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 ;

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。

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.

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.

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.

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.

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.

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.

甘蔗(Saccharum officinarum L.)品种遗传差异的AFLP分子标记分析

采用AFLP技术 ,从 6 4对引物中筛选出 5对带型分布均匀、多态性丰富且分辨能力强的引物 ,对两个在生产上广泛推广应用的甘蔗品种的AFLP指纹图谱进行分析 ,计算了两品种间的遗传相似性和遗传距离。结果表明 ,5对引物在 2个甘蔗品种间均存在显著的差异 ,其中多态性位点占总扩增位点的 10 2 % ,区分率达 10 0 %。这对应用AFLP技术鉴定甘蔗品种的真伪提供了依据。本文还对甘蔗AFLP分析体系的关键技术以及银染的安全性和可靠性进行了讨论。

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。

用AFLP分子标记技术鉴别甘蔗(Saccharum of ficinarum L.)品种

本研究采用AFLP技术 ,从 6 4对引物中筛选出 5对带型分布均匀、多态性丰富且分辨能力强的引物 ,对两个在生产上广泛推广应用的甘蔗品种的AFLP指纹图谱进行分析 ,计算了两品种间的遗传相似性和遗传距离。结果表明 ,5对引物在 2个甘蔗品种间均存在显著的差异 ,其中多态性位点占总扩增位点的 10 2 % ,区分率达 10 0 %。研究结果对生产上应用AFLP技术鉴定甘蔗品种的真伪性提供了依据。本文对甘蔗AFLP分析体系的关键技术以及银染技术的安全性和可靠性进行了讨论。

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。

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。

甘蔗的分类──Ⅱ．Saccharum L．属

Ｓａｃｃｈａｒｕｍ Ｌ．属的分类，经历了较大的变化。现在受到普遍接受的是Ｂｒａｎｄｅｓ（１９５８）的分类方法。他把ＳａｃｃｈａｒｕｍＬ．属分为６个种。本文讨论该属分类的历史衍变过程，各个种的形态特征、遗传特点、及对甘蔗育种的重要性。

Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane(Saccharum officinarum L.)

Gamma ray-induced in vitro mutagenesis and selection for salt(NaC l) tolerance were investigated in sugarcane(Saccharum officinarum L.). Embryogenic callus cultures were irradiated(10 to 80 Gy) and subjected to in vitro selection by exposure of irradiated callus to NaC l(0, 50, 100,150, 200, and 250 mmol L-1). Increasing NaC l concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+and K+concentration. Higher accumulation of proline and glycine betaine was observed in NaC l stressed callus irradiated at 20 Gy. Na+concentration increased and K+concentration decreased with increasing salt level. Irradiated callus showed50–60% regeneration under NaC l stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%,number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

甘蔗割手密种LRRII-RLK基因家族演化和表达分析

LRRII-RLK是一种类受体激酶(receptor-likekinase,RLK),该基因家族在植物中广泛存在,在植物的生长发育和生物胁迫中发挥着重要的作用,但目前在甘蔗中未见对该基因家族的系统鉴定和分析。本研究对甘蔗的LRRII-RLK基因进行分类,并研究了其结构和功能。结合割手密种基因组和发育组织、叶片发育梯度、昼夜节律和生物胁迫反应转录组数据,在甘蔗割手密种中共鉴定到27个LRRII-RLK基因,分布在17条染色体上。系统进化分析发现,它们分别属NIK、SERK和LRRII-C分支,其中处于LRRII-C分支的SsLRRII-RLK基因出现了显著扩增。顺式元件分析显示,它们的启动子共包含32类顺式元件,其主要参与植物光反应、响应胁迫反应、调控植物激素和植物生长发育。这些基因共有35对共线性基因对,具有多个保守基序,主要通过片段复制的方式进行扩增,且根据Ka/Ks比率发现,它们在演化过程中经历了严格的纯化选择作用。此外,SsLRRII-RLK基因表达分析显示,它们在甘蔗不同组织中表达量不同,其中部分基因在昼夜不同时期表达量呈现差异,这表明它们参与了植物生长和光合作用的调节。值得注意的是,SsLRRII-RLK基因的表达模式在不同病害(甘蔗梢腐病和花叶病)胁迫下呈现差异,因此,我们认为这些基因在病毒复制和发病机制过程中发挥作用。本研究结果有助于深入了解甘蔗LRRII-RLK基因的演化过程,且为LRRII-RLK基因在甘蔗生长发育、光合作用以及在病害胁迫中的功能研究提供初步的理论基础。

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。