玉米分子标记技术的研究进展

主要介绍了AFLP、RAPD、RFLP和SSR分子标记技术在玉米育种中的应用，以及近年来分子标记技术在玉米遗传多样性，QTL定位等研究方面的应用。应用分子标记技术，不仅对玉米种质资源的发现、鉴定、分类整理和开发利用有一定作用，而且能进一步挖掘与玉米高产、优质高抗有关的基因，从而进行基因的定位和标记，提高玉米育种的效率。同时运用该技术对玉米抗逆性基因进行精确定位，为这些抗逆基因的克隆、转化及利用打下良好的基础，为分子标记辅助选择育种提供了条件，加快育种进程。     

玉米品质相关的opaque基因研究进展

玉米中的opaque突变体改变了胚乳的蛋白特性,导致胚乳表现柔软且不透明的粉质状。粉质胚乳的高赖氨酸营养特性引起人们的极大关注,研究人员先后发现了13个opaque胚乳突变体,只有o2的分子机理研究较为清楚,对提高赖氨酸含量的作用最大。为了研究胚乳中储藏物质合成、装配、转运的调控机理,从而将这些有益突变基因应用于农业生产,研究人员继而发现了多个胚乳修饰的主效位点及基因(Opm)。本文综述玉米opaque突变体及相关基因的研究进展,对当前育种中利用该类基因培育优质蛋白玉米(QPM)的研究状况进行分析,为高赖氨酸玉米的分子标记辅助选择(MAS)和基因聚合育种提供参考。     

辅助选择优质蛋白玉米种质的ELISA方法

基于玉米胚乳中赖氨酸含量与EF-1α含量呈显著正相关,利用ELISA技术定量检测胚乳中EF-1α含量,进而估测胚乳中赖氨酸含量,建立了辅助选择优质蛋白玉米种质的ELISA技术体系.通过对653份QPM家系的检测,表明该方法具有简便、快速、准确的特点,可有效辅助选择优质蛋白玉米种质。     

优质蛋白玉米育种研究进展

普通玉米蛋白质品质差,以改良玉米蛋白质品质为目标的优质蛋白玉米(QPM)育种一直是玉米研究的重要课题。本文简要回顾了利用o2基因改善玉米蛋白质品质的育种历程,重点介绍了QPM的营养价值、遗传基础、种质选育和应用以及分子标记应用于QPM辅助育种,分析了目前QPM种质遗传基础狭窄、杂种优势研究滞后和QPM品种抗逆性差、适应性窄等问题,提出了针对这些问题的解决策略,并展望了QPM的重大应用前景。     

玉米育种理论技术新拓展与商业育种实践

一套功能健全的商业育种技术体系是商业育种研发流水线的保障。新世纪伊始,各种玉米育种新技术层出不穷,例如分子标记辅助选择技术、单倍体育种技术、品种精确评价技术和高通量数据采集技术等,这些技术大大提高了商业育种研发流水线的效率。本文从我国玉米种业的研发现状出发,分析育种新技术在加速商业育种进步中的作用,提出切实可行的玉米种质资源创新体系,该体系整合了包括分子标记辅助选择技术、单倍体育种技术、品种精确评价技术、高通量数据采集技术和育种大数据管理技术在内的先进商业育种技术,构建了玉米规模化种质资源创新体系,可以更有效地加速我国玉米商业育种的进程。     

分子标记技术在玉米基因定位和辅助选择中的应用

本文评述了近一、二十年利用分子标记技术研究玉米重要基因的作图和定位及分子标记辅助选择等,并提出了我国在该领域的发展策略。     

具抗稻瘟病基因Pi25杂交稻恢复系的分子标记辅助选育

 从组合中156/谷梅2号所衍生的重组自交系群体中选择携带抗稻瘟病基因Pi25的3个株系,分别与高产恢复系9308和3 11配组,通过单粒传法获得的6个F6重组自交系群体用于研究。在Pi25先前定位的遗传图谱上,新增加与该基因紧密连锁的分子标记RM3330和A7,并结合RM3330和A7分子标记辅助选择的结果,共筛选到了109个Pi25基因纯合的株系。用现有的与恢复基因连锁的标记对这109个株系进行二次筛选,最终获得20个Pi25基因和恢复基因均纯合的株系。对育性恢复力鉴定试验表明,选育出的抗病恢复系具有较好的恢复能力,并已应用于育种实践;人工稻瘟病接种试验证实,所用标记不同,分子标记辅助选择的效率差异显著,单个标记辅助选择符合率均不高,而采用目标基因两侧连锁的标记同时进行辅助选择,则可以明显提高分子标记辅助选择符合率。     

分子标记与玉米遗传育种

建立在分子遗传学基础上的分子标记技术的快速发展,推动了玉米研究在各个领域的发展。本文从分子标记图谱的构建、自交系亲缘关系和遗传多样性评价、QTL分析及分子标记辅助选择等方面介绍了分子标记技术在玉米遗传育种中的应用。     

马铃薯青枯病抗性AFLP图谱的初步研究

通过温室单株抗病性的鉴定,选出了三个能够作为马铃薯青枯病分子标记辅助选择抗病育种的亲本。利用AFLP分子标记的方法检测了它们基因组的DNA的多态性,并构建了其AFLP图谱,且有望应用于标记辅助的选择育种。     

玉米叶片基因组快速提取方法研究

SSR(Simple Sequence Repeat)分子标记技术已经成为玉米常规育种某些性状辅助选择的重要手段,需要对大量个体样本进行标记分析。高效、快速提取植物组织基因组DNA是关键的技术环节。本研究以高油玉米回交世代群体25～30d单株幼叶为材料,建立以EDTA、CTAB、KCl为主要试剂,结合组织研磨棒液氮研磨为主要步骤的基因组DNA快速提取方法,并探讨了将提取的DNA应用于SSR分子标记辅助选择的可行性。     

Quality Protein Maize: Overcoming the Hurdles

SUMMARY

Quality Protein Maize (QPM), a nutritionally enhanced maize, was developed by researchers from CIMMYT using too genetic systems–opaque-2 and genetic modifiers. The use of these two genetic systems overcame the highly complex problems that were inherent in the original soft endosperm opaques. This review describes the ever-evolving breeding options and strategies for the development of QPM with examples from the CIMMYT maize program, where much of the research and practical breeding work has been done. The soft endosperm opaque-2 materials developed earlier had poor agronomic performances and lacked producer and consumer acceptance. To overcome these constraints, subsequent research explored various options, with and without high lysine mutants. Like other institutions, CIMMYT researchers tried and critically examined the merits and demerits of different strategies. Of all the strategies available, the selection for modified kernels in which CIMMYT scientists had gained information, experience, and confidence seemed viable. To implement this strategy, modified opaque-2 donor stocks were built and were subsequently used for expanding the QPM developmental efforts. A large volume of QPM germplasm was developed using different breeding options, which were later merged and reorganized into a fixed number of pools and populations to permit working in homozygous opaque-2 genetic backgrounds. The development of QPM hybrids was the next turning point in the mid-80s. During the QPM developmental process, serious problems inherent in the opaques were circumvented and since then, rapid progress has been made. There is a renewed interest in QPM and several countries have recently released QPM varieties and hybrids. To further accelerate the QPM developmental process, to enhance its popularity amongst nations and its farmers, and to meet future challenges, innovative ideas and the tools of biotechnology will be needed.     

青饲(贮)玉米育种与优质蛋白玉米种质的商业化创新利用

结合近30年来我国优质蛋白玉米(QPM)的发展演变,通过对国外青饲(贮)玉米部分标准的分析与国内优质蛋白玉米(QPM)、青饲(贮)玉米发展现状的研究,提出促进我国优质蛋白玉米种质的经济实用化(商业化)创新利用,加快青饲(贮)玉米、粮饲型玉米育种发展步伐的优先序:青饲(贮)玉米--粮饲型玉米--优质玉米--优质蛋白玉米。     

Nixtamalized Flour From Quality Protein Maize (<Emphasis Type="Italic">Zea mays</Emphasis> L). Optimization of Alkaline Processing

Quality of maize proteins is poor, they are deficient in the essential amino acids lysine and tryptophan. Recently, in México were successfully developed nutritionally improved 26 new hybrids and cultivars called quality protein maize (QPM) which contain greater amounts of lysine and tryptophan. Alkaline cooking of maize with lime (nixtamalization) is the first step for producing several maize products (masa, tortillas, flours, snacks). Processors adjust nixtamalization variables based on experience. The objective of this work was to determine the best combination of nixtamalization process variables for producing nixtamalized maize flour (NMF) from QPM V-537 variety. Nixtamalization conditions were selected from factorial combinations of process variables: nixtamalization time (NT, 20–85 min), lime concentration (LC, 3.3–6.7 g Ca(OH)₂/l, in distilled water), and steep time (ST, 8–16 hours). Nixtamalization temperature and ratio of grain to cooking medium were 85 °C and 1:3 (w/v), respectively. At the end of each cooking treatment the steeping started for the required time. Steeping was finished by draining the cooking liquor (nejayote). Nixtamal (alkaline-cooked maize kernels) was washed with running tap water. Wet nixtamal was dried (24 hours, 55 °C) and milled to pass through 80-US mesh screen to obtain NMF. Response surface methodology (RSM) was applied as optimization technique, over four response variables: {In vitro} protein digestibility (PD), total color difference ( E), water absorption index (WAI), and pH. Predictive models for response variables were developed as a function of process variables. Conventional graphical method was applied to obtain maximum PD, WAI and minimum E, pH. Contour plots of each of the response variables were utilized applying superposition surface methodology, to obtain three contour plots for observation and selection of best combination of NT (31 min), LC (5.4 g Ca(OH)₂/l), and ST (8.1 hours) for producing optimized NMF from QPM.     

“Antioxidant activity and characterization of protein fractions and hydrolysates from normal and quality protein maize kernels”

Maize is the main crop cultivated worldwide with more than 1 billion metric tons produced annually and is one of the most relevant sources of protein for human consumption in developing countries. Proteins and peptides isolated from maize exert relevant antioxidant activity which is increased by enzymatic hydrolysis. However, there is limited information about the antioxidant potential of proteins isolated from Quality Protein Maize (QPM) varieties and their hydrolysates. The aim of this research was to determine the differences in protein profile and antioxidant activity of protein fractions and hydrolysates between a hybrid white maize (Asgrow 773) and a QPM variety (CML-502). The biophysical evaluation and the total protein quantification by Kjeldahl and fractions by ninhydrin were consistent with the changes due to the breeding process of the QPM material. The antioxidant potential of the hydrolysates obtained from albumins and globulins had a 3-fold increase in both maize varieties. The prolamins hydrolysates presented an increase of 7-fold for the normal variety and 2-fold for the QPM variety. The results of this research allow indicate that the QPM varieties are a source of antioxidant peptides and promising candidates in the search for proteins and peptides with other bioactivities.     

几个供体对优质蛋白玉米(QPM)近等基因系构建效果的比较

对CA335、CA339、山东2548和齐205这4个国内常用优质蛋白玉米自交系的赖氨酸含量、子粒表型、醇溶蛋白积累及opaque-2基因的序列进行分析,对不同自交系作为opaque-2突变基因(o2)基因供体的分子标记选择效果进行比较。结果表明,4个自交系属于两类不同的o2突变体类型,CA335和CA339为一类,山东2548和齐205为一类。两类突变体作为o2供体选择效果有较大差异,CA335和CA339是优良供体,齐205和山东2548则不是理想供体。对于优质蛋白玉米育种,供体的选择非常重要,好的QPM自交系不一定是好的供体。     

Collaborative breeding with farmers can be effective

Plant breeding efficiency is increased by better taking into account the needs of the clients. This can sometimes be helped by involving farmers in selection in the segregating generations. We describe a rice-breeding programme in Nepal where the use of simple breeding methods optimised the benefits from such collaboration. Farmers grew in their fields large populations that were bulks derived from the F₂ generations of single crosses. They selected within them starting in a generation where selfing was sufficiently advanced for the between-plant heritability to be high. Poorer bulks were soon rejected by farmers. A high proportion selected within the bulks and a significant proportion did so over several generations. An increasing number of farmers grew the best of the selected bulks because they spread from farmer to farmer without the need of intervention from scientists. Varieties selected from these bulks were preferred by farmers in on-farm trials and had a superior combination of yield and maturity compared with the later breeders’ selection from the same bulk. Levels of participation were higher with the better performing bulks and even when training was not provided, provided the germplasm performed acceptably well sufficient farmers selected in the bulks to make collaborative breeding cost effective. Collaborative breeding can be successful but demands different types of resources than those for on-station breeding so its cost effectiveness will vary with circumstances. It can be used to increase the probability of selecting segregants that perform well across environments by using methods that pool bulks selected by different farmers and it is also extremely useful for decentralising breeding programmes.     

Protein quality and endosperm modification of quality protein maize (Zea mays L.) under two contrasting soil nitrogen environments

Quality protein maize (QPM) breeding involves the combined use of the opaque-2 (o2) gene and the genetic modifiers of the o2 locus to develop cultivars with modified kernel endosperm, and increased concentrations of lysine and tryptophan. This study was designed to assess grain yield performance, endosperm modification, and protein quality and quantity under two contrasting soil nitrogen environments. A 15-parent diallel cross was evaluated under one low nitrogen stress and one optimal nitrogen environment each at Harare (Zimbabwe) and Bako (Ethiopia). Most QPM hybrids showed higher protein quality levels than the best non-QPM check under both conditions. Protein concentration tended to vary across nitrogen levels, but not endosperm type. Significant differences were found for the test of main effect (nitrogen-level) for endosperm modification and tryptophan concentration. This indicated that QPM maintains quality even under low soil nitrogen, a widespread condition in Africa. General combining ability (GCA) mean squares were highly significant for most protein quality traits for each environment and across environments whereas specific combining ability (SCA) mean squares were not significant in most cases. This indicated that additive gene effects were primarily responsible for variation of most traits evaluated and hence progeny performance can adequately be predicted on the basis of parental performance. Inbred lines P2, P4 and P12 had desirable GCA effects for endosperm modification while P1 and P3 had the best GCA for tryptophan concentration in grain. The current study suggests that hybrids with desirable endosperm modification, protein quality and stable performance under low nitrogen stress and optimal conditions can be produced with careful selection.     

大豆M型细胞质雄性不育恢复基因标记定位

鉴于与恢复基因紧密连锁的分子标记在分子辅助选择育种中的应用前景,采用SSR标记法定位大豆CMS恢复系WR016的恢复基因.根据(W931A×WR016)F1、F2的植株育性,分析表明大豆M型不育系统为单基因配子体不育.由于大豆M-CMS恢复系WR016的恢复基因定位在A1连锁群上,利用A1连锁群上的大豆SSR引物对不育系W931A和恢复系WR016构建的F,分离群体进行分析,获得了与恢复基因连锁的三个标记Satt684、Satt276和Sat545,遗传距离分别为29.5cM、10.7 cM和14.1 cM.虽然10.7 cM还是一个较远的距离,但为进一步精确定位恢复基因,并最终克隆恢复基因打下了基础.