回交育种分析花生脂肪酸含量的变化

采用亲缘关系甚远的高油酸亲本Sunoliec95R与低油酸亲本汕油162配置杂交组合（F1）,并回交2代（BC1和BC2）,结果表明,在花生回交种中油酸及油酸／亚油酸值均有不同程度的升高,轮回亲本中控油基因已部分转入到回交种中。选育出一批高低油酸、高低O/L值种仁,以期作为花生品质育种选配亲本的参考,为广西丰产种质拓宽与改良提供一定的理论依据。     

花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答

Δ~(12)-脂肪酸脱氢酶基因(FAD2)催化油酸生成亚油酸,是决定油酸亚油酸比值的关键基因。高油酸花生对低温更加敏感,暗示FAD2在低温响应中发挥作用。为探索花生FAD2的低温胁迫应答,本研究分析了花生FAD2-1A/B的基因结构,从普通油酸花生品种豫花9326中克隆了FAD2-1A/B启动子和内含子,并通过转化拟南芥验证了功能及其对冷胁迫的应答。结果表明, Ah FAD2-1A/B包含2个外显子和1个位于5'-UTR的内含子; Ah FAD2-1A/B基因启动子功能较弱,仅Ah FAD2-1B启动子在子叶期幼苗的子叶叶尖中观察到蓝色; Ah FAD2-1假基因5'侧翼序列具有启动子活性,可调控基因在子叶、下胚轴、种子中表达。Ah FAD2-1内含子序列具有启动子的功能,驱动基因在幼苗下胚轴及子叶中表达,同时还有提高基因表达效率和扩大表达范围的功能,是Ah FAD2-1基因表达调控必需元件;包含5'-UTR内含子的Ah FAD2-1A/B启动子的调控序列功能受低温胁迫抑制。     

CRISPR/Cas9编辑花生FAD2基因研究

油酸脱氢酶((35)12FAD或FAD2)是催化油酸(OA)的C12位上脱氢生成双不饱和亚油酸(LA)的关键酶,它控制油酸、亚油酸的含量及其比值(O/L)。研究表明, AhFAD2是油酸生成亚油酸的关键基因,决定花生种子中油酸和亚油酸的相对含量。本研究根据AhFAD2基因序列,设计了相应sgRNA序列,并构建了旨在敲除FAD2A和FAD2B两个花生油酸脱氢酶的CRISPR/Cas9基因编辑载体。经花生基因转化后,通过对转基因花生突变位点基因组序列分析找出基因突变体。对靶基因分析发现,16株转基因花生含有29个FAD2A基因突变,其中16个突变引起蛋白质序列变化;11株转基因花生含有30个FAD2B基因突变,其中17个突变引起蛋白质序列变化。FAD2A和FAD2B蛋白质序列的变化可影响花生油酸脱氢酶的活性,改变油酸催化脱氢,使亚油酸合成受阻,油酸含量增加。本研究为研究花生脂肪酸合成及高油酸花生育种提供了宝贵的基因突变体材料。     

低温胁迫对普通和高油酸花生种子萌发的影响

高油酸花生以其营养价值高和耐储藏等特点深受广大消费者和加工企业的喜爱。近年来,随着高油酸花生品种在我国的推广应用,高油酸花生在高海拔、高纬度地区的发芽期耐寒性成为关注热点。为探究花生种子的油酸含量与其萌发期耐寒性的相关性,本研究调查6组不同遗传背景的花生品种及其高油酸回交后代品系(BC4F8)在低温条件下的发芽率和发芽指数发现,在低温胁迫下花生的萌发期耐寒性与其油酸含量无显著相关性。在泉花551高油酸后代品系(Quanhua551-HO)低温发芽率显著低于其普通油酸含量亲本(Quanhua551-NO)的组合中,追踪分析8种主要脂肪酸在低温胁迫萌发过程中含量的变化发现,在低温胁迫下Quanhua551-NO中油酸含量显著减少且亚油酸含量显著增加,而Quanhua 551-HO中也表现出了油酸含量减少、亚油酸含量增加的趋势,但是未达到显著水平。进而分析上述2种材料中低温胁迫下各脂肪酸脱氢酶(fattyaciddesaturase2,FAD2)基因的表达模式发现,Quanhua551-NO中AhFAD2-1A/B受低温诱导显著上调表达,而AhFAD2-4A/B显著下调表达;在Quanhua551-HO中AhFAD2-4A/B受低温诱导持续显著上调表达,而AhFAD2-1A/B显著下调表达,推测由于高油酸花生中AhFAD2-1A/B编码蛋白失活, AhFAD2-4A/B在低温诱导下高量表达,部分弥补了AhFAD2-1A/B缺失的功能。综上所述,花生种子中油酸含量并不是决定其萌发期耐寒性的关键因素。     

以分子标记辅助连续回交快速提高花生品种油酸含量及对其后代农艺性状的评价

高油酸是花生重要的品质性状,高油酸花生及其制品具有较好的品质稳定性和较高的营养和保健价值。我国高油酸花生的育成品种类型较少,遗传背景不够丰富,育种手段比较单一。针对上述问题,本研究开发了AS-PCR-MP高油酸分子标记检测方法,优化了KASP分子标记检测体系,利用分子标记辅助连续回交,结合近红外品质快速检测技术及南繁加代技术,以河南省大面积推广的豫花15、远杂9102、豫花9327、豫花9326四个不同类型品种为轮回亲本, 5年内连续回交4代、自交4代,定向获得了4个轮回亲本遗传背景的BC4F4和BC4F5稳定高油酸改良材料24个。调查分析了BC4F4和BC4F5单株的13个农艺性状与轮回亲本的相似度,并利用轮回亲本与非轮回亲本之间的差异SNP的KASP分子标记进行了BC4F4和BC4F5株系的轮回亲本遗传背景检测。结果表明,轮回亲本的遗传背景在BC4F5的比例为79.49%～92.31%。本研究为快速高效改良花生油酸含量探索了新的方法,获得的新品系拓展了高油酸花生的遗传背景,获得的一系列近等基因系可作为遗传研究材料进一步加以利用。     

分子标记辅助选育高产高油酸花生新品种舜花 14 号

舜花 14 号是以普通油酸含量的花育 19 号作母本及轮回亲本,高油酸花生开农 176 作父本,经 TaqMan 探针及 KASP分子标记辅助选育而成的高产高油酸大花生品种。2019 年开始参加山东省高油酸花生联合体新品种多点比较试验,荚果平均产量 5601.90kg/hm2,较对照花育 25 号平均增产 8.33%。2022 年单粒精播高产攻关试验实打验收荚果产量 9728.40kg/hm2,首创高油酸花生实打验收山东省高产纪录。品种籽仁蛋白质含量 26.4%,含油量 50.67%,油酸含量 80.6%,亚油酸含量 2.99%,O/L 比值 26.96。2022 年 9 月通过国家非主要农作物品种登记,登记编号：GPD 花生（2022）370149。     

大花生花育22号栽培技术

花育22号,原代号花20-1,系山东省花生研究所以8014为母本,钴60辐照海花1号干种子M1代为父本,辐射与杂交相结合,经系谱法选育而成。2003年通过黄海品种审定委员会审定。     

利用近红外技术和转座子标记鉴定花生杂交F_1真杂种

为探讨花生耐低温的遗传机理,实现耐低温分子标记辅助选择育种的长远目标,并培育耐低温的高油酸品种,搭配了2个普通油酸×高油酸杂交组合。首先通过近红外技术初筛,选择油酸含量预测值高于40%的F1单粒种子,进一步经转座子标记鉴定,分别获得了32粒和24粒真杂种。两对转座子标记引物Ah TE0398和Ah TE0167鉴定结果完全一致,也与FAD2等位基因特异PCR(AS-PCR)结果一致,证实了转座子标记鉴定技术的可靠性。     

花生新品种花育22号种植表现及高产配套技术

<正>花育22号是山东花生研究所以8014为母本,辐照海花1号干种子M1代为父本,辐射与杂交相结合,经系谱法选育而成的早熟大花生。2003年通过山东省农作物品种审定委员会审定。为了筛选出适宜烟台地区种植的花生新品种,烟台市种子管理站自2008年引进并开展新品种比较试验和适应性试验,该品种表现出高产、优质、多抗的特性,在烟台地区得到大面积推广,近4年,累计推广13.63万hm2,为我市花生产业的提升做出巨大贡献。     

苜蓿雄性不育系MS-4回交早代雌蕊育性分析

以苜蓿雄性不育系MS-4为母本,4个具有优良性状的苜蓿材料为父本,进行杂交和连续回交,转育苜蓿雄性不育系。本文对回交早代不育株雌蕊育性进行了分析,结果表明：BC1A1-21和BC1A1-22雌蕊育性提高且雄性不育性稳定。     

胚珠培养获得秘鲁番茄与栽培番茄种间杂种具有非温敏抗根结线虫基因Mi-3

以含有Mi-3抗根结线虫基因的秘鲁番茄(Lycopersicon peruvianum)LA2823为父本,栽培番茄（Lycopersicon esculentum)高代优良自交系为母本,授粉后20～30d摘取幼果。将从幼果中分离出的直径大于2mm的胚珠放置在营养分化培养基上培养。2～4周后胚芽、胚根分别由两端伸出。培养的胚珠还可以通过体细胞胚发生途径产生胚芽。获得的种间杂种对根结线虫的抗性表型与野生种父本完全相同,在植物学性状上亦与其父本秘鲁番茄更相近。以栽培番茄为母本进行回交,成熟果实与母本相似,但其种子即使放置在MS培养基上也不能萌发。对回交种子胚败育现象进行了探讨。     

甘蓝型油菜BnFAD2-C5基因启动子及内含子在表达水平的功能分析

富含油酸的菜籽油具有重要的经济价值,使得高油酸育种和形成机理的研究成为热点。油酸脱氢酶基因(FAD2基因)是控制油酸含量的关键酶基因。本文针对BnFAD2-C5基因展开研究,根据油菜和甘蓝的同源性,克隆了1257 bp启动子序列,利用GUS和GFP作为报告基因分别构建含有不同片段长度的启动子和内含子的缺失载体并转化拟南芥,经GUS染色检测发现–319 ~ –1 bp为该研究中最小启动子;采用Western技术分析启动子和内含子不同区域的功能,发现BnFAD2-C5启动子区域–1257 ~ –1020 bp和–319 ~ –1 bp能够诱导报告基因在转基因拟南芥种子发育中期高效表达,BnFAD2-C5内含子具有增强启动子转录水平的功能,该功能主要由631~1033 bp区域调控。     

采用SSR标记辅助选育具有Xa22(t)的云南高原粳稻新种质

以高原粳稻新品种滇粳优1号为轮回亲本,携带Xa22(t)基因的云南地方稻种扎昌龙为供体亲本,回交后代BC3F1290株和BC3F2290个株行为供试材料,采用与Xa22(t)紧密连锁标记RM224及其它11对SSR．标记进行辅助选择。290株BC3F1个体在RM224位点上杂合基因型个体的比率为21．04％;其它11对SSR．标记位点的轮回亲本纯合基因型个体比率平均为93．07％,但不同染色体标记位点上纯合基因型的比率不同。用云南高原粳稻上的白叶枯病优势菌株YH24对亲本、BC3F1单株和BC3F2株行接种鉴定,BC3F1中RM224位点上杂合基因型的61个植株表现为抗至中抗,RM224位点上滇粳优1号纯合基因型单株都表现为感病;由RM224位点杂合基因型BC3F1单株衍生的61个BC3F2代株行表现为抗、感分离。     

Effects of high oleic acid soybean on seed yield,protein and oil contents,and seed germination revealed by near‐isogeneic lines

Typical soybean oil is composed of palmitic, stearic, oleic, linoleic and linolenic acids. High oleic acid content in soybean seed is a key compositional trait that improves oxidative stability and increases oil functionality and shelf life. Using a marker‐assisted selection method, near‐isogenic lines (NILs) of G00‐3213 for the high oleic trait were developed and yield tested. These NILs have various combinations of FAD2‐1A and FAD2‐1B alleles that were derived from the same backcrossing populations. The results indicated that G00‐3213 NILs with both homozygous mutant FAD2‐1A and FAD2‐1B alleles produced an average of 788 g/kg oleic acid content. The results also demonstrated that possessing these mutant alleles did not cause a yield reduction. Furthermore, seed germination tests across 12 temperatures (12.8–32.0°C) showed that modified seed composition for oleic acid in general did not have a major impact on seed germination. However, there was a possible reduction in seed germination vigour when high oleic seeds are planted in cold soil. The mutant FAD2‐1A and FAD2‐1B alleles did not hinder either seed or plant development.     

棉籽油分和3种主要脂肪酸含量QTL分析

本研究对棉籽油分、棕榈酸、油酸和亚油酸含量进行了不同遗传体系的QTL分析,为相关性状挖掘出更多有用的基因信息。分别于2017年和2018年,利用陆地棉亲本HS46 (P₁)和MARCABUCAG8US-1-88 (P2)所构建的188个重组近交系分别与双亲杂交构建F1群体BC (P₁)和BC(P₂)。基于这些回交群体种子,采用专为种子性状设计的母体和胚核基因组QTL定位的混合线性遗传模型及QTLNetwork-CL-2.0-Seed软件,对棉籽油分、棕榈酸、油酸和亚油酸含量进行QTL定位分析。共检测到7个控制棉籽油分含量、3个控制棕榈酸含量、2个控制油酸含量和3个控制亚油酸含量的QTL,均具有显著或极显著的源自母体和胚2个核基因组的加性主效应,其中有7个QTL的表型变异贡献率大于10%。研究结果可为这些性状的分子标记辅助选择育种提供更为可靠的参考,为这些性状的分子遗传机制研究提供理论基础。     

反义RNA介导GmFAD2-1B基因沉默增强大豆种子中油酸的高效累积

油酸含量是评价大豆油食用品质和稳定性的重要指标。本研究采用农杆菌介导转化法,将反义GmFAD2-1B基因导入栽培大豆品种,获得油酸含量显著提高的转基因大豆新品系。Southern杂交检测表明,外源GmFAD2-1B基因片段已导入大豆基因组,其插入拷贝数为1~5个。q RT-PCR检测表明,外源GmFAD2-1B主要在大豆种子中表达,并导致种子中内源GmFAD2-1 m RNA表达水平显著降低,而根、茎、叶、花组织中内源GmFAD2-1 m RNA表达水平无显著变化。脂肪酸组分分析表明,12份转基因大豆种子油酸含量为27.38%~80.42%,其中,L40和L72油酸含量分别为68.91%~80.42%和65.98%~80.22%,较对照品种Williams 82(17.8%~22.0%)提高2.65倍以上,亚油酸降低至4.84%~14.55%,饱和脂肪酸降低至10.34%~11.16%,但总脂肪和总蛋白含量与对照品种相比没有显著变化。农艺性状分析表明,转基因大豆在熟期、株高、叶形、花色、结荚高度、百粒重等方面与对照品种也没有显著差异。     

冀豆12遗传背景导入系蛋白、脂肪含量分布特征

以高蛋白品种冀豆12为受体亲本,不同来源、不同蛋白脂肪含量的大豆种质资源为供体亲本,构建了28个组合BC2F1后代群体,分析冀豆12遗传背景导入系后代蛋白、脂肪含量分布特征。结果表明,28个后代群体均有蛋白含量超高亲个体,超高亲个体比例介于4.0%～68.2%之间,超高亲比例≥40%的组合有18个,占64.3%,BC2F1后代群体蛋白含量以超高亲和偏高亲类型组合为主。而脂肪含量分布特征恰相反,BC2F1后代群体脂肪含量以超低亲和偏低亲类型组合为主,超高亲个体比例介于0～67.4%,超高亲个体比例≥40%的组合有7个,占25.0%,9个组合无超高亲后代。表明以冀豆12为遗传背景通过有限回交易选育高蛋白含量品种,而不易选育高脂肪含量品种。本研究结果为利用冀豆12培育高蛋白品种提供了依据。     

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F₁, from which F₂ and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F₁ plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F₂ populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F₁ hybrids and used commercially to prevent downy mildew at the cotyledon stage. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro selection for oleic and linoleic acid content in segregating populations of microspore derived embryos of Brassica napus

Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62% C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection for high oleic acid can be started already during an early stage of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular marker analysis of the linkage drag around the FAE1 loci of Brassica juncea during conventional backcross breeding

A post-transfer investigation was carried out to analyze the process of gene introgression in a conventional backcross-breeding program taking low erucic acid as target trait in Brassica juncea. FAE1 locus is involved in the elongation of oleic acid (C18:1) to erucic acid (C22:1). A high concentration of erucic acid in the seeds of Brassica species has been reported to be nutritionally undesirable. Molecular markers like AFLP markers, microsatellite markers, and gene-based SNP markers were used to determine the size of the donor parent chromosomal segments retained around the FAE1 genes in the individuals selected from different backcross generations. The genotype of the individuals was inferred from the genotype of the markers and the graphical genotypes were constructed using GGT software. Molecular marker analysis led to the identification of rare recombinants near the target locus with reduced size of introgressed segment from the donor parent. Based on the present study, we propose that marker-assisted backcross breeding in B. juncea could prove to be a promising tool for the transfer of many quality traits from unadapted East European germplasm to Indian cultivars.