Characterization of meiotic chromosome behavior in the autopolyploid Saccharum spontaneum reveals preferential chromosome pairing without distinct DNA sequence variation

Autopolyploidy and allopolyploidy may represent an evolutionary advantage and are more common in plants than assumed. However, less attention has been paid to autopolyploidy than to allopolyploidy,and its evolutionary consequences are largely unclear, especially for plants with high ploidy levels. In this study, we developed oligonucleotide(oligo)-based chromosome painting probes to identify individual chromosomes in S. spontaneum. Using fluorescence in situ hybridization(FISH), we investigated chromosome behavior during pachytene, metaphase, anaphase, and telophase of meiosis I(MI) in autotetraploid,autooctoploid, and autodecaploid S. spontaneum clones. All autopolyploid clones showed stable diploidized chromosome behavior;so that homologous chromosomes formed almost exclusively bivalents during MI. Two copies of homologous chromosome 8 with similar sizes in the autotetraploid clone showed preferential pairing with each other with respect to the other copies. However, sequence variation analysis showed no apparent differences among homologs of chromosome 8 and all other chromosomes. We suggest that either the stable diploidized pairing or the preferential pairing between homologous copies of chromosome 8 in the studied autopolyploid sugarcane are accounted for by unknown mechanisms other than DNA sequence similarity. Our results reveal evolutionary consequences of stable meiotic behavior in autopolyploid plants.

甘蔗细茎野生种(Saccharum spontaneum L.)的遗传多样性和系统演化研究

利用 RAPD技术 ,采用 2 5个随机引物对来自中国不同生态环境的 195份甘蔗细茎野生种材料进行了地理群体结构的遗传多样性研究。结果表明 :甘蔗细茎野生种的种内遗传变异较大 ,各地理类群的遗传分化明显 ,具有丰富的遗传多样性。基于分子聚类分析 ,推论中国甘蔗细茎野生种的起源演化方式为 :起源于云南 ,然后由云南→四川→贵州→广西→广东→海南→福建→江西 ;

甘蔗属割手密种(Saccharum spontaneum)‘云南82-114’的F_2(BC_1)代同异法综合评价及同一度分类

为综合评价甘蔗属割手密种(Saccharum spontaneum)‘云南82-114’的F2(BC1)代亲本材料,采用基于集对论的同异分析法,对65份亲本材料进行了分析。结果表明,同异联系度排名前10位的材料是:云瑞09-23、云瑞09-44、云瑞09-36、云瑞09-74、云瑞09-65、云瑞09-35、云瑞09-14、云瑞09-58、云瑞09-51、云瑞09-67。超过对照新台糖22号(ROC22)的材料有33份,超过对照粤糖93-159(YT93-159)的材料有36份。在等价矩阵中取截距λ=0.910,65份‘云南82-114’的F2(BC1)代亲本材料可分为26个类型。同异联系度排名靠前、又聚为不同类型的亲本材料,通常是某些优良性状表现突出的亲本材料,这为亲本材料的进一步筛选和利用提供了依据。

两个割手密(Saccharum spontaneum L.)无性系对UV-B辐射响应的形态和生理差异

采用田间试验方法,研究了连续两年大田条件下,增强UV-B辐射(UV-B,280￣310nm,5.0kJ·m-2)对两个割手密(Saccharum spontaneum L.)无性系(Ⅰ91-48和Ⅱ91-81)形态和生理的影响,以及两个割手密无性系对UV-B辐射响应的种内差异。结果表明,两年中,在增强UV-B辐射下,Ⅰ91-485个生育时期的株高(除2004年成熟后期外)和分蘖数,以及分蘖期和伸长初期的茎径显著增加,而Ⅱ91-81株高在伸长初期和伸长后期,分蘖数在伸长初期、伸长后期、成熟初期和成熟后期(除2004年伸长后期外),以及茎径在分蘖期、伸长初期和伸长后期显著降低。两年的成熟后期,UV-B辐射导致Ⅰ91-48生物量、叶面积指数和节间长显著增加,Ⅱ91-81生物量、叶面积指数和节间长显著降低,而两个无性系节数无显著变化。两年中,UV-B辐射显著降低两个割手密无性系伸长后期和成熟初期叶展开角度。在UV-B辐射下,两年的5个生育时期中,Ⅰ91-48的类黄酮含量显著增加,丙二醛含量无显著变化,而Ⅱ91-81的类黄酮含量显著降低(除2003成熟后期外),丙二醛含量显著增加。总体上,UV-B辐射导致Ⅰ91-48叶绿素a、叶绿素b和叶绿素(a+b)含量在两年的分蘖期、伸长初期和伸长后期显著增加,而Ⅱ91-81叶绿素a、叶绿素b和叶绿素(a+b)含量在两年的分蘖期、伸长初期、伸长后期和成熟初期显著降低。割手密无性系Ⅰ91-48和Ⅱ91-81对UV-B辐射的响应具有种内差异。

甘蔗属割手密(Saccharum Spontaneum),近缘属斑茅(Sclerostachya)及河八王(Narenga)的染色体数目研究

本文对来自云南、四川、福建、广东、贵州、江西的１０６份甘蔗野生种割手密无性系，以及甘蔗近缘属斑茅无性系２８份与河八王无性系３份的染色体进行观察计数。发现割手密染色体数目类型十分丰富有２ｎ＝６０、６４、７０、７２、７８、８０、９０、９２、９６、１０４、１０８，１１种类型，其中以２ｎ＝６４、８０、９６，３种类型出现频率最高，说明我国的割手密资源十分丰富。斑茅与河八王的染色体数目较为固定，分别为２ｎ＝６０，３０各１种类型。

Optimization of protoplast isolation,transformation and its application in sugarcane (Saccharum spontaneum L.)

Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transformation.The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however,quick and effective transformation systems are still lacking for sugarcane.Here,we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S.spontaneum.The yield of viable protoplasts was approximately 1.26×107 per gram of leaf material,and the transformation efficiency of 80.19%could be achieved under the optimized condition.Furthermore,using this approach,the nuclear localization of an ABI5-like bZIPs transcription factor was validated,and the promoter activity of several putative DNase I hypersensitive sites(DHSs)was assessed.The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays.A highly efficient S.spontaneum mesophyll cell protoplast isolation and transient transformation method was developed,and it shall be suitable for in vivo functional gene analysis in sugarcane.

割手密(Saccharum spontaneum L.)幼叶的组织培养

研究了从割手密幼叶诱导愈伤组织的条件,试验表明,培养基以MS附加2,4-D1～4mg/L为佳,所接种的幼叶以4cm以内为适宜的区段。不同材料在诱导愈伤组织和分化成苗方面都表现出明显的差异,说明云南的割手密在生理和遗传上存在着丰富的种内变异.

Energy Productivity Potential Assessment of Saccharum spontaneum L. Germplasm Resources

[Objective]The energy productivity potential of Saccharum spontaneum L.germplasm resources as energy plants was discussed in this paper.[Method]The energy productivity potential of Saccharum spontaneum L.germplasm resources collected from the wild was assessed from the aspects of cellulose,semicellulose,lignin and crude ash contents and caloric value.[Result]The total content of cellulose and semicellulose ranged from 41.75%to 69.13%,the lignin content ranged from 2.16%to 11.75%,the crude ash content ranged from 4.79%to 9.34%and the caloric value ranged from 16.00 to 17.69 MJ/kg among the 30 Saccharum spontaneum L.germplasms.Compared to other plants,Saccharum spontaneum L.had higher contents of cellulose and semicellulose,a higher caloric value but lower contents of lignin and crude ash.[Conclusion]From the perspective of producing fuel ethanol with lignocellulose,Saccharum spontaneum L.has a great potential for development as an energy plant.

Study on Saccharification of Saccharum spontaneum L. by Sulphuric Acid Hydrolysis

In order to improve the saccharification rate by acid hydrolysis in the technique for production of ethanol from biomass, in this study, Saccharum spontaneum L. was used as the experimental material to investigate the rules and technological conditions for two-step acid-hydrolysis saccharification by single factor experiment and orthogonal experiment. According to the results, concentrated sulfttric acid hydrolysis was the first step, with the liquid-solid ratio of 42： 1, sulfuric acid concentration of 70%, hydrolysis time of 20 rain, and hydrolysis temperature of 55 ℃ ; dilute sulphuric acid hydrolysis was the second step, with the liquidsolid ratio of 115： 1, sulfuric acid concentration of 5.5%, hydrolysis time of 155 min, and hydrolysis temperature of 100℃. In two-step acid hydrolysis process, the yield of reducing sugar was 48.78%. The results indicated that two-step acid-hydrolysis saccharification required mild conditions, simple operation and low cost, and led to high reducing sugar yield, exhibiting a broad application prospect.

Molecular Markers Associated with Starch Content and Implications for Sugarcane Introgression Breeding Using Saccharum spontaneum

Introgression with Saceharum spontaneum intended to broaden the genetic base of sugarcane resulted in an increase in desirable genes for broader adapt ability as well as undesirable genes for high starch content. Markers could provide a quick and efficient method of screening parental genotypes for low starch. The objectives of this study were to identify potential markers associated with starch in S. spontaneum population and evaluate their potential for screening for starch content. Data for starch content and （simple sequence repeats） SSR markers were collected from 51 S. spontaneum clones grown in replicated pots. The mixed procedure of statistical analysis system （SAS） was used to determine markers significantly associated with starch. Thirty-nine out of 357 polymorphic markers were significantly （P 〈 0.05） associated with starch content. Eighteen were positively associated and 21 were negatively associated. The presence of a positive marker produced 39% more starch than absence while the absence of a negative marker produced 57% more starch than presence. Selecting parents using negative markers may be more efficient than selecting using positive markers.

Nutritional Value Evaluation of Saccharum spontaneum L. Germplasm Resources

The contents of dry matter, crude fat, crude protein, neutral detergent fiber, acid detergent fiber, crude ash, Ca and P in 43 Saccharum spontaneum L.germplasms during the vegetative period were determined. Among them, the crude protein, crude fat, neutral detergent fiber, acid detergent fiber, P and Ca contents were treated as the judging indicators. The nutritional value evaluation was carried out with the analytic hierarchy process(AHP). The results showed A49(Guangxi)had the highest nutritional value. Its dry matter, crude protein, crude fat, crude ash,neutral detergent fiber, acid detergent fiber, Ca and P contents were 32.35%,7.20%, 4.06%, 8.07%, 75.81%, 50.72%, 0.23% and 0.16% respectively. While A3(Guangdong) had the lowest nutritional value. Its dry matter, crude protein, crude fat, crude ash, neutral detergent fiber, acid detergent fiber, Ca and P contents were32.24%, 4.62%, 0.51%, 6.38%, 46.40%, 40.02%, 0.15% and 0.09% respectively.The crude protein content differed significantly among different germplasms. A48 had the highest crude protein content(9.11%), and A14 had the lowest crude protein content(3.72%). Based on the evaluation results, the 43 Saccharum spontaneum L.germplasms were divided into 3 groups: high-nutritional value type, moderate-nutritional value type and low-nutritional value type. We hoped to provide a theoretical reference for the application of Saccharum spontaneum L. as a forage grass.

Genetic Research and Utilization Reference of Saccharum spontaneum L.

[Objectives]The genetic characteristics of wild germplasm resources of Saccharum spontaneum L.were further investigated to provide a scientific theoretical basis for the improved hybridization of sugarcane varieties.[Methods]The first flowering date data of 112 wild resources were statistically analyzed by broad heritability for 3 consecutive years,and pollen quantity and yield were investigated to provide reference for hybridization.[Results]The broad heritability of the first flowering date of S.spontaneum was 0.079.The differences in the first flowering date between the S.spontaneum resources were in the range of 4-49 d,with an average of 13.65 d.The inter-annual changes within 20 d accounted for 90 %,and only 4.5% exceeded 30 d.The first flowering dates of some S.spontaneum resources were obviously postponed after being treated.[Conclusions]In the hybridization season,the hybridization plan must be adjusted according to the actual situation of the flowering period to make full use of it.

云南割手密（Saccharum spontaneum）染色体数目及其地理分布研究

对８７份云南割手密无性系进行了染色体数目和植物学性状的观察，发现云南割手密染色体数目有２ｎ＝６０、６４、７０、８０四种类型。讨论了染色体倍性与植物学性状及地理分布的关系。

割手密SnRK2基因家族全基因组鉴定及干旱胁迫表达分析

【目的】探究割手密SnRK2基因家族成员在干旱胁迫中的调控机制,为抗旱性甘蔗品种的选育提供侯选基因。【方法】以全基因组数据为基础从割手密中鉴定SnRK2基因,并对其进行生物信息学分析和干旱胁迫下的表达分析。【结果】在割手密基因组中共鉴定出11个SnRK2基因家族成员,命名为SsSnRK2.1-SsSnRK2.11,且这些基因不均匀地分布于8条染色体上。SnRK2蛋白的氨基酸残基数为227~580,分子质量为25683.53~64695.8 kD,等电点为4.62~8.94,且均为亲水性蛋白。系统发育树可将其分为3个亚组,且同亚组中的保守基序基本相似,外显子数量以7~9个为主。SsSnRK2基因家族成员的启动子中含有多种激素类和逆境胁迫响应类的作用元件。割手密SsSnRK2基因家族成员的表达具有组织特异性。所有的SsSnRK2基因均能不同程度地响应干旱胁迫。【结论】割手密SnRK2基因家族在响应干旱胁迫过程中发挥重要作用,可为割手密的抗逆性研究提供一定的理论依据。

30份割手密种质资源苗期抗旱性综合评价

【目的】依据生理生化指标综合评价不同基因型割手密种质资源的苗期抗旱性,为甘蔗抗旱性育种的亲本选育提供参考。【方法】以30份割手密野生种质为试验材料进行盆栽试验,测定割手密苗期干旱胁迫处理前后的6项生理生化指标,采用主成分分析、模糊隶属函数分析和聚类分析对割手密的抗旱性进行综合评价。【结果】与正常供水相比,干旱胁迫下各割手密种质材料的过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性增强,丙二醛(MDA)、可溶性糖(SS)、脯氨酸(Pro)含量增加,但各指标的变化程度因材料不同而存在一定差异。相关性分析结果表明,在干旱胁迫下,不同割手密种质材料叶片的过氧化氢酶活性和脯氨酸含量之间呈显著正相关(P<0.05),超氧化物歧化酶活性和脯氨酸之间呈极显著正相关(P<0.01);过氧化物酶活性和超氧化物歧化酶活性之间呈显著负相关;过氧化氢酶活性与丙二醛含量之间也呈显著负相关,其他指标之间无显著相关性(P>0.05)。主成分分析表明,SOD活性、POD活性、CAT活性和Pro含量、SS含量、MDA含量可作为割手密抗旱性评价的重要指标。通过模糊隶属函数分析和聚类分析可将30份割手密种质材料划分为4类,其中高度抗旱材料1份(90⁃53),中高度抗旱材料2份,中度抗旱材料14份,低度抗旱材料13份。【结论】CAT活性、SOD活性、POD活性、MDA含量、SS含量、Pro含量可作为割手密苗期的抗旱性评价指标。30份参试割手密种质中,90⁃53的抗旱性最强,2015⁃22、88⁃301次之,2015⁃104的抗旱性最弱。

割手密抗旱相关基因SsREMO-1a的表达与功能分析

【目的】获得割手密SsREMO-1a基因全序列,开展组织特异表达、qPCR分析、亚细胞定位研究,研究转SsREMO-1a基因拟南芥的表型变化以及CAT基因的表达模式。【方法】以海南甘蔗野生种(割手密Sp-24)为材料,通过PCR、纯化、克隆和测序验证后获得完整的割手密SsREMO-1a基因序列。对苗期正常生长的割手密进行干旱胁迫处理,于胁迫处理后0、1、2、3、4和5 d收集幼嫩叶片、根和茎等组织进行组织特异表达分析和qPCR分析。采用烟草亚细胞定位技术、基因遗传转化技术等对SsREMO-1a的表达情况进行组织定位和功能分析。构建pBI221-SsREMO-1a-GFP融合表达载体,通过农杆菌转化烟草进行细胞定位分析。构建过量表达载体pCAMBIA 1304-SsREMO-1a,利用农杆菌转化法将SsREMO-1a导入拟南芥,观测干旱胁迫后0、1、2、3、4和5 d植株表型以及CAT-1基因表达量。【结果】SsREMO-1a基因全长均为738 bp,开放读码框(ORF)564 bp,编码187个氨基酸;组织特异性表达表明,SsREMO-1a在根、茎、叶中均有表达;qPCR分析结果表明,该基因受干旱胁迫的诱导并呈显著上调表达;亚细胞定位结果表明,SsREMO-1a蛋白定位于细胞膜上;共获得11个转基因拟南芥株系,抗旱性鉴定表明转基因植株能够提高植株的抗旱能力;转基因植株CAT-1基因表达量显著升高。【结论】SsREMO-1a能够提高植株的抗旱能力,对CAT-1基因具有一定的调控作用。

甘蔗割手密种转录因子NAP亚家族的鉴定及SsNAP2a参与叶片衰老的功能分析

NAP(NAC-like,activated by apetala3/pistillata)是转录因子NAC基因家族中一类参与调控植物生长发育、叶片衰老及响应激素和非生物胁迫应答的亚家族。本研究利用甘蔗割手密种基因组数据和生物信息学方法,首先,基于比较基因组学对NAP亚家族成员进行鉴定、系统进化分析、保守结构域及顺式调控元件预测;其次,克隆获得割手密种SsNAP2的等位基因SsNAP2a,分析该基因在不同生长发育阶段的表达及其在激素和非生物胁迫下的表达特征;最后,利用瞬时表达和亚细胞定位分析SsNAP2a基因的功能。结果表明,在割手密种基因组中共鉴定5个NAP亚家族成员,亚细胞定位预测所有成员编码蛋白均定位于细胞核上,这些基因的Ka/Ks比值均小于1,表明纯化选择在演化中起到关键作用。系统聚类分析表明,5个代表性的被子植物(拟南芥、菠萝、水稻、玉米和高粱)与已报道的12个物种及甘蔗的NAP亚家族成员,共计46个,分为4个Clade,其演化的顺序Clade I>Clade II>Clade III>Clade IV。此外,在SsNAP亚家族成员的启动子区域预测到较多响应脱落酸和茉莉酸、低温等逆境胁迫的顺式作用元件,推测其参与多种激素和非生物胁迫相关应答通路。进一步,从割手密种SES208中克隆到SsNAP2a基因,该cDNA全长序列1173 bp(GenBank登录号为OQ335094),编码390个氨基酸残基,其与等位基因SsNAP2编码蛋白的序列相似性为97.70%,有10个氨基酸残基的差异,表明同源8倍体的割手密种等位基因序列差异较大。qRT-PCR分析表明,SsNAP2a基因在割手密种不同生长发育阶段中组成型表达,尤其在衰老的蔗皮和根中的表达量最高;在乙烯利(ethylene,ET)、脱落酸(abscisic acid,ABA)、4℃低温和40℃高温处理下,其表达量呈现显著诱导表达;而在8%聚乙二醇(polyethyleneglycol,PEG)胁迫下显著下调表达。亚细胞定位表明,SsNAP2a融合蛋白定位在细胞核上。瞬时过表达SsNAP2a基因7 d后,本氏烟(Nicotianabenthamiana)叶片有明显的卷曲、皱缩等衰老现象,ET合成相关基因(NtEFE26,NtAccdeaminase)显著上调表达,而水杨酸、茉莉酸和ABA合成相关基因(NtPR-1a/c、NtPR3和NtAREB1)显著下调表达,表明SsNAP2a基因参与多种激素信号传导途径诱发叶片衰老。以上研究结果为挖掘甘蔗NAP亚家族基因成员参与叶片衰老的生物学功能奠定了研究基础,也为培育甘蔗抗衰老分子育种提供候选的基因资源。

四川割手密(Saccharum spontancum L.)同工酶研究

以四川省的48个割手密为材料,采用垂直板状聚丙烯酰胺凝胶电泳进行3种同工酶分析。结果表明,在酯酶、过氧化物酶、谷氨酸草酰乙酸转氨酶中,以过氧化物酶分析效果最佳,可作为甘蔗野生资源种群划分的生化指标。根据过氧化物酶谱特征,供试样本出现19个类群,反映了四川割手密的多态性、遗传差异和地理分布的特点。

广西割手密核心种质构建与关联分析

为发掘甘蔗育种优异野生基因资源,以来自广西的333份割手密为材料,应用12对SSR引物的分子标记数据和28个表型性状资料构建广西割手密核心种质,并进行关联分析。关联分析结果显示,广西割手密茎径、节间长度、曝光前颜色、脱叶性和毛群共5个表型性状与8个位点显著相关;茎径与叶长、叶宽、节间长度、节数和锤度5个性状均表现出极显著相关;株高、茎径、节间长度、节数4个表型性状之间呈现显著或极显著的正相关;锤度与茎径和节间长度均表现为极显著的负相关。核心种质抽样按照总资源比例的0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9进行梯度筛选,当抽样比例达到30%以上时,即可包含100%等位基因覆盖度。遗传多样性评价和主成分分析结果显示,构建割手密核心种质所筛选材料具有丰富的遗传多样性,且基于核心种质绘制的主成分图与原始种质的分布图趋势相吻合。结果表明,根据30%的抽样比例筛选出99个割手密样本构建核心种质,遗传多样性评价和主成分分析所构建的核心种质具有较好的代表性。

24份农艺性状优异割手密黑穗病抗性鉴定及Bru1基因分子检测

【目的】筛选抗黑穗病和褐锈病的割手密种质,为甘蔗黑穗病和褐锈病抗病亲本选育提供理论参考。【方法】采用人工浸渍接种法及与褐锈病基因Bru1紧密连锁的2个分子标记,分别对24份割手密进行甘蔗黑穗病和褐锈病抗性鉴定。【结果】结果表明,试验的割手密材料表现出较强的抗黑穗病能力,有16份材料表现1级高抗,5份材料表现2级抗病,3份材料表现3级抗病;分子检测结果显示,有7份材料含有Bru1基因,占参试材料的29.17%。【结论】本研究筛选出的高抗黑穗病且兼含Bru1基因的种质,是培育抗黑穗病和抗褐锈病的甘蔗品种(亲本)的良好抗源材料。