摘要
体细胞杂交是转移野生种有益性状如抗病性等到栽培种中的重要方式。体细胞杂种由于可能带有一些野生种不利性状,在应用中往往需要不断自交或者通过与栽培种不断回交以消除遗传累赘。为了更深入地揭示马铃薯体细胞杂种的遗传特点以及更好地利用体细胞融合所创造的遗传资源,本研究以前期实验室获得的44个体细胞杂种和一个体细胞杂种的回交与自交后代为材料,系统分析了体细胞杂种及回交、自交后代的遗传组分,对主要目标性状青枯病抗性进行了评价,并对体细胞杂种的一些重要农艺性状和植物学性状进行了观察。主要研究结果如下：
1.体细胞杂种的遗传组分分析与表型评价。研究以44个体细胞杂种(Solanum tuberosum cv.3#+S. chacoence acc. C9701)为材料,通过108对SSR引物对其遗传组分进行分析。结果显示,44个体细胞杂种总共扩增出12576个位点,每个杂种平均285.8个位点。双亲共有位点在杂种中的保留率较高,49个共有位点在杂种中几乎全部保留。栽培种融合亲本特异位点平均保留90.02%,野生种融合亲本特异位点平均保留83.59%。体细胞杂种间双亲遗传信息保留程度存在差异,最高的栽培种亲本和野生种亲本遗传信息保留率分别可达到98.11%和90.83%,最低只有84.91%和72.48%,表明即使是对称融合产生的体细胞杂种,其遗传信息仍然发生了部分丢失。本研究还证明,在44个体细胞杂种中,栽培种遗传组分比野生种遗传组分比例高,以分子标记位点保留率进行的统计分析显示,二者在后代中的保留率差异达到显著性水平(P=0.048)。
44个体细胞杂种中37个系生长正常,7个系生长极其缓慢,长势极弱。在武汉气候环境下,29个体细胞杂种能够开花,占65.9%,其中2个系可以自交形成浆果。39个体细胞杂种能够形成块茎,占88.6%。大部分植物学性状变异幅度较大,部分性状出现超亲分离。基于形态学的聚类分析显示,61.4%的杂种后代趋于栽培种亲本。
2.体细胞杂种3C28-1回交和自交后代的倍性与遗传组分分析。本研究对体细胞杂种3C28-1的自交和与栽培种鄂马铃薯1号(E1)的回交后代进行了相关遗传研究。采用染色体压片法对88个回交后代的染色体计数结果显示,染色体数为48左右且经卡方测验符合四倍体的系有14个(15.91%),染色体数为60的系1个,为典型的五倍体(1.14%)。其余73个系的染色体数在50-56之间,卡方测验既不符合四倍体也不符合五倍体,为非整倍体(82.95%)。
从184对SSR引物中筛选出94对可在3C28-1和E1中扩增出清晰、且具有多态性的SSR引物用于回交后代遗传组分分析。结果显示,90个回交后代共扩增出16758个多态性位点,平均每个后代186.2个位点。进一步分析显示,多态性位点中12625为本研究所涉及的栽培种(体细胞融合亲本3#和回交亲本E1)和野生种(体细胞融合亲本C9701)的共有位点,其余4133个为特异位点。野生种特异位点为1343个,栽培种为2790个(包括3#特异位点1223个和E1特异位点1567个)。这些位点在群体中的比例分析显示,共有遗传信息在群体中占75.34%,野生种特异信息占8.01%,栽培种特异信息占16.65%。栽培种特异信息中,轮回亲本E1的信息占有比例为9.35%。以栽培种和野生种的特异位点数在总特异位点数作为各自遗传组分,比较分析回交群体与体细胞杂种的栽培种与野生种遗传组分的变化,结果表明,经过一次回交后,后代中栽培种遗传组分从体细胞杂种亲本3C28-1的36.2%上升至54.7%,而野生种组分从63.8%下降至45.3%。
本研究通过同源四倍体测交的遗传分析模式计算标记的分离方式,进而推测体细胞杂种可能的遗传方式。83个3C28-1特异位点中,62个位点(74.7%)符合四倍体的二体遗传或四体遗传,其余21个位点不符合四倍体遗传可能的任何分离模式。在符合四倍体遗传模式的62个位点中,25个位点(30.1%)符合二体遗传或四体遗传方式,37个(44.6%)符合四体遗传方式。而符合四体遗传的位点中,16个位点表现出染色单体随机分离的方式。通过t测验发现,3C28-1中的3#和C9701特异位点遗传方式上并没有差异,说明3C28-1中3#和C9701的特异位点以相同的方式遗传给后代。
3C28-1自交后代的遗传组分分析表明,自交群体中栽培种组分和野生种组分没有出现显著的分离现象。
3.体细胞杂种及其回交和自交后代的青枯病抗性评价。本研究采用试管苗伤根接种方法进行青枯病鉴定评价。结果显示,15.9%的体细胞杂种后代具有青枯病抗性,但抗病水平均低于抗病野生种融合亲本C9701。15.5%的3C28-1的回交后代具有青枯病抗性,其中5个株系和C9701抗性水平一致,表现为高抗。46.4%的3C28-1的自交后代具有不同程度青枯病抗性,其中9个系和C9701处于同一抗性水平。这些结果表明,野生种S. chacoense的青枯病抗性已通过原生质体融合转移到栽培种马铃薯中,体细胞杂种的抗性能正常遗传。通过青枯病抗性、农艺性状、野生种的遗传组分等综合分析,共筛选到3个具有育种潜质的体细胞杂种,同时获得了3个具有利用价值的回交品系。
4.分子标记与青枯病抗性相关分析。为了筛选与青枯病抗性相关的SSR标记,本研究利用109个体细胞融合抗性亲本C9701的特异SSR标记进行标记与体细胞杂种的青枯病抗性相关性分析。结果显示,本研究涉及的SSR标记中,共有3个SSR标记和体细胞杂种的青枯病抗性呈显著相关,它们是STI0002.108、 STI0056.173和STI0057.195。采用单标记检测分析法,将C9701的特异SSR标记与回交后代青枯病抗性进行了相关分析,结果表明,有2个SSR标记在回交群体中与青枯病抗性呈显著相关性,它们是STI0056.173和STI0046.190。 STI0056.173是在体细胞杂种和体细胞杂种的回交后代中均与青枯病抗性具有显著的相关性,表明STI0056.173可能与抗病基因具有较近的遗传距离,可作为青枯病抗性辅助选择标记。
Somatic hybridization is an important avenue for transferring the alien traits from wild species into cultivar, such as resistance against pathogens. However, undesirable traits may be co-introduced with the target traits to cause so called genetic drag, which needs inbreeding, or more often the backcrossing, to overcome. Forth-four somatic hybrids have been previously obtained from protoplast fusion between Solanum tuberosum cv.3#and S. chacoense acc. C9701, and the backcrossing and inbred progenies derived from one of the hybrids (3C28-1) have been obtained. For the purposes of better gain of the knowledge about potato somatic hybrids and efficient use of these uniquely created germplasms, in present research, their genetic components, inheritance mode were investigated by employing the SSR markers. Additionally, the morphology characteristics and the resistance to bacterial wilt of the somatic hybrids and the derived progenies were evaluated. The main results are as follow:
1. Genetic components and morphological characters of the somatic hybrids. One hundred and eight SSR markers were used to clarity the genome components of the somatic hybrids. The results showed that totally12576SSR alleles were amplified in the44somatic hybrids with an average of285.8in each. Nearly all of the fourth-ninety common SSR alleles of cultivated parent3and the wild parent C9701were retained in the hybrids while90.02%unique alleles of cultivated parent3#and83.59%unique alleles of wild parent C9701were present in the hybrids. There were differences in number of the parent-specific alleles among the hybrids. The highest allele proportion of the cultivated fusion parent that were retained in the hybrids was98.11%and that of the wild fusion parent was90.83%, whereas the lowest were observed as84.91%and72.48%, respectively. The results suggested that there could be some loss of the parental genetic information even in the hybrids of symmetric fusion. The genomic dosage of3#and C9701in the hybrids was further analyzed after arcsine transformation of the percent of polymorphic alleles of each fusion parent appeared in the hybrids by paired samples t-test. The result showed that the hybrids retained a higher proportion of nuclear DNA dosage from cultivated parent than wild parent (p=0.048).
The plant growth of37out of44somatic hybrids tested were normal while7of them were poor. Twenty-nine hybrids (65.9%) could flower under Wuhan geographic conditions and two of them could produce inbred berries, suggesting most of the hybrids could be further improved when it is necessary for a breeding program. There were88.6%of the hybrids produced tubers although they exhibited remarkable variation in botanic traits, by which61.4%of the hybrids could be clustered into one clade.
2. Genetic components of the backcrossing and inbred progenies. The ploidy level of88backcrossing progenies were analyzed by root tip chromosome counting. The results demonstrated that the chromosome number of14clones was48(tetraploid) and1was60(pentaploid). The rest73clones had chromosome number between50-56, which could not be statistically confirmed tetraploid or pentaploid, were assigned aneuploids.
Ninety-four SSRs yielded clear and repeatable polymorphic bands between3C28-1and E1were selected to clarify the nuclear genome components of the backcrossing progenies. The results showed that16758SSR bands were amplified in90backcrossing clones with an average of186.2in each. Of the polymorphic alleles,12625were common in both cultivate and wild species, accounting for75.34%. Of the rest4133unique alleles,1343were wild species-specific (8.01%) and2790cultivate species-specific (16.65%). Further analysis indicated that the1223alleles were unique of3#(7.3%) and1567were unique of El (9.35%). The genome components of the backcrossing clones showed that the genome of cultivate species increased from36.2%(percentage the unique alleles of the cultivate fusion parent3#retained in somatic hybrid3C28-1) to54.7%(percentage of sum of unique alleles of 3#and the backcross parent E1in the backcrossing population), while that of the wild species decreased from63.8%to45.3%after one cycle of backcross.
Inheritance pattern of the somatic hybrids was analyzed with the mode of the test-cross for tetraploid. The inheritance pattern was determined by following the segregation of83unique SSR alleles of3C28-1and testing all the possible segregation in the hybrids. The results demonstrated that62SSR alleles (74.7%) fit the expected segregation ratios of tetraploid, in which25cases (30.1%) displayed disomic or tetrasomic inheritance, while other37(44.6%) undoubtedly displayed tetrasomic inheritance. Of the tetrasomic-inherited SSR alleles,16displayed random chromatid inheritance and21did not fit any possible segregation. The percentage of3#and C9701specific SSR alleles fitting the segregation ratios in the hybrids was further analyzed after arcsine transformation by paired samples t-test. No significant difference (P=0.824) was detected between the segregation ratios of the unique alleles of3#and C9701, suggesting that there was no bias toward any genome components of the somatic hybrids which may have similar inheritance pattern.
The results of genetic component analysis for the inbred progenies showed that there were no obvious changes in the proportion of genome components retained from the cultivar and wild fusion parents.
3. Evaluation of bacterial wilt resistance. The results demonstrated that15.9%of the somatic hybrids were resistant to bacterial wilt though their resistance levels were lower than the wild parent C9701;15.5%of the backcrossing progenies were resistant and5of them exhibited same resistance level as C9701;46.4%of the inbred progenies were resistant and9of them had same resistance level as C9701. These results suggest that the bacterial wilt resistance of S. chacoense has been transferred to cultivated potatoes through protoplast fusion and the resistance of the somatic hybrids is inheritable. Three somatic hybrids and2backcrossing clones which have potentials for further use in potato breeding were selected based on their genome components, plant morphology and the disease resistance.
4. Coefficient analysis of molecular makers and bacterial wilt resistance. To identify the SSR alleles associated with bacterial wilt resistance in potato, all of the108unique SSR alleles from C9701were analyzed in relation to the resistance of somatic hybrids tested. The results showed that3nuclear alleles (STI0002.108, STI0056.173and STI0057.195) were significantly related to bacterial wilt resistance. Furthermore, all the44unique SSR alleles of C9701identified in the backcrossing population were analyzed in relation to the resistance of the90backcrossing clones by single marker analysis. The results showed that2alleles (STI0046.190and STI0056.173) had significant effects on the resistance. Noticeably, STI0056.173was identified with significant correlation to the resistance in both somatic hybrids and the backcrossing population derived from one resistant somatic hybrids, implying that STT0056.173might be closer to the resistance locus than other SSR alleles and could be used for marker-assist selection of potato bacterial wilt resistance introgressed from C9701.
引文
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