【作者】 蔡青；

【导师】 黄兴奇；

【作者基本信息】 云南大学 ， 植物学， 2013， 博士

【摘要】 现代甘蔗品种来源于甘蔗属Saccharum L.热带种Saccharum officinarum L.与细茎野生种Saccharum spontaneum L.的属内种间杂交后代,血缘基础狭窄,育种难有较大突破。利用甘蔗近缘属野生资源斑茅Erianthus arundinaceus (Retz.) Jesw.,与甘蔗进行属间杂交,可拓宽遗传基础、增强品种抗性、促进育种突破性进展,是国内外研究的重点和热点。滇蔗茅Erianthus rockii Keng是我国特有种,是蔗茅属中具有开发利用潜力的新资源。目前,斑茅和滇蔗茅的杂交利用取得了一些进展,但缺乏全面和系统化的遗传结构和遗传背景分析,使杂交利用的有效性、针对性和利用范围受到一定影响,不利于进一步推进资源的开发利用进程和深入研究。本文从地理分布、表型变异、染色体类型和分子标记四个层次,对我国目前收集保存的斑茅和滇蔗茅资源进行全面和系统化的遗传多样性研究,旨在了解其遗传背景,探明地理种群与表型、染色体和DNA分子水平上的遗传多样性变化规律,为资源的保护和利用提供指导,为杂交利用亲本选择提供科学依据。同时,通过开展斑茅、滇蔗茅种质创新研究,鉴定杂交利用的有效性,评价杂交后代的育种利用价值,为拓宽甘蔗选育基础提供科学指导。通过研究,得到以下结果：1斑茅的遗传多样性与种质创新研究(1)我国斑茅主要分布于长江以南,地理种群丰富,表型性状多样并在不同海拔区表现不同我国收集保存的314份斑茅资源来源于云南、四川、贵州、广东、广西、福建、海南、江西、浙江、西藏、陕西、湖北、湖南、河南共14个省区145个县,主要分布区为长江以南地区,海拔-24-2627m,跨越热带、亚热带至温带三种气候类型,显示出较强的生态适应性和地理种群多样性。云南、四川是斑茅分布较为密集的地区,以云南分布最广,除昆明和曲靖外全省均有分布。其次是福建和海南,其中海南全岛一半以上区域均有分布。表型性状变异分析显示：108份资源的5个数量性状平均变异系数为28.62%,变异幅度16.22～40.01%。变异系数最大为叶宽(41.01%),最小为叶长(16.22%),其他依次为株高(22.08%)、锤度(20.42%)和茎径(17.27%)。说明数量性状遗传变异主要来自于叶宽,其次为株高和锤度,茎径和叶长则较其它性状相对稳定。地理分布与表型性状多样性呈现一定规律,各海拔区表型变异为：中、低海拔区的锤度变异差异最大(21.56%与9.97%)；其次为中、高海拔区的株高(23.24%与12.8%)；各海拔区叶宽变异相近(41.79%、37.02%和37.97%),均呈现较高的多样性。质量性状Shannon-Wiener多样性指数显示：叶鞘背毛群性状多样性指数最高(0.9917),显示该性状为质量性状主要变异。其次为叶色和叶姿(0.8566,0.4705),脱叶性则变异极小。表型性状聚类结果显示：表型性状相似材料各自聚类,各类群均有来源不同的地理种群,呈现出各表型性状类群资源在地理分布上的多样性。(2)斑茅染色体数目有3种类型并与地理分布和表型呈现一定相关性鉴定出斑茅染色体数共有3种类型：2n=60、40、30。云南有2n=60、40两种类型,贵州、福建有2n=60、30两种类型。2n=60是主要类型,地理分布广、表型变异大；2n=40数量少,为云南滇西高大型斑茅；2n=30为来自贵州、福建的中小型斑茅。(3)AFLP分子标记遗传多样性丰富,并存在地区差异207份中国斑茅和部分国外斑茅材料AFLP分子标记分析结果显示：3个AFLP引物共扩增出287个条带,其中286个多态条带,多态率99.3%。与参试的国外材料相比,中国斑茅具有较高的遗传多样性,印度尼西亚则较低。菲律宾与中国的遗传相似性最高,而印度与中国较低。在中国斑茅中,云南斑茅遗传多样性最高,以云贵川为主的西南省区材料、和以福建、广西、广东和江西为主的东南省区的材料相对分开,各自聚为一类,说明亲缘关系与地理分布呈现出一定相关性。(4)创制了甘蔗与斑茅杂交组合并获得真实性杂交后代和优良创新种质通过光周期技术,诱导热带种、品种和品系开花或提早开花,调节亲本花期相遇,与斑茅F1、BC1、BC2进行回交或杂交,创制甘蔗与斑茅杂交组合19个获得实生苗1332株,经分子标记鉴定出真实性杂交后代11份。不同亲本组合类型的杂交配制成功率不同,分析初步表明：斑茅、滇蔗茅F]、BC1材料较适于用作母本,斑茅BC2作为父本略好于作为母本,而BC3材料则用作父母本均产生初生苗。经农艺性状综合评价,筛选出2份优良创新种质(BC207-106、BC307-131),获得品种权保护申请受理,并提供国家产业体系育种利用。2滇蔗茅的遗传多样性与种质创新(1)滇蔗茅地理种群丰富,滇西是主要分布区,表型变异丰富多样我国收集保存的60份滇蔗茅来源于云南、贵州、广西、四川4个省19个县。在云南收集到的55份滇蔗茅采集地主要分布于滇西地区,海拔范围693m-2340m,垂直高差1647m。表型性状变异分析显示：41份材料6个数量性状的平均变异系数为21.23%,变异幅度12.71-34.3%,由大到小依次为：株高(34.3%)、锤度(24.1%)、茎径(21.1%)、节间长度(19.2%)、叶宽(15.9%)和叶长(12.7%)。表明滇蔗茅性状变异主要来自于株高,其次为锤度,节间长度、叶宽和叶长变异较小。滇蔗茅表型多态水平在不同海拔区有差异,中海拔区株高变异系数比高海拔区大(39.3%、19.5%),说明中海拔区的滇蔗茅株高类型比高海拔区丰富；其次为锤度(25.6%与19.9%)；各海拔区茎径的多态性水平相近(21.1%和21.7%)。6个质量性状Shannon-Wiener多样性指数分析结果表明,曝光后节间颜色多样性指数大于1(1.3113),具有较高的多态性,其次为叶姿、叶色,茎形和节间形状则变异极小。表型性状聚类分析显示：不同类群含有采集地或海拔不同的资源,说明表型性状相似的资源,在不同地理分布区均有分布,呈现出各表型性状类群的资源在地理分布上的多样性。(2)滇蔗茅的染色体类型有1种：2n=30采用花粉母细胞鉴定技术,鉴定出来源于云南、贵州、四川的滇蔗茅染色体数均为2n=30,原产地水平距离和地理位置相差大,海拔、表型性状具有差异。(3)滇蔗茅的分子标记遗传多样性丰富对39份云南滇蔗茅和2份四川、贵州材料进行AFLP分子标记遗传多样性分析,结果显示,滇蔗茅的AFLP分子标记多态性高,多态率为73.1%。云南滇蔗茅中,遗传多样性最高的为德宏材料,其次为保山材料。德宏与保山的滇蔗茅遗传相似性较高,遗传关系较近。聚类分析的结果显示,研究材料亲缘关系与原产地呈现出一定相关性,来自云南保山、德宏各县市的材料大部份聚在一起,形成较为集中的一群。(4)热带种与滇蔗茅的属间杂交中染色体以n+n方式传递采用GISH分析滇蔗茅F1的染色体组分,结果显示：滇蔗茅F103-316的染色体数目为54,其中来自母本的染色体为39、父本的染色体为15,说明热带种与滇蔗茅属间杂交的染色体传递与甘蔗属内杂交“高贵化”传递方式(2n+n)不同,热带种与滇蔗茅的属间杂交中,染色体以n+n方式传递。(5)创制滇蔗茅F1,选出第一个滇蔗茅优良创新种质通过光周期诱导调节花期和人工杂交技术,以滇蔗茅F1、BC1为母本与品种或品系杂交,成功创制杂交组合12个,获得实生苗857株。其中1个组合为热带种与滇蔗茅的原种杂交组合,获得了62株实生苗,是开展属间远缘杂交研究以来获得实生苗数量最大的一个组合,并经分子鉴定全部为真实性杂交种。结果初步表明：以滇蔗茅F1、BC1为母本的杂交组合获得了较多实生苗。通过鉴定评价,筛选出滇蔗茅优良创新种质BC106-279已申请了品种权保护。更多还原

【Abstract】 Modern sugarcane cultivars are derived from a limited number of inter-specific hybridization events between Saccharum officinarum L. and Saccharum spontaneum L.. The genetic base hence is narrow and difficult to have breakthrough in sugarcane breeding.Intergeneric hybridization between Erianthus arundinacens (Retz.) Jesw. and sugarcane (S. officinarum) is a way for broadening the genetic basis so as to increase the ability of stress resistant. Erianthus rockii Keng, which is originated from China, is another new source of species used for the intergenetic hybridization. A number of attemps in crossing have been performed by using E. arundinaceus or E. rockii with sugarcane, and several hybrids have been identified as genuine. However, lack of the knowledge from the genetic diversity analysis for these two germplasm resources, influences the utilization of targeting to the clones, as well as the more number of clones used in crossing.This study repored the results of the genetic diversity assessment for E. arundinaceus and E. rockii by four analysis levels including:geographic distribution, phenotypic variations, chromosome types and AFLP molecular makers. The study was to learn the genetic base of these species, and to provide the basic information of the relationships between and within existing germplasm collections, for the purpose of conservation of genetic diversity and for targeted sampling of clones for future sugarcane introgression breeding programs.This study meanwhile reports the intergeneric crosses between Saccharum and E. arundinaceus or E. rockii. A series of intergeneric progenies were verified by molecular makers and GISH analysis. The aim was to verify the introgression of E. arundinaceus and E. rockii germplasm into sugarcane, for identifying and evaluating the value of Erianthus germplasm utilization in sugarcane breeding. The innovated germplasm hybrids of BC2, BC3from E. arundinaceus and BC1from E. rockii were selected and being used as new parents in sugarcane variety improvement. The main results from the study were as following:1The genetic diversity of E. arundinaceus and germplasm innovation(1) The geographic distribution were diverse and the phenotypic variations were obvious in elevationsThe information from the data records of the collecting origins was analyzed to assess the geographic distribution for the E. arundinaceus collections in the whole country. The result showed that:the314clones were from14provinces and145counties. The horizontal distribution was in the east longitude of97°36’～121°38and the northern latitude of18°14’～32°28’; the vertical distribution was in the elevation of-24-2627m. The majoy distribution of E. arundinaceus was the south of the Yangze river region, which crossed the tropical and subtropical to the temperate zones. This revealed the ecological adaptability of E. arundinaceus to the diverse geography. Yunnan, Sichuan, Fujian and Hainan were the concentrated regions of the collections.The9phenotypic variations from the108clones preserved in the National Field-Genebank for Sugarcane Germplasm were analyzed. The results showed that:the average variation coefficient from5quantitative characters was28.62%from a range of16.22～40.01%, in which the highest was from the Lamina width (41.01%) and the lowest was from the Lamina length (16.22%). The others were the Stalk length (22.08%), the Brix (20.42%) and the Stalk diameter (17.27%). This meant that the main phenotypic variation of E. arundinaceus was from the Lamina width.In the different elevation regions, the phenotypic variation coefficients were obvious:the Brix was21.56%in the middle latitude area and9.97%in the low area; the Stalk length was23.24%in the middle and12.8%in the high latitude areas. The variation coefficient of Lamina width was higher in the high latitude area than in the middle and low latitude area; the variation coefficient of Stalk length was higher in the middle and low latitude areas than in the high latitude area. The Shannon-Wiener coefficient from4qualitative characters showed that the Hair group57was the highest (0.9917).(2) Three types of chromosome number of E. arundinaceus were observed:2n=60、40、30The cytological observation of pollen mother cells showed:there were three types of chromosome number observed:2n=60、40、30. The type of60and40were observed in Yunnan materials, and the60and30were observed in Guizhou and Fujian materials. Most materials were the main type of60and had wide geographic distribution with more phenotypic variations. Several materials were observed as40and30, the former was originated in the western of Yunnan province and the latter was from Guizhou and Fujian. The type of60presented the varied types of the agronomic characters, the type of40presented higher stalk length, and the type of30presented as shorter stalk length.(3) The diversity of AFLPs was high and differences existed among areasFurther analyses from the DNA level was conducted based on the above studies. The207materials of E. arundinaceus accessions mainly from China, and part of other countries including:Indonesia, India, New Guinea, Philippines and Vietnam, were sampled for genetic diversity analysis using AFLP markers. The results showed that:(i) the highest diversity level was found within the Chinese collection and lowest within the Indonesian collection;(ii) the genetic similarity showed that the materials from Philippines were found more similar to the Chinese collection, while the ones from India were opposite.(iii) There was a general division amongst clones from China based on those collected from eastern versus western parts of the country, and clones from Yunnan province (western China) were the most diverse.(4) Crosses combination between sugarcane and E. arundinaceus spp. were successful conducted to produce seedlings identified to be genuine hybrids, and2innovated germplasm clones were selected. Hybrid utilization of E. arundinaceus germplasm was performed by manual crossing with S. officinarum clones or sugarcane cultivars, which were inducted flowering by photoperiodic induction.19crosses combination between sugarcane and F1, BC1or BC2of E. arundinaceus were successful conducted to produce1332seedlings,11of them were identified to be genuine hybrids. The2innovated germplasm clones (BC207-106、BC307-131) were selected and applied the Plant Protection Rights.2The genetic diversity of E. rockii and germplasm innovation(1) The geographic distribution were diverse and the western part of Yunnan province was the main diverse area, the phenotypic variations were obvious in elevationsThe information from the data records of the collecting origins was analyzed to assess the geographic distribution for60E. rockii collections. The result showed that: the horizontal distribution was in the east longitude of95°53’～103°25and the northern latitude of22°30’～28°40’;40of55Yunnan clones were originated in the western of Yunnan. The vertical distribution was in the elevation of-693～2340m. The ecosystem was rich in this area so as to form the diverse geographic distribution.The phenotypic variation was mainly from the quantitative traits of Stalk length and Brix, and the qualitative trait of Internode colour exposed. The variation coefficient of Stalk length was higher in the middle latitude area than in the high latitude area; the variation coefficient of Brix was higher in the middle latitude areas than in the high latitude area. The Shannon-Wiener of the qualitative characters showed that the Internode colour exposed was the highest (1.3113).(2) Only one type of chromosome number of E. rockii was observed:2n=30The cytological observation of pollen mother cells showed:only one type of chromosome number was observed:2n=30. Total4materials were identified as this type and their geographic locations were far distance. Also, the elevations were obviously different as well as the phenotypic characters.(3) The diversity of AFLPs was high level in E. rockii collectionFurther analyses from the DNA level was conducted based on the above studies. The39materials of E. rockii accessions mainly from Yunnan, and2from Sichuan and Guizhou, were sampled for genetic diversity analysis using AFLP markers. The results showed that:(i) the highest diversity level was found within the Dehong collection in Yunnan, and the second was the Baoshan collections;(ii) the genetic similarity showed that the clones from Dehong was found more similar to the Baoshan;(iii) the genetic relationship was basically match to the geographic area, the ones from Baoshan and Dehong were clustered to be a major group.(4) The chromosome transmission from the parents of S. officinarum and E. rockii to F1was found as" n+n"GISH methods was conducted to analyze the chromosome composition in F1(03-316) arising from the cross between Vietnam Niuzhe (S. officinarum) and Yunnan92-20(E. rockii). The aim was to characterize the genome structure of hybrids derived from E. rockii, for providing information that could be used in designing effective introgression breeding strategies. The results showed that:(i) the chromosome numbers of Yunnan92-20(E. rockii) was identified as2n=30; the chromosome numbers of F1was observed as54, in which39from the male and15from the female;(ii) the chromosome transmission was n+n by the male parentand the female parent into F1generation. This first reported by GISH that the chromosome transmission from the intergeneric hybridization of S. officinarum and E. rockii was n+n, which was not as the2n+n of "Nobilization"as the interspecific hybrids among the species in Saccharum genera(5) Crosses combination between S. officinarum and E. rockii were successful conducted to produce seedlings identified to be genuine F1hybrids, and a innovated germ plasm clone was selected.Hybrid utilization of E. rockii germplasm was performed by manual crossing with S. officinarum clones or sugarcane cultivars, which were inducted flowering by photoperiodic induction.12crosses combination between sugarcane and E. rockii clone&F1were successful conducted to produce857seedlings,62of F1and3BC1were identified to be genuine hybrids. E. rockii BC106-279was selected to be an elite innovation germplasm to obtain the acception of PBR.更多还原