHNSTON JS, 2012. Genome size determination using flow cytometry of propidium iodide-stained nuclei [M]//ORGOGOZO V, ROCKMAN M. Molecular methods for evolutionary genetics. Totowa: Humana Press: 3-12.

    • HOSHINO T, 1993. Cytological studies of Chinese Cyperaceae (1). Chromosome counts of nine species collected from Jilin, Liaoning and Hebei provinces [J]. J Jap Bot, 68(2): 65-69.

    • HUO KS, CAO QH, WANG Y, et al. , 2019. Genome survey analysis of relative wild species Ipomoea littoralis in sweetpotato [J]. Chin J Trop Crops, 40(10): 2001-2005. [霍恺森, 曹清河, 王珧, 等, 2019. 甘薯近缘野生种Ipomoea littoralis全基因组Survey分析 [J]. 热带作物学报, 40(10): 2001-2005. ]

    • JINGADE P, HUDED A, MISHRA MK, 2021. First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis [J]. Braz J Bot, 44(2): 381-389.

    • KAUR N, DATSON PM, MURRAY BG, 2012. Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae) [J]. Bot J Linn Soc, 169(3): 555-564.

    • LAZAREVIĆ M, KUZMANOVIĆ N, LAKUŠIĆ D, et al. , 2015. Patterns of cytotype distribution and genome size variation in the genus Sesleria Scop. (Poaceae) [J]. Bot J Linn Soc, 179(1): 126-143.

    • LEITCH IJ, JOHNSTON E, PELLICER J, et al. , 2019. Plant DNA C-values Database [EB/OL]. Release 7. 1. https: //cvalues. science. kew. org/.

    • LI R, ZHU H, RUAN J, et al. , 2010. De novo assembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 20(2): 265-272.

    • LI XJ, 2015. Ecological adaptation of Scirpus mariqueter in heterogeneous environments of estuaries [D]. Shanghai: Fudan University: 1-70. [李昕骥, 2015. 海三棱藨草在河口异质性生境下的生态适应性 [D]. 上海: 复旦大学: 1-70. ]

    • MA ZJ, LI B, JING K, et al. , 2003. Effects of tidewater on the feeding ecology of hooded crane (Grus monacha) and conservation of their wintering habitats at Chongming Dongtan, China [J]. Ecol Res, 18(3): 321-329.

    • MGWATYU Y, STANDER AA, FERREIRA S, et al. , 2020. Rooibos (Aspalathus linearis) genome size estimation using flow cytometry and k-mer analyses [J]. Plants, 9(2): 270.

    • MOWFORTH MA, 1986. Variation in nuclear DNA amounts in flowering plants: an ecological analysis [D]. Sheffield: University of Sheffield: 1-10.

    • NISHIKAWA K, FURUTA Y, ISHITOBA K, 1984. Chromosomal evolution in genus Carex as viewed from nuclear DNA content, with special reference to its aneuploidy [J]. Jpn J Genet, 59(5): 465-472.

    • OU SH, SONG GY, 1992. The special situation of Sea-bulrush (Scirpus mariqueter) population in Shanghai [C]// OU SH. A collection of research papers on population characteristics of Scirpus mariqueter and its silt-promoting efficiency in the coastal tidal flats in Shanghai. Shanghai: J Shanghai Norm Univ (Nat Sci): 1-3. [欧善华, 宋国元, 1992. 海三棱藨草种群在上海的特殊地位 [C]//欧善华. 上海市海岸带滩涂海三棱藨草种群特征及其促淤效能研究论文集. 上海: 上海师范大学学报(自然科学版): 1-3. ]

    • PRICE HJ, 1988. DNA content variation among higher plants [J]. Ann Mo Bot Gard, 75(4): 1248-1257.

    • PRICE HJ, CHAMBERS KL, BACHMANN K, 1981. Geographic and ecological distribution of genomic DNA content variation in Microseris douglasii (Asteraceae) [J]. Bot Gaz, 142(3): 415-426.

    • ROZEN S, SKALETSKY H, 2000. Primer3 on the WWW for general users and for biologist programmers [M]// MISENER S, KRAWETZ SA. Bioinformatics methods and protocols. Totowa: Humana Press: 365-386.

    • SMITH EM, GREGORY TR, 2009. Patterns of genome size diversity in the ray-finned fishes [J]. Hydrobiologia, 625(1): 1-25.

    • SUN SC, GAO XM, CAI YL, 2001. Variations in sexual and asexual reproduction of Scirpus mariqueter along an elevational gradient [J]. Ecol Res, 16(2): 263-274.

    • TANG J, WANG FZ, 1961. Cyperaceae, genus Scirpus [M]// Delectis Florae Reipublicae Popularis Sinicae Agenadae Academiae Sinicae Edita. 1 ed. Beijing: Science Press: 7-34.

    • TATANOV IV, 2007. Hybrida intergenericus novus × Bolboschoenoplectus Tatanov (Cyperaceae) [J]. Novosti Sist Vyssh Rast, 39: 150-158.

    • VINDELOV LL, CHRISTENSEN IJ, NISSEN NI, 1983. Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards [J]. Cytometry, 3(5): 328-331.

    • WANG Y, XIAO Y, LIU W, et al. , 2015. Operation skills of flow cytometer for detecting nuclear DNA contents in higher plant cells [J]. Plant Sci J, 33(1): 126-131. [汪艳, 肖媛, 刘伟, 等, 2015. 流式细胞仪检测高等植物细胞核DNA含量的方法 [J]. 植物科学学报, 33(1): 126-131. ]

    • WU YF, XIAO FM, XU HN, et al. , 2014. Genome survey in Cinnamomum camphora L. Presl [J]. J Plant Genet Resour, 15(1): 149-152. [伍艳芳, 肖复明, 徐海宁, 等, 2014. 樟树全基因组调查 [J]. 植物遗传资源学报, 15(1): 149-152. ]

    • XI LL, LI JB, ZHU KL, et al. , 2020. Variation in genome size and stomatal traits among three Sorbus species [J]. Plant Sci J, 38(1): 32-38. [郗连连, 李嘉宝, 朱凯琳, 等, 2020. 花楸属3种植物的基因组大小与叶气孔特征分析 [J]. 植物科学学报, 38(1): 32-38. ]

    • YANG M, LV XN, FAN W, et al. , 2013. Interspecific hybridization between Scirpus mariqueter Ts. Tang & F. T. Wang and S. planiculmis F. Schmidt and their selfing [J]. Aquat Bot, 110: 92-96.

    • YANG M, 2010. The study on species’ biology of Scirpus × mariqueter and its genetic structure in China [D]. Shanghai: Fudan University: 1-97. [杨梅, 2010. 海三棱藨草的物种生物学和遗传结构研究 [D]. 上海: 复旦大学: 1-97. ]

    • YANG SL, 1998. The role of Scirpus marsh in attenuation of hydrodynamics and retention of fine sediment in the yangtze estuary [J]. Estuar Coast Shelf Sci, 47(2): 227-233.

    • YUAN XZ, LU JJ, LIU H, 2002. Influence of characteristics of Scirpus mariqueter community on the benthic macro-invertebrate in a salt marsh of the Changjiang Estuary [J]. Acta Ecol Sin, 22(3): 326-333. [袁兴中, 陆健健, 刘红, 2002. 河口盐沼植物对大型底栖动物群落的影响 [J]. 生态学报, 22(3): 326-333. ]

    • ZAHRADNÍČEK J, CHRTEK J, FERREIRA MZ, et al. , 2018. Genome size variation in the genus Andryala (Hieraciinae, Asteraceae) [J]. Folia Geobot, 53(4): 429-447.

    • ZHANG H, CHEN YY, YE JY, et al. , 2017. Monthly variation of fish species composition and quantity in the habitat of Phragmites australis and Scirpus mariqueter in Dongtan wetland of Changjiang estuary [J]. Mar Fish, 39(5): 500-507. [张衡, 陈渊戈, 叶锦玉, 等, 2017. 长江口东滩湿地芦苇和海三棱藨草生境下的鱼类种类组成和数量的月变化 [J]. 海洋渔业, 39(5): 500-507. ]

    • ZHOU XY, 2009. The study of the relationship between nuclear DNA content of Hippophae L. species and their hybrid origin [D]. Lanzhou: Lanzhou University: 1-30. [周香艳, 2009. 沙棘核DNA含量与杂交种起源的相关性研究 [D]. 兰州: 甘肃农业大学: 1-30. ]

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    • FANG YX, 1992. The chromosome numbers of three species of genus Scirpus [C]// OU SH. A collection of research papers on population characteristics of Scirpus mariqueter and its silt-promoting efficiency in the coastal tidal flats in Shanghai. Shanghai: J Shanghai Norm Univ (Nat Sci): 49-52. [方永鑫, 1992. 藨草属三种植物的染色体 [C]//欧善华. 上海市海岸带滩涂海三棱藨草种群特征及其促淤效能研究论文集. 上海: 上海师范大学学报(自然科学版): 49-52. ]

    • GONG N, 2011. Variations in the nuclear DNA content of the homoploid hybrid species in Senecio [D]. Lanzhou: Lanzhou University: 1-30. [弓娜, 2011. 千里光属同倍体杂交物种核DNA含量变异 [D]. 兰州: 兰州大学: 1-30. ]

    • GREILHUBER J, DOLEŽEL J, LYSÁK M A, et al. , 2005. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents [J]. Ann Bot, 95(1): 255-260.

    • HARE EE, JOHNSTON JS, 2012. Genome size determination using flow cytometry of propidium iodide-stained nuclei [M]//ORGOGOZO V, ROCKMAN M. Molecular methods for evolutionary genetics. Totowa: Humana Press: 3-12.

    • HOSHINO T, 1993. Cytological studies of Chinese Cyperaceae (1). Chromosome counts of nine species collected from Jilin, Liaoning and Hebei provinces [J]. J Jap Bot, 68(2): 65-69.

    • HUO KS, CAO QH, WANG Y, et al. , 2019. Genome survey analysis of relative wild species Ipomoea littoralis in sweetpotato [J]. Chin J Trop Crops, 40(10): 2001-2005. [霍恺森, 曹清河, 王珧, 等, 2019. 甘薯近缘野生种Ipomoea littoralis全基因组Survey分析 [J]. 热带作物学报, 40(10): 2001-2005. ]

    • JINGADE P, HUDED A, MISHRA MK, 2021. First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis [J]. Braz J Bot, 44(2): 381-389.

    • KAUR N, DATSON PM, MURRAY BG, 2012. Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae) [J]. Bot J Linn Soc, 169(3): 555-564.

    • LAZAREVIĆ M, KUZMANOVIĆ N, LAKUŠIĆ D, et al. , 2015. Patterns of cytotype distribution and genome size variation in the genus Sesleria Scop. (Poaceae) [J]. Bot J Linn Soc, 179(1): 126-143.

    • LEITCH IJ, JOHNSTON E, PELLICER J, et al. , 2019. Plant DNA C-values Database [EB/OL]. Release 7. 1. https: //cvalues. science. kew. org/.

    • LI R, ZHU H, RUAN J, et al. , 2010. De novo assembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 20(2): 265-272.

    • LI XJ, 2015. Ecological adaptation of Scirpus mariqueter in heterogeneous environments of estuaries [D]. Shanghai: Fudan University: 1-70. [李昕骥, 2015. 海三棱藨草在河口异质性生境下的生态适应性 [D]. 上海: 复旦大学: 1-70. ]

    • MA ZJ, LI B, JING K, et al. , 2003. Effects of tidewater on the feeding ecology of hooded crane (Grus monacha) and conservation of their wintering habitats at Chongming Dongtan, China [J]. Ecol Res, 18(3): 321-329.

    • MGWATYU Y, STANDER AA, FERREIRA S, et al. , 2020. Rooibos (Aspalathus linearis) genome size estimation using flow cytometry and k-mer analyses [J]. Plants, 9(2): 270.

    • MOWFORTH MA, 1986. Variation in nuclear DNA amounts in flowering plants: an ecological analysis [D]. Sheffield: University of Sheffield: 1-10.

    • NISHIKAWA K, FURUTA Y, ISHITOBA K, 1984. Chromosomal evolution in genus Carex as viewed from nuclear DNA content, with special reference to its aneuploidy [J]. Jpn J Genet, 59(5): 465-472.

    • OU SH, SONG GY, 1992. The special situation of Sea-bulrush (Scirpus mariqueter) population in Shanghai [C]// OU SH. A collection of research papers on population characteristics of Scirpus mariqueter and its silt-promoting efficiency in the coastal tidal flats in Shanghai. Shanghai: J Shanghai Norm Univ (Nat Sci): 1-3. [欧善华, 宋国元, 1992. 海三棱藨草种群在上海的特殊地位 [C]//欧善华. 上海市海岸带滩涂海三棱藨草种群特征及其促淤效能研究论文集. 上海: 上海师范大学学报(自然科学版): 1-3. ]

    • PRICE HJ, 1988. DNA content variation among higher plants [J]. Ann Mo Bot Gard, 75(4): 1248-1257.

    • PRICE HJ, CHAMBERS KL, BACHMANN K, 1981. Geographic and ecological distribution of genomic DNA content variation in Microseris douglasii (Asteraceae) [J]. Bot Gaz, 142(3): 415-426.

    • ROZEN S, SKALETSKY H, 2000. Primer3 on the WWW for general users and for biologist programmers [M]// MISENER S, KRAWETZ SA. Bioinformatics methods and protocols. Totowa: Humana Press: 365-386.

    • SMITH EM, GREGORY TR, 2009. Patterns of genome size diversity in the ray-finned fishes [J]. Hydrobiologia, 625(1): 1-25.

    • SUN SC, GAO XM, CAI YL, 2001. Variations in sexual and asexual reproduction of Scirpus mariqueter along an elevational gradient [J]. Ecol Res, 16(2): 263-274.

    • TANG J, WANG FZ, 1961. Cyperaceae, genus Scirpus [M]// Delectis Florae Reipublicae Popularis Sinicae Agenadae Academiae Sinicae Edita. 1 ed. Beijing: Science Press: 7-34.

    • TATANOV IV, 2007. Hybrida intergenericus novus × Bolboschoenoplectus Tatanov (Cyperaceae) [J]. Novosti Sist Vyssh Rast, 39: 150-158.

    • VINDELOV LL, CHRISTENSEN IJ, NISSEN NI, 1983. Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards [J]. Cytometry, 3(5): 328-331.

    • WANG Y, XIAO Y, LIU W, et al. , 2015. Operation skills of flow cytometer for detecting nuclear DNA contents in higher plant cells [J]. Plant Sci J, 33(1): 126-131. [汪艳, 肖媛, 刘伟, 等, 2015. 流式细胞仪检测高等植物细胞核DNA含量的方法 [J]. 植物科学学报, 33(1): 126-131. ]

    • WU YF, XIAO FM, XU HN, et al. , 2014. Genome survey in Cinnamomum camphora L. Presl [J]. J Plant Genet Resour, 15(1): 149-152. [伍艳芳, 肖复明, 徐海宁, 等, 2014. 樟树全基因组调查 [J]. 植物遗传资源学报, 15(1): 149-152. ]

    • XI LL, LI JB, ZHU KL, et al. , 2020. Variation in genome size and stomatal traits among three Sorbus species [J]. Plant Sci J, 38(1): 32-38. [郗连连, 李嘉宝, 朱凯琳, 等, 2020. 花楸属3种植物的基因组大小与叶气孔特征分析 [J]. 植物科学学报, 38(1): 32-38. ]

    • YANG M, LV XN, FAN W, et al. , 2013. Interspecific hybridization between Scirpus mariqueter Ts. Tang & F. T. Wang and S. planiculmis F. Schmidt and their selfing [J]. Aquat Bot, 110: 92-96.

    • YANG M, 2010. The study on species’ biology of Scirpus × mariqueter and its genetic structure in China [D]. Shanghai: Fudan University: 1-97. [杨梅, 2010. 海三棱藨草的物种生物学和遗传结构研究 [D]. 上海: 复旦大学: 1-97. ]

    • YANG SL, 1998. The role of Scirpus marsh in attenuation of hydrodynamics and retention of fine sediment in the yangtze estuary [J]. Estuar Coast Shelf Sci, 47(2): 227-233.

    • YUAN XZ, LU JJ, LIU H, 2002. Influence of characteristics of Scirpus mariqueter community on the benthic macro-invertebrate in a salt marsh of the Changjiang Estuary [J]. Acta Ecol Sin, 22(3): 326-333. [袁兴中, 陆健健, 刘红, 2002. 河口盐沼植物对大型底栖动物群落的影响 [J]. 生态学报, 22(3): 326-333. ]

    • ZAHRADNÍČEK J, CHRTEK J, FERREIRA MZ, et al. , 2018. Genome size variation in the genus Andryala (Hieraciinae, Asteraceae) [J]. Folia Geobot, 53(4): 429-447.

    • ZHANG H, CHEN YY, YE JY, et al. , 2017. Monthly variation of fish species composition and quantity in the habitat of Phragmites australis and Scirpus mariqueter in Dongtan wetland of Changjiang estuary [J]. Mar Fish, 39(5): 500-507. [张衡, 陈渊戈, 叶锦玉, 等, 2017. 长江口东滩湿地芦苇和海三棱藨草生境下的鱼类种类组成和数量的月变化 [J]. 海洋渔业, 39(5): 500-507. ]

    • ZHOU XY, 2009. The study of the relationship between nuclear DNA content of Hippophae L. species and their hybrid origin [D]. Lanzhou: Lanzhou University: 1-30. [周香艳, 2009. 沙棘核DNA含量与杂交种起源的相关性研究 [D]. 兰州: 甘肃农业大学: 1-30. ]