Comparative Analysis of Genetic Diversity in Landraces of Waxy Maize from Yunnan and Guizhou Using SSR Markers

Waxy maize landraces are abundant inYunnan and Guizhou of China. Genetic diversity of waxy maize landraces from Yunnan and Guizhou were analyzed using SSR markers. We screened 38 landraces with 50 primers that generated 3 to 6 polymorphic bands, with an average of 4.13 bands. Shannon＇s information indices for genetic diversity of the 14 waxy maize landraces from Yunnan varied from 4.9571 to 42.1138 and averaged 26.5252; Shannon＇s information indices for genetic diversity of the 24 waxy maize landraces from Guizhou varied from 22.0066 to 40.6320 and averaged 32.3156. For the 14 waxy maize landraces from Yunnan, the within-landrace genetic diversity accounted for 45.40% and the among-landrace genetic diversity accounted for 54.60% of the total genetic diversity observed. For the 24 waxy maize landraces from Guizhou, the within-landrace genetic diversity accounted for 50.76% and the among-landrace genetic diversity accounted for 49.24% of the total observed. Some individual landraces possessed as much as 96.86% of the total genetic diversity occurring among landraces within origins. Differentiation between geographic origins accounted for only 3.14% of the total genetic diversity. Both Yunnan and Guizhou would be the diversity centers and the original centers of waxy maize.     

云南糯玉米种质资源的研究与利用

云南省是糯玉米的起源中心和遗传多样性中心,糯玉米种质资源丰富,品种类型多样,分布广泛。全省16个地州(市)78个县(市)都分布有糯玉米地方品种,西双版纳傣族自治州勐海县至今仍种植有被公认为具有一系列原始性状的地方糯玉米品种四路糯。从糯玉米地方品种的生态型或多样性、支链淀粉含量变化、过氧化物同工酶带型特征、分子标记揭示的遗传多样性等方面,回顾了相关研究机构和研究者对云南省糯玉米种质资源的研究,分析了勐海四路糯玉米与缅甸糯玉米、锡金原始玉米的关系,以及糯玉米在发展云南高原特色农业中的作用和面临的主要问题。     

一种三角帆蚌肌浆网Ca2+-ATP酶基因cDNA的全长克隆与表达分析

采用RACE技术,从三角帆蚌(Hyriopsis cumingii)鳃组织中成功克隆得到一种肌浆网Ca²⁺-ATP酶(sarco/endoplasmic reticulum calcium ATPase,SERCA)基因的全长cDNA序列,共3 326 bp,包含201-bp 5’-UTR区域、3 060-bp编码框(ORF)和65-bp 3’-UTR。ORF共编码1 019个氨基酸,预测无信号肽。该基因氨基酸序列呈现出典型的Ca²⁺-ATP酶特征,由Cation_ATPase_N、E1-E2_ATPase、Hydrolase、Cation_ATPase_C四种类型结构域组成,其内含SERCAs的常见结构组成包括磷酸化区域、异硫氰酸荧光素位点、FSBA结合位点、受磷蛋白结合区以及毒胡萝卜素位点。分析显示,该基因序列高度保守且与海洋软体动物具有最高同源性。荧光定量PCR检测,该基因在三角帆蚌外套膜、斧足、鳃、肝胰腺、性腺等5个组织中均有表达,且在鳃、外套膜、肝胰腺组织中表达较高。不同Ca²⁺浓度处理试验的结果表明,随水体中Ca²⁺浓度逐渐升高,该基因在外套膜中的表达水平呈先下降后上升趋势,并在Ca²⁺浓度为60 mg·L^-1时达到最低值,80 mg·L^-1时达到最高值。同时在60 mg·L^-1 Ca²⁺浓度条件下,外套膜中SERCA基因的表达量随时间推移先上升,并于48 h时达到最高,而后逐渐下降。上述结果为进一步深入研究SERCA基因的功能及其调控机理奠定了基础。     

滇杨优树遗传多样性的AFLP分析

以收集于云南和四川的52株滇杨优树为试验材料,并以1株大叶杨、1株北京杨和2株黑杨做类外对照,采用AFLP分子标记技术进行基因组DNA水平检测。结果表明,筛选出的8对EcoRⅠ+3/MseⅠ+3引物组合对52株滇杨优树共扩增出264条带,其中多态性条带174条,多态带百分率为64.52%,检测到有效等位基因数（Ne）为1.184,基因多样度（H）为0.121,Shannon信息指数为0.201,平均遗传相似系数为0.877;对56份杨树样本共扩增出325条带,其中多态性条带共247条,多态带百分率为75.21 %,检测出有效等位基因数（Ne）为1.185,基因多样度（H）为0.122,Shannon信息指数为0.209,平均遗传相似系数为0.876。UPGMA聚类分析结果显示,除滇杨优树QXB007与QXJ030外,其余杨树分析样本均能够被鉴别。基于采集地和遗传相似系数的模糊聚类分析表明,52株滇杨优树除开远采集地外,丽江采集地样本与其他采集地样本之间均有交集,说明丽江可能是滇杨的分布中心和起源中心。该研究结果为滇杨人工选择育种、遗传改良、种质资源收集与保存等提供了理论依据。     

糯玉米、优质蛋白玉米花培反应率的研究初报

用82-1培养基对14份糯玉米和10份优质蛋白玉米的花培反应率进行了离体培养研究。结果表明,14份糯玉米材料中有13份具有花培反应,占参试糯玉米材料的92.86%,其中反应率最高的是作登糯玉米(出愈率3.16%);10份优质蛋白玉米材料中有5份具有花培反应,占参试优质蛋白玉米材料的50.00%,其中反应率最高的是CML170(出愈率0.65%)。3份糯玉米材料即田东作登糯、都安良昌糯、天峨糯能产生正常绿苗,占参试糯玉米材料的21.43%,正常绿苗率分别为4.08%、9.09%、25.00%;1份优质蛋白玉米材料(CML170)产生正常绿苗,正常绿苗率占参试优质蛋白玉米材料的10.00%。试验结果还表明,糯玉米材料的出愈率以及绿苗率均比优质蛋白玉米材料高;糯玉米与优质蛋白玉米的花培反应同普通玉米一样存在基因型依赖性。     

水稻地方品种Pi-ta的3'-UTR遗传多态分析

【目的】分析Pi-ta的3'-UTR区遗传变异与该基因抗性功能之间的关系,了解Pi-ta的抗性决定机制,为培养更持久的抗性品种提供依据。【方法】以遗传多样性极高的云南水稻地方品种为研究对象,收集了137个云南地方水稻品种。育苗后提取三叶一心期的水稻幼苗总DNA,设计引物扩增了Pi-ta的3'-UTR区的DNA序列,并扩增了关键功能位点6 640到终止密码子第6 675处这一段的DNA序列。通过双向序列测定获得了137条3'-UTR区的DNA序列并提交至Gen Bank,通过变异位点检测分析云南水稻地方品种Pi-ta的3'-UTR区的遗传多样性程度,并基于最大简约法构建单倍型网络图,分析不同单倍型之间的谱系关系。同时,联合编码区关键抗病位点6 640的碱基状态对3'-UTR单倍型的分布进行分析,讨论3'-UTR区与Pi-ta抗性功能之间的关系。【结果】云南水稻地方品种Pi-ta的3'-UTR区呈现出高度的遗传多样性,长度为1.1 kb的3'-UTR区共有12个SNP位点,由这些SNP可将137个品种划分成7个单倍型。不同单倍型之间没有重组的信号。Pi-ta的3'-UTR对应的DNA编码区长为1 120bp,是植物基因3'-UTR平均长度(200 bp)的5倍多,G+C含量相对较低,为40.43%,不存在插入或缺失导致的长度多态性。Pi-ta的3'-UTR序列中存在多个非保守的潜在poly A位点,此外,Pi-ta的3'-UTR区还存在非常高频率的TTTT序列,提示Pi-ta在转录终止时可能具有复杂的调控机制;而对Pi-ta的不同转录本的分析也表明3'-UTR对应于DNA编码区序列时呈现复杂多变的剪切方式,3'-UTR这种选择性拼接可能与抗性决定作用有关。对遗传多态的进一步分析表明,3'-UTR的SNP高度多态性都出现在感病品种中,所有抗性品种只共享一种单倍型。有趣的是,唯一的3'-UTR抗性单倍型与Pi-ta编码区唯一的抗性单倍型相对应,也即是6 640G所在单倍型也是3'-UTR唯一抗性单倍型。这表明3'-UTR与其编码区是紧密关联的,在功能上和所受到的选择压力方面是连续和一致的。Pi-ta的抗性单倍型区域已从编码区扩展到了3'-UTR区,在研制广谱抗性品种引入Pi-ta时需要同时保证其3'-UTR区不能有额外的SNP,必须是抗性单倍型特有的SNPs。【结论】Pi-ta的3'-UTR与其编码区紧密连锁,抗性品种的3'-UTR受到纯净化选择,维持单一单倍型,3'-UTR对于Pi-ta的抗性功能具有不可或缺的作用。     

玉米高无机磷突变体的选育和特性研究

采用⁶⁰Coγ射线辐射诱变,创造出2个玉米高无机磷突变体lpa-zhong1和lpa-zhong2,研究了其突变的等位性和遗传控制类型,同时对野生型亲本和突变体的种子发芽率、磷组分含量、微量金属元素含量以及农艺性状和产量性状做了初步研究。结果表明,两个突变是等位的,且突变受单隐性基因所控制;与野生型亲本相比,突变造成种子发芽率降低;总磷、无机磷含量极显著增加,植酸含量显著下降;金属元素含量、农艺性状无显著变化;除了百粒重显著下降之外,其他产量性状差异不显著。     

Susceptibility breakpoint for cefquinome against Escherichia coli and Staphylococcus aureus from pigs

Cefquinome is the only fourth-generation cephalosporin used solely for veterinary applications.In this study,we established the wild-type cut-off (CO_(WT)) and pharmacokinetic/pharmacodynamic cut-off (CO_(PD)) of cefquinome against Escherichia coli and Staphylococcus aureus.A total of 210 E.coli and 160 S.aureus isolates were collected from pigs in Guangdong Province between 2014 and 2018.The minimum inhibitory concentrations (MICs) were determined using a microdilution broth method.MIC_(50) and MIC_(90) were 0.06 and 0.25μg m L~(–1) for E.coli and 0.5 and 1μg m L~(–1) for S.aureus,respectively.Statistical analysis and the ECOFFinder Program showed that the CO_(WT )for cefquinome against E.coli and S.aureus were0.125 and 2μg m L~(–1),respectively.The resistance rates were 11.9%for E.coli and 6.25%for S.aureus.Based on a5 000-subject Monte Carlo simulation,the CO_(PD) value for cefquinome against E.coil and S.aureus was 0.25μg m L~(–1) under the recommended dose (2 mg kg~(–1),twice a day for 3 days),confirming that infections caused by strains with MIC≤0.25μg m L~(–1) could be effectively treated.Following adjustment of the dosing regimen to 4.5 mg kg~(–1),effective treatment (90)was achieved for S.aureus infections with MIC_(90) 1μg m L~(–1).This susceptibility breakpoint determination is significant for resistant surveillance and cefquinome dosage guidance against E.coli and S.aureus in pigs.     

Grain zinc and iron concentrations of Chinese wheat landraces and cultivars and their responses to foliar micronutrient applications

Grain zinc (Zn) and iron (Fe) concentrations and their responses to foliar application of micronutrients in 28 Chinese wheat landraces and 63 cultivars were investigated in a two-year field experiment.  The average grain Zn and Fe concentrations were 41.8 mg kg^–1 (29.0−63.3 mg kg^–1) and 39.7 mg kg^–1 (27.9−67.0 mg kg^–1), respectively.  Compared with cultivars, landraces had greater grain Zn (11.0%) and Fe (4.8%) concentrations but lower harvest index (HI), grain weight per spike (GWS), grain number per spike (GNS) and thousand grain weight (TGW).  Both Zn and Fe concentrations were negatively and significantly correlated with HI, GWS, and GNS, while showed a poor association with TGW, suggesting that lower HI, GWS, and GNS, but not TGW, accounted for higher Zn and Fe concentrations for landraces than for cultivars.  Grain Zn concentrations of both cultivars and landraces significantly increased after foliar Zn spray and the increase was two-fold greater for landraces (12.6 mg kg^–1) than for cultivars (6.4 mg kg^–1).  Foliar Fe spray increased grain Fe concentrations of landraces (3.4 mg kg^–1) and cultivars (1.2 mg kg^–1), but these increases were not statistically significant.  This study showed that Chinese wheat landraces had higher grain Zn and Fe concentrations than cultivars, and greater increases occurred in grain Zn concentration than in grain Fe concentration in response to fertilization, suggesting that Chinese wheat landraces could serve as a potential genetic source for enhancing grain mineral levels in modern wheat cultivars.     

贵州糯玉米地方品种的SSR遗传多样性分析

为糯玉米种质扩增改良提供依据,利用15对糯玉米核心引物,采用SSR标记技术,对贵州糯玉米品种种质资源的遗传多样性进行了分析。结果表明:15对核心引物在36份糯玉米材料中共检测出205个等位基因,平均每对引物检测出13.66个,每个位点的SSR多态信息量(PIC)平均为0.87;36个糯玉米品种间平均遗传相似性系数为0.64±0.072,变异系数为11.30%;聚类分析与主坐标分析结果一致,36份糯玉米材料共划分为3个类群。     

Investigating seed mineral composition in Korean landrace maize(Zea mays L.) and its kernel texture specificity

Mineral malnutrition affects billions of people all over the world and biofortification of staple crops provides a potential way to alleviate dietary mineral deficiencies. For example, nutritional quality is an important breeding target for fresh waxy maize(Zea mays L.), which is widely consumed in Asian countries. Successful improvement of mineral composition will require comprehensive profiling of the mineral composition of maize varieties and an understanding of the capacity for maize grains to accumulate minerals. Here, using inductively coupled plasma absorption emission spectrometry, we quantified 12 minerals from the seeds of 47 maize varieties, including 25 Korean landraces. We also compared the mineral contents in varieties with different seed starch profiles: waxy maize(which contains 100% amylopectin), dent maize(roughly 75% amylopectin and 25% amylose), and flint maize(similar to dent maize). The amounts of potassium, phosphorus, and sulfur were correlated with seed texture, waxy maize having higher amounts of phosphorus and potassium than dent maize and lower amounts of sulfur than flint maize or dent maize. In addition, a positive relationship was detected between the amount of phosphorus and that of potassium, magnesium, and manganese. These results provide information on maize seed mineral composition and indicate that it could be affected by starch composition. Furthermore, the landraces that exhibit high mineral contents could be used as germplasm materials for breeding programs aimed at producing biofortified maize cultivars.     

waxy Locus and Its Mutant Types in Maize Zea mays L.

The waxy （wx） locus of Zea mays L. encodes a granule-bound starch synthase （GBSS I = waxy protein） required for the synthesis of amylose in endosperm and pollen grain. This review covers recent advances in understanding the waxy locus in maize, focusing particularly on the new information on mutant type and mutation mechanisms. The results showed that the insertion and deletion played an important role in the generation of spontaneous wx-mutations. The current status of utilizing waxy locus has been summarized and the perspectives of the further studies on this locus have also been proposed.     

糯玉米萌发期耐低温品种资源的综合评价

为构建不同糯玉米品种萌发期耐低温评价体系，以48个糯玉米品种为试验材料，测定低温胁迫下糯玉米的10个萌发及生长指标，通过主成分分析法、隶属函数法、聚类分析和回归分析，对糯玉米品种耐低温性进行综合评价与分类。结果表明，主成分分析将10个测定指标转化为根系生长因子和种子萌发因子2个相互独立的综合指标，建立了糯玉米耐低温综合得分模型Y=0.647 7Y₁+0.144 2Y₂（其中，Y₁、Y₂为2个主成分，Y₁代表的主要指标包括相对根干重、相对根表面积、相对根体积、相对根长，反映低温胁迫下糯玉米根系生长的状态；Y₂代表的主要指标包括相对苗干重、相对发芽率、相对活力指数、相对发芽势，反映低温胁迫下糯玉米的萌发情况）。通过隶属函数法对48个糯玉米品种的耐低温能力进行排序，筛选出5个耐低温能力较强的品种，依次为‘润彩糯686’‘景坡82’‘润糯175’‘润糯605’和‘天贵糯932’。聚类分析将48个糯玉米品种划分成4个类群。回归分析表明，相对根干重、相对苗长、相对发芽势、相对发芽率、相对苗干重和相对根表面积可作为糯玉米耐低温评价的重要指标。多方法关联分析的综合评价方法可对不同耐低温能力的糯玉米品种进行有效筛选分类，为糯玉米耐低温评价及耐低温种质资源的筛选提供参考。     

糯玉米自交系遗传多样性及其产量、农艺性状与SSR分子标记的关联研究

[目的]研究糯玉米自交系的遗传多样性,确定其亲缘关系远近,为糯玉米的新品种选育提供依据。[方法]以84个糯玉米自交系为试验材料,利用分布在玉米全基因组上的71个SSR标记对供试材料的遗传多样性进行分析,并对其产量及农艺性状与SSR标记进行了关联分析。[结果]150对SSR引物中有71对在糯玉米自交系中能扩增出多态性条带,共检测到342个等位基因,每对引物可检测到2～11个数目不等的等位基因,多态性信息量为0.249～0.876;糯玉米自交系间的遗传距离为0.02～0.32,均值为0.178,4个自交系可划分为8个组别;玉米10个连锁群上71个SSR位点的2 485个组合,都存在一定程度的连锁不平衡;共检测出21个SSR座位与10个产量、农艺性状显著相关。[结论]该试验阐明了所选糯玉米自交系的遗传多样性及相互间的亲缘关系,为有目的的组配糯玉米杂交种及其新品种选育提供了参考。     

Mutator诱导的玉米白化突变体插入位点的遗传分析及代谢途径的构建

【目的】利用Mutator(Mu)诱变群体获得、验证叶色白化突变体的Mu因子插入位点;解析玉米叶色变异相关基因及其代谢网络。【方法】以含有活性MuDR转座子的W22∷Mu为父本,与玉米自交系综31(Z31)杂交产生的M2和M3家系群体为材料,经表型性状的遗传分析和Mu插入位点的分离获得白化突变体,经生物信息学方法解析白化突变体产生的相关基因,同时构建产生白化突变代谢网络。【结果】对870个M2家系的16000余单株和M3种植的36个家系近700单株进行考察,获得了遗传稳定的白化突变体41株。经Mu-TAIL-PCR分析获得35条Mu因子插入序列;对靶位点序列分析,获得的14个靶位点可能与叶绿素合成及光合作用有关;利用其中6个靶位点构建了5条玉米叶色突变产生白化的叶绿素代谢网络途径。【结论】初步证实了Mu转座子插入的27个靶位点与叶色白化突变体的关联;建立了6个靶位点的玉米叶色突变产生白化的叶绿素代谢网络途径。     

黑龙江省稻瘟病菌无毒基因AVR-Pib、AVR-Pik和AvrPiz-t的检测与分析

【目的】检测黑龙江省稻瘟病菌（Magnaporthe oryzae）无毒基因AVR-Pib、AVR-Pik和AvrPiz-t在不同地区、年份间流行菌株的分布情况与变异机制,了解其等位基因的致病表型,为黑龙江省抗瘟品种布局提供参考依据。【方法】利用NCBI中公布的无毒基因序列对3个无毒基因AVR-Pib、AVR-Pik和AvrPiz-t的基因全长和编码序列（coding sequence,CDS）分别设计特异性引物,将2016年和2017年采自黑龙江省不同地区335个稻瘟病菌单孢分离菌株的DNA进行PCR扩增,通过琼脂糖凝胶电泳检测分析,并挑选不同带型和不同地区代表菌株的PCR产物进行测序。测序结果与相应无毒基因序列进行碱基与氨基酸序列的比较分析,并利用水稻抗性单基因系,对不同变异类型的稻瘟病菌菌株进行致病型测定。【结果】在PCR电泳检测中AVR-Pib、AVR-Pik和AvrPiz-t均出现特异性条带,说明这3个无毒基因在黑龙江省均有分布,并以不同分布频率与突变类型出现。3个无毒基因平均扩增频率分别为75.52％、87.16％和85.67％。其中AVR-Pib通过电泳检测与PCR产物测序检测出4种带型（无带、高带、中高带与低带）和5种变异类型AVR-Pib（1-1、1-2、2、3、3-1）,基因型AVR-Pib-1-1、AVR-Pib-1-2、AVR-Pib-2和AVR-Pib-3-1为新发现的变异类型,其中基因型AVR-Pib-1-1和AVR-Pib-1-2均为转座子Pot2的插入,但插入位点不同;基因型AVR-Pib-2在阅读框上游存在小片段的插入;基因型AVR-Pib-3-1碱基序列与原序列比对有4处差异,即32（C/G）35（T/A）36（T/A）38（T/A）,导致氨基酸翻译提前终止。对检测到的5种等位基因型进行致病型测定,分析发现除正常基因型AVR-Pib-3外,其他变异基因型无毒功能均已丧失。而AVR-Pik经PCR产物测序检测出7种等位基因型,AVR-Pik（D、A、B、C、E、F、F2）,碱基序列的变化均导致氨基酸错义突变,7种等位基因型均已见报道。无毒基因AvrPiz-t通过电泳检测和PCR产物测序分析,发现2种带型（高带和正常带型）与4种基因型AvrPiz-t（A、B、C、D）,其中AvrPiz-t-A为正常基因型;基因型AvrPiz-t-B在191位处有碱基A的插入,导致氨基酸翻译提前终止;基因型AvrPiz-t-C为新发现的等位基因型,其特点在于与A类型存在17（T/C）和19位处碱基C的插入,发生移码突变翻译提前终止;高带型对应的无毒基因型AvrPiz-t-D经测序验证为Pot3转座子的插入。4种基因型菌株分别接种水稻单基因系发现,AvrPiz-t-A可以被Piz-t识别表现无毒,AvrPiz-t（B、C、D）均不能被Piz-t所识别而表现为有毒性。【结论】黑龙江省稻瘟病菌无毒基因AVR-Pib、AVR-Pik和AvrPiz-t分布较为广泛,且变异类型丰富,研究结果可为选育和推广携带相应抗病基因的水稻品种提供参考。     

Expression profiles of Cry1Ab protein and its insecticidal efficacy against the invasive fall armyworm for Chinese domestic GM maize DBN9936

The fall armyworm(FAW) Spodoptera frugiperda, which originated in the Americas, is advancing across China and threatening the nation's maize crops. Currently, one widely used tool for its control is genetically modified(GM) Bacillus thuringiensis(Bt) maize. Sufficient content of Bt protein in appropriate plant parts is crucial for enhancing resistance against insect pests. In this study, we conducted a systematic investigation of Cry1 Ab levels in Chinese domestic GM maize DBN9936, which has recently obtained a biosafety certificate, and evaluated its efficacy against FAW. Quantification of expression levels of Cry1 Ab, via ELISA, indicated a spatio-temporal dynamic, with significant variation of mean Cry1 Ab, ranging from 0.76 to 8.48 μg g–1 FW with the Cry1 Ab protein level ranked as: V6–V8 leafR1 leafR4 leafR1 silkVT tasselR4 kernel. Among the nine locations, the Cry1 Ab levels in DBN9936 of the Xinxiang, Langfang, and Harbin fields were significantly lower than those from Wuhan and Shenyang, and were slightly, but not significantly lower than those from the other four fields. Furthermore, the artificial diet–Cry1 Ab mixture and plant tissue feeding bioassays revealed that DBN9936 has high efficacy against FAW. The insecticidal efficacy of different tissues against FAW larvae reached 34–100% with a descending order of lethality as follows: VT leafR4 leafR1 huskR1 silkVT tasselR4 kernel. Taken together, our results showed that Bt-Cry1 Ab maize DBN9936 has potential as a promising strategy to manage FAW.