Genetic Diversity of Peanut (Arachis hypogea L.) Cultivars as Revealed by RAPD Markers

The objectives of this study were to evaluate the genetic diversity of the peanut accessions using Random Amplified Polymorphic DNA (RAPD) molecular marker and to evaluate RAPD markers to be used in peanut as genetic markers and improve such techniques as suitable strategies for peanut germplasm characterization. Twenty peanut accessions were included in this study and were subjected to RAPD molecular markers analysis. Twenty-seven RAPD primers produced 210 amplification products of which 80 (36.4%) were polymorphic. In conclusion, this study reported a successful fingerprinting of peanut accessions using RAPD markers and demonstrated the usefulness of these markers in estimating the extent of genetic variation in peanut germplasm.

Construction of Genetic Linkage Map Based on SSR Markers in Peanut(Arachis hypogaea L.)

Molecular genetic maps of crop species can be used in a variety of ways in breeding and genomic research such as identification and mapping of genes and quantitative trait loci （QTLs） for morphological, physiological and economic traits of crop species. However, a comprehensive genetic linkage map for cultivated peanut has not yet been developed due to the extremely low frequency of DNA polymorphism in cultivated peanut. In this study, 142 recombinant inbred lines （RILs） derived from a cross between Yueyou 13 and Zhenzhuhei were used as mapping population in peanut （Arachis hypogaea L.）. A total 652 pairs of genomic-SSR primer and 392 pairs of EST-SSR primer were used to detect the polymorphisms between the two parents. 141 SSR primer pairs, 127 genomic-SSR and 14 EST-SSR ones, which can be used to detect polymorphisms between the two parents, were selected to analyze the RILs population. Thus, a linkage genetic map which consists of 131 SSR loci in 20 linkage groups, with a coverage of 679 cM and an average of 6.12 cM of inter-maker distance was constructed. The putative functions of 12 EST-SSR markers located on the map were analyzed. Eleven showed homology to gene sequences deposited in GenBank. This is the first report of construction of a comprehensive genetic map with SSR markers in peanut （Arachis hypogaea L.）. The map presented here will provide a genetic framework for mapping the qualitative and quantitative trait in peanut.

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut(Arachis hypogaea L.) Seeds

Phosphoenolpyruvate carboxylase（PEPC;EC 4.1.1.31） catalyses phosphoenolpyruvate（PEP） to yield oxaloacetate,which is involved in protein biosynthesis.Pyruvate kinase（PK;EC 2.7.1.40） catalyzes PEP to yield pyruvate,which is involved in fatty acid synthesis.In this study,five PEPC genes（AhPEPC1,AhPEPC2,AhPEPC3,AhPEPC4,and AhPEPC5） from peanut have been cloned.Using a quantitative real-time RT-PCR approach,the expression pattern of each gene was monitored during the seed development of four peanut varieties（E11,Hebeigaoyou,Naihan 1,and Huayu 26）.It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging（DAP）;whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou.In Naihan 1 and Huayu 26,the expression levels of the five genes remained relatively high in the first stage.The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development.The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter.These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.

Drought-induced responses of organic osmolytes and proline metabolism during pre-flowering stage in leaves of peanut (Arachis hypogaea L.)

Peanut （Arachis hypogaea L.）, an improtant oil crop, usually encounters drought stress in the process of growth and development, especially at pre-flowering stage. In order to gain insight into the drought tolerance potentials based on osmolyte accumulation and metabolism of proline aspects of peanut, pot experiments were conducted with a split-plot design in Tai＇an, Shangdong Province, China in 2013 and 2014. Pre-flowering drought （PFD） stress and optinum irrigation （control, CK） were served as the main plots and the two peanut cultivars Shanhua 11 and Hua 17 served as sub-plots. Shanhua 11 was drought-tolerant cultivar and Hua 17 was drought-sensitive. The content of soluble sugars, soluble protein, free proline and other free amino acids, the activities of enzymes involved in proline metabolism, and malondialdehyde （MDA） content and ion leakage were all investigated in the two cultivars at pre-flowering stage. Results showed that PFD stress significantly increased the levels of soluble protein, free proline and free amino acid, and increased Al-pyrroline-5-car- boxylate synthetase （P-5-CS, EC 2.7.2.11） activity in the leaves of drought-tolerant and drought-sensitive cultivars. The activity of proline dehydrogenase （proDH） （EC 1.5.99.8） decreased under PFD stress in both cultivars. The leaves of the tolerant cultivar maintained higher increments of osmolyte levels, lower increments of MDA content and ion leakage, as well as a higher increased proportion of P-5-CS activity and higher inhibited proportion of proDH activity under water stress compared with the drought-sensitive cultivar. The study suggests that proline accumulation in peanut leaves under PFD can be explained by the higher enhanced activities of P-5-CS and higher inhibition of proDH. The results will provide useful information for genetic improvement of peanut under drought tolerance.

Phytochemical Constituents and Determination of Resveratrol from the Roots of <i>Arachis hypogea</i>L.

Two triterpenoid saponins, 6, 7, together with five known compounds were isolated from the roots of Arachis hypogea L. collected in Vietnam. Their structures were established by spectroscopic analysis (1D, 2D NMR and HRMS) as well as by comparison with those reported in the literature. Furthermore, the high content of resveratrol in the roots opens a new potential resource for pharmaceutical companies to prepare medicines from the wasted peanut roots.

Identification of main effect and epistatic QTLs controlling initial flowering date in cultivated peanut(Arachis hypogaea L.)

Initial flowering date(IFD)is closely related to mature period of peanut pods.In present study,a population of recombinant inbred lines(RIL)derived from the cross between Silihong(female parent)and Jinonghei 3(male parent)was used to map QTLs associated with IFD.The RIL population and its two parental cultivars were planted in two locations of Hebei Province,China from 2015 to 2018(eight environments).Based on a high-density genetic linkage map(including 2996 SNP and 330 SSR markers)previously constructed in our laboratory,QTLs were analyzed using phenotypic data and the best linear unbiased prediction(BLUP)value of initial flowering date by inclusive composite interval mapping(ICIM)method.Interaction effects between every two QTLs and between individual QTL and environment were also analyzed.In cultivated peanut,IFD was affected by genotypic factor and environments simultaneously,and its broad sense heritability(h2)was estimated as 86.8%。Using the IFD phenotypic data from the eight environments,a total of 19 QTLs for IFD were detected,and the phenotypic variation explained(PVE)by each QTL ranged from 1.15 to 21.82%.Especially,five of them were also detected by the BLUP value of IFD.In addition,12 additive QTLs and 35 pairs of epistatic QTLs(62 loci involved)were identifed by the joint analysis of IFD across eight environments.Three QTLs(qIFDB04.1,qIFDB07.1 and qIFDB08.1)located on chromosome B04,B07 and B08 were identified as main-effect QTL for IFD,which had the most potential to be used in peanut breeding.This study would be helpful for the early-maturity and adaptability breeding in cultivated peanut.

Cloning, Expression Pattern Analysis and Subcellular Localization of Resveratrol Synthase Gene in Peanut (<i>Arachis hypogaea</i>L.)

Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.

Gomparative analysis of DGAT3（diacylglycerol acyltraiisferase 3） gene from different peanut （Arachis hypogaea L.） varieties

Cultivated peanut is one of the primary sources of vegetable oil and protein in developing countries. DG〉A73 family in peanut cotyledons has no membrane-bound regions suggesting that cytosol is one of the sites for triacylglycerol （TAG） biosynthesis in oilseeds. According to functional annotation and classification of 5 cDNA libraries, 12 unigenes were found with relation of peanut DGAT3 in different organs. Three clones of unigenes, OCP- contig168t OCPcontig12101-3 and OCPcontigl2388-1 were selected for sequencing. Full length sequence of DGAT3 was obtained, showing over 98% sequence similarity with peanut DGAT3 gene AY875644 or EU183333. Upon cluster analysis, DGAT3 of 40 culti- vars were divided into 3 types, namely AhDGATS-1, AhDGAT3-2 and AhDGAT3-3. Coding regions are 1023, 1038 and 1026 base pairs which encoded proteins with 340-, 345- and 341-amino acids, respectively. DGAT3-3 might be a novel gene type among the DGAT3 family which provides great help for studying DGAT3 gene evolution in peanut.

不同土壤类型对花生(Arachis hypogaea L.)籽实镉积累特性的影响

为了在不同土壤环境下选育出低镉(Cd)积累型花生品种以提高花生品质,以‘白沙1016号’品种花生(Arachis hypogaea L.)为供试作物,棕壤和潮土为供试土壤,采用盆栽试验的方法,研究了2种类型土壤对花生籽实Cd积累特性的影响。结果表明:2种类型土壤条件下的花生籽实生物量差异显著(P<0.05);在供试土壤Cd处理范围内(≤10.0 mg/kg),籽实生物量均随2种土壤Cd处理浓度增加呈"低促高抑"现象。花生籽实对土壤Cd的生物富集系数均随2种土壤Cd处理增加呈显著降低趋势(P<0.05),且土壤类型间差异不显著。花生籽实Cd含量和Cd生物富集量均随2种土壤Cd处理的增加而显著增加(P<0.05),但两者在棕壤介质中均显著大于潮土(P<0.05)。土壤类型对花生籽实Cd积累差异的影响主要体现在籽实Cd含量和生物富集量方面。而土壤pH、氧化还原电位(Eh)等均是影响籽实对Cd积累差异的重要因素。

花生栽培种与野生种(Arachis oteroi)人工杂交双二倍体的创制和鉴定

花生野生种是改良花生栽培种的重要基因资源。为了利用野生花生的抗性基因,本研究利用花生栽培品种豫花9331与二倍体野生种A.oteroi人工杂交,借助胚拯救和染色体秋水仙素加倍,创制一个双二倍体杂种Am E-4,并利用荧光原位杂交和分子标记技术准确鉴定了该双二倍体。观察结果表明,Am E-4的叶片与豫花9331存在显著差异,而主茎高、侧枝长和总分枝数等性状与豫花9331差异不显著。Am E-4开花期较豫花9331推迟60 d,结实性与荚果发育状况较差,不利于Am E-4的育种利用。同时,开发了57个追踪Am E-4中A.oteroi染色体的显性或共显性SSR标记,为创制和鉴定花生栽培种A.oteroi易位系或渐渗系奠定分子基础。

Inoculation of <i>Rhizobiums sp</i>Strains to Improve Soil Fertility: A Peanut Trial in Covèand Ouessè(Benin)

Thought the increasing demand Arachis hypogaea L. (groundnut), its yields remain low with increasingly using chemical fertilizers. To reduce the costs for chemical fertilizers inquisition and their long-term toxic effects on soils, microbial bio-fertilizers could be an accessible alternative to peanut farms. Thus, the aim of this study was to assess the performance of rhizobia strains on peanut varieties production. The experiments were conducted in two agro-ecological zones of Benin, in a peasant environment peasant-researcher control or under peasant and researcher control. The experimental device used was a complete random block with nine repetitions and two factors namely inoculation (with Rhizobium sp and without Rhizobium sp) and mineral fertilizer (with N15P15K15 and without N15P15K15). The effects of these factors divided into four treatments were evaluated on the plants vegetative, symbiotic and production parameters. In addition, an evaluation of each treatments’ comparative advantages was carried out. The results showed that the association Rhizobium sp and N15P15K15 induced groundnut plants best vegetative and productive parameters. The best comparative advantages in economic terms were also recorded with the same combination (Rhizobium sp + N15P15K15). Considering the technical performance, the recorded treatments effects can be classified as follows: Control Rhizobium sp 15P15K15 Rhizobium sp + N15P15K15. Thus, the association Rhizobium sp + N15P15K15 induced both the best plants vegetative and productive parameters and the best comparative advantage from an economic point of view. The results also showed that the plants’ response to inoculation, the application of manure and their combination was more marked in the bar soil zone (Covè) than in the cotton zone (Ouessè). Considering the negative effects linked to the use of chemical fertilizers, the use of Rhizobium sp could be an interesting path to increase the groundnut production.

Interaction of Seedling Germination, Planting Date, and Flumioxazin on Peanut Physiology under Irrigated Conditions

Diclosulam and flumioxazin applied preemergent (PRE) results in direct peanut exposure to these herbicides prior to seedling emergence. Flumioxazin has been reported to induce injury in adverse weather (i.e. cool-wet soil conditions) at crop emergence. Research at Ty Ty and Plains, Georgia evaluated the physiological effects of PRE herbicides to emerging peanut in 2018 and 2019. Peanut seed with variable germination and different planting dates were evaluated as additional factors. Peanut plant physiological measurements included electron transport (ETR), net assimilation rate (Anet), quantum yield of PSII (ΦPSII), and stomatal conductance to water vapor (GSW). Data were obtained from V3 to R1 peanut growth stages using a LiCOR 6800, along with stand counts and plant width measures. In 2018, diclosulam reduced peanut ETR when measured across multiple growing degree days (GDD) after planting, compared to the nontreated control (NTC). Flumioxazin reduced peanut ETR compared to the NTC, at several sample timings for each planting date. In 2018 and 2019 at both locations, flumioxazin impacted Anet less than ETR, but was consistently similar to/or greater than the NTC. Peanut ΦPSII responded similarly as Anet at each location and yr. GSW was variable in both years;however flumioxazin treated plants had higher GSW rates than other treated plants. Peanut stand counts, plant widths, and pod yields noted few differences compared to the physiological measures. Though some peanut plant physiological differences were noted when measured at varying GDD’s after planting with the different PRE treatments, planting date, and seed vigor, no specific trends were observed. Growers will often observe peanut injury from flumioxazin early in the season. However, it is transient and does not affect yield.

Molecular characterization of a peanut variety and its derivatives based on SSR and COP analysis

Despite the economic importance of the peanut, no studies have been carried out to deter-mine the correlation between genetic distances based on molecular markers and on coefficient of parentage （COP） data. In this study, simple sequence repeat （SSR） and pedigree data were used to assess the ge-netic distance between the Fuhuasheng variety and its derivative cultivars. A total of 39 SSR polymor-phism primers were used, and 151 bands were obtained, with an average of 2. 04 bands in each primer. The genetic SSR - based distance （GD） values ranged from 0. 02 to 0. 81, while the COP - based GD ranged from 0. 25 to 0. 98. Certain Fuhuasheng loci displayed higher transmission rates. These loci or nearby chromosomal regions might be associated with desirable traits in Fuhuasheng variety, thus being frequently selected in breeding programs. Therefore, it can be suggested that COP analysis should be the preferred method for estimating genetic diversity invarieties with available complete pedigree information and parents. In this case, marker analysis would provide the best estimations.

HPLC法测定花生不同部位中白藜芦醇的含量

目的 :探讨测定花生不同部位中白藜芦醇含量的方法。 方法 :采用高效液相色谱 (HPLC)法测定花生的根、茎、花、果壳、种衣、叶、仁 7个部位中白藜芦醇的含量。 结果 :花生的根、茎、花、果壳、种衣 5个部位中含有白藜芦醇 ,其中花生根中含量最高。方法回收率为 98.7% ,RSD为 2 .39% ,花生叶、花生仁中不含白藜芦醇。 结论 :采用HPLC法测定花生中白藜芦醇的含量 ,方法简便 。

花生主要品质性状的QTLs定位分析

以郑8903×豫花4号的215个重组自交系群体(RILs)为材料,采用2008年原阳种植材料(F8)的品质检测数据,运用WinQTLCart 2.5软件进行与花生蛋白质、脂肪及脂肪酸含量相关的QTLs定位分析。研究结果显示,检测到2个与蛋白质含量相关的QTLs,遗传贡献率分别为4.82%和9.66%;2个与脂肪含量相关的QTLs,遗传贡献率分别为5.25%和8.24%。与油酸、亚油酸、硬脂酸和山嵛酸含量相关的QTL各检测到1个,遗传贡献率分别为5.13%、8.28%、24.14%和7.88%;2个与花生酸含量相关的QTLs,遗传贡献率分别为7.12%和18.32%。

利用RIL群体和自然群体检测与花生含油量相关的SSR标记

以远杂9102×中花5号杂交后代衍生的重组近交系F8代家系为材料,在含油量测试的基础上,选用10份低油材料(平均含油量52.91%)、12份高油材料(平均含油量58.85%)以及亲本进行SSR引物筛选,通过631对SSR引物扩增,筛选出来源于7对引物的13个有显著差异的片段可以有效区分低油材料和高油材料。以这7对差异引物在F8RIL群体中扩增,对20份低油家系材料(含油量<55%)和45份高油家系材料(含油量>56%)进行统计分析,获得1个与花生含油量相关的分子标记2A5-250/240,其中,标记2A5-250为低油材料(含油量<55%)所拥有,相符率为95.0%,标记2A5-240为高油材料(含油量>56%)所拥有,相符率为88.9%。用SSR标记2A5-250/240检测11份高油(平均含油量为55.93%)栽培种花生和11份低油(平均含油量为48.41%)栽培种花生,结果表明,标记2A5-240与高油栽培种花生的符合率为63.6%,2A5-250与低油栽培种花生的符合率为90.9%。在19份高油(平均含油量为58.60%)野生花生中,10份野生花生能检测到标记2A5-240。综合分析RIL群体和自然群体的研究结果表明,标记2A5-250/240可用于花生含油量分子标记辅助选择。

花生锈病抗性的AFLP标记

利用抗、感锈病花生品种为亲本配制杂交组合远杂9102×ICGV 86699,以其F2分离群体为研究材料,利用BSA法和AFLP技术,获得了与锈病抗性连锁的2个分子标记,与抗性基因间的遗传距离分别为10.90cM和7.86cM。利用获得的分子标记对其F3进行了验证,分子标记与抗性鉴定结果具有较高的一致性。

花生AFLP遗传图谱构建及青枯病抗性QTL分析

青枯病抗性分子标记能为花生抗青枯病育种提供辅助选择技术,以抗青枯病品种远杂9102与感病品种Chico杂交构建的重组自交系群体(RIL)为材料,用66对具多态性的引物(EcoR I/MseI引物组合35对,MluI/MseI引物组合14对,PstI/MseI引物组合17对)对其进行扩增,共检测到324个多态性位点。应用JoinMap(3.0软件对这些多态性位点进行遗传连锁分析,构建了一张栽培种花生的AFLP遗传连锁图。该图谱包含98个AFLP标记,涉及20个连锁群,覆盖总距离285 cM,标记间平均图距为2.90 cM。结合RIL群体的青枯病抗性鉴定结果,利用分析软件QTLNetwork(2.0共检测到与青枯病抗性相关的3个QTL(qBWr1、qBWr2和qBWr3)。其中,qBWr1和qBWr2均位于第4连锁群,qBWr3位于第14连锁群。这3个QTL形成2对具有加性×加性上位性互作效应的QTL区段(qBWr1/qBWr3和qBWr2/qBWr3),贡献率分别为12.81%和16.56%,共解释青枯病抗性总变异的21.62%。

花生晚斑病抗性AFLP标记

以抗感晚斑病组合"中花5号×ICGV 86699"的F2分离群体为材料,经田间抗性鉴定明确抗性亲本ICGV 86699的抗性受隐性单基因或主效基因控制;AFLP分析结合BSA法筛选到与晚斑病抗性连锁较紧密的AFLP标记3个,即E35/M51、E37/M48和E41/M47,它们与抗性间的图距分别为7.40cM、7.40cM和8.67cM;所获得的3个标记间连锁紧密,位于同一连锁群上。这3个标记在具有野生种亲缘的7个抗病花生品种或材料中均能检测到,而在栽培种以多粒型为代表的抗病材料中均未检测到,表明ICGV 86699的抗性基因与栽培种花生中的抗性基因不同。这是国内外有关花生晚斑病抗性分子标记的首例报道。

花生重组近交系(RIL)根部性状的遗传分析

利用花生RIL群体．分析了主根长、侧根数、根基粗（1cm和3cm处）、根体积、主根鲜重、干重、侧根鲜重和干重、主侧根瘤数等11个花生根部性状的遗传力，估算基因对数及性状间的相互关系，根据偏度系数（g1）和峰度系数（g2）的估算控制性状基因互作情况。结果表明：在11个研究性状中，有6个性状在2个亲本间差异显著或极显著。但不论性状在亲本间的差异显著与否，在RIL群体中基因型间的性状差异均表现为连续变异和明显的超亲分离。同时主根粗（1cm）和主根长的变异系数较小，分别为11．27％和11．218％。11个花生根部性状都是受多基因控制的数量性状，如影响侧根根瘤数、侧根鲜重和侧根干重的基因均在10对左右；而其它性状的基因估计在5—7对左右，尤其是控制侧根数的基因最少为5对左右。而在RIL群体中．除侧根干重的遗传力最高．达0．569，其次是侧根根瘤和侧根数分别达0．545和0．542外，其它性状的遗传力均较低。同时控制主根长和主根粗（1cm）的基因间存在重叠作用；而控制侧根根瘤、侧根鲜重和侧根干重基因间存在互作，表现为互补作用；控制其它性状的基因间互补或重叠作用不明显或者不存在。主根干重和侧根干重与根体积、主根粗（1cm）和主根粗（3cm）显著相关，根体积与主根粗（3cm）极显著相关，主根鲜重和侧根鲜重与根体积的相关表现不一致。