【作者】 李健；

【导师】 蔡一林；

【作者基本信息】 西南大学 ， 作物遗传育种， 2020， 博士

【摘要】 青稞是藏区最重要的粮食作物,是保证藏区粮食安全的重要支撑。因此,青稞育种工作在解放初期就已全面开展,近70年来,选育出了一百多个优良品种,有力的推动了青稞总产、单产的提高。随着藏区社会、经济的长足发展以及青稞精深加工的开展,市场对优质青稞的需求激增,优质也逐渐成为青稞育种的重要方向。β-葡聚糖具有降血脂、调控血糖、提高人体免疫力的作用,因此,高β-葡聚糖含量是优质青稞品种选育的热点方向,但是优异种质资源少,研究基础薄弱,技术手段落后成为了阻碍高β-葡聚糖含量青稞新品种选育的主要障碍。加强青稞种质资源遗传多样性和品质性状的评价,挖掘优异种质,探索高β-葡聚糖含量的成因,研发高效的育种方法可以为藏区高β-葡聚糖含量青稞新品种选育提供优异种质和技术支撑。本研究以1950s后,影响力高、推广面积大的105个藏区青稞育成品种和3个引进品种为研究对象,采用亲缘系数分析法对藏区青稞育成品种的遗传多样性进行评价,从中筛选遗传距离较远,田间综合农艺性状较好的育成品种。对中选育成品种和青稞种质的β-葡聚糖含量、总淀粉含量进行分析,从中筛选出β-葡聚糖含量差异显著的优异种质,以β-葡聚糖含量与Hv Csl F6基因的相关性分析为切入点,探索优异青稞种质高β-葡聚糖含量的成因并对其进行验证。通过上述研究,取得如下结果:（1）108个品种共形成5778对组合,16.49%的品种组合间存在亲缘关系,各组合亲缘系数的变异范围为0.000-0.750,组合平均亲缘系数为0.054,显著高于国内大麦主栽品种以及北美大麦品种;不同时期,参试育成品种的组合平均亲缘系数存在显著差异,1970s和1980s 2个时期,参试育成品种的组合平均亲缘系数较高,分别达到0.131和0.115;1990s和最近十年,参试育成品种的组合平均亲缘系数较低,分别为0.045和0.037;不同地区,参试育成品种的组合平均亲缘系数同样存在明显区别,其中,甘肃省最高,达到0.260,西藏自治区、青海省、四川省则较低,分别为0.098、0.131和0.155;基于亲缘系数分析和聚类分析,从108个品种中筛选出9个骨干亲本。9个骨干亲本及其67个大面积推广的衍生品种占参试育成品种总量的72.38%,外引大麦种质利用率低,导致藏区青稞育成品种遗传多样性低,遗传基础相对狭窄。（2）203份青稞种质资源的β-葡聚糖平均含量为5.11%,变异范围为3.22%-9.50%,β-葡聚糖含量在4.00%-7.00%之间的分布频率达到96.06%;参试青稞种质的淀粉平均含量为50.46%,变异范围为42.28%-59.91%,淀粉含量在45.00%-55.00%之间的分布频率达到93.10%;西藏、青海、四川、甘肃的青稞种质资源的β-葡聚糖平均含量分别为5.23%、5.07%、5.05%和5.21%,各地青稞种质资源的β-葡聚糖平均含量间不存在显著差异;西藏、青海、四川、甘肃的青稞种质资源的淀粉平均含量分别为50.30%、50.86%、50.53%、48.91%;来源于青海省和甘肃省的青稞种质资源的淀粉平均含量间存在显著差异;根据种质资源评价的结果,从中筛选出β-葡聚糖含量差异显著的11份青稞种质,其β-葡聚糖含量变幅为4.00%-9.50%,包含3份育成品种和8份优异种质;（3）11份青稞种质的Hv Csl F6基因存在16个SNP多态性位点,其中两个位于5’UTR,8个位于第一内含子,5个位于第二内含子,1个位于第三外显子;Hv Csl F6基因系统发育树分析结果表明,分属不同类群种质的β-葡聚糖平均含量间没有显著差异;因此,11份青稞种质Hv Csl F6基因的SNP位点与籽粒的β-葡聚糖含量间不存在相关性;（4）在授粉后第22天,青稞种质Hv Csl F6基因在胚乳中的表达丰度与成熟籽粒的β-葡聚糖含量显著正相关;在同为糯质、非糯种质的条件下,Hv Csl F6基因在胚乳中的表达丰度与成熟籽粒的β-葡聚糖含量间存在显著、极显著正相关关系;因此,“在胚乳发育中期Hv Csl F6基因高表达”是导致青稞种质高β-葡聚糖含量的重要原因;（5）糯质青稞种质与非糯青稞种质相比,在授粉后第22天,糯质种质的Hv Csl F6基因表达丰度较低,但是糯质种质的β-葡聚糖含量却高于或近似于非糯种质的β-葡聚糖含量;因此,“糯质”可能是导致青稞种质高β-葡聚糖含量的另一重要原因,并且“糯质”与“在胚乳发育中期Hv Csl F6基因高表达”存在“叠加效应”,该效应导致Z999的β-葡聚糖含量达到9.50%;（6）以糯质种质Z1191和非糯育成品种ZQ25为亲本,构建由101个F₆代重组自交系组成的验证群体;通过两年田间实验并对群体β-葡聚糖含量、淀粉含量、直链淀粉占比以及千粒重进行分析,结果显示,非糯亲本ZQ25和糯质亲本Z1191的β-葡聚糖平均含量分别为6.83%和5.78%,群体β-葡聚糖平均含量为6.51%,变幅为4.11%-9.08%;非糯品系的β-葡聚糖含量变幅为4.11%-7.79%,平均值为5.62%;糯质品系的变异范围为6.69%-9.08%,平均值为8.11%;验证群体β-葡聚糖含量与直链淀粉占比呈极显著负相关,相关系数达到-0.83;因此,“糯质”是导致青稞种质高β-葡聚糖含量的另一重要因素。糯质品系的β-葡聚糖平均含量分别较糯质亲本、非糯亲本提升40.31%,18.74%,较非糯品系的β-葡聚糖平均含量提升44.31%且呈极显著差异;3份糯质后代品系的β-葡聚糖含量大于9.00%,分别较糯质亲本、非糯亲本提升55.71%和31.77%,且与Z999非常接近;该结果表明在糯质后代品系中存在“糯质”与“在胚乳发育中期Hv Csl F6基因高表达”的叠加效应。利用糯质青稞种质与高β-葡聚糖含量的非糯青稞种质组配是选育高β-葡聚糖含量青稞品种的可行方法。更多还原

【Abstract】 In Qinghai-Tibet plateau,hulless barley is the most important staple food,therefore the breeding activity of hulless barley has been carried out for nearly 70 years.More than one hundred varieties were bred at this period,promoting the yield increase of hulless barley.With the rapid development of Tibetan society and economy,the need of high quality hulless barley has been increased.Therefore,quality has become the main breeding target of barley breeding.β-glucan has been believed to greatly reduce the risk of serious human diseases,including type II diabetes,cardiovascular disease and colorectal cancer.To breed varieties with highβ-glucan content has become a hot target in the hulless barley breeding activity.The lack of excellent germplasm resources and the effective breeding methods has hindered the progress.In order to promote the breeding process,it has been necessary to evalute the genetic diversity and the quality characters of hulless barley germplasm resources,select the excellent germplasms and explore the efficient breeding method.In this study,105 bred hulless barley varieties grown commonly and broadly across Qinghai-Tibet plateau,which has been surveyed since 1950,and 3 introduced varieties were selected as representative materials.The genetic diversity of the bred hulless barley varieties in Qinghai-Tibet plateau was estimated by coefficient of parentage（COP）.Based on the results of COP analysis and local field performance,the selected bred varieties and some hulless barley accessions were selceted to evalute the quality traits.Based on evaluation results,11 accessions with differentβ-glucan content were selected as research materials to analyse the association between theβ-glucan content and the Hv Csl F6 gene.The following results have been obtained:（1）5778 variety combinations were produced by 108 varieties.16.49%of variety combinations were genetically related.The COP of all combinations ranged from 0.000to 0.750,with an average of 0.054,which was significantly higher than that of the main barley varieties grown in the rest of China and North American.At different era,there was significant difference about the average COP of the combinations.This index was higher at 1970s and 1980s,reaching 0.131 and 0.115,respectively,while this index was lower at the 1990s and the recent decade,reaching 0.045and 0.037,respectively.At different areas,the average COP of the combinations made by the varieties from Gansu Province was highest,reaching 0.260,while this index for Tibet Autonomous Region,Qinghai Province,and Sichuan Province were similar,reaching 0.098,0.131 and 0.155,respectively.Based on the COP analysis,9 bred varieties were identified as important core parents in Qinghai-Tibet plateau,from which,67 varieties had been derived.9 core parents and their 67 derivatives account for 72.38%of investigated bred varieties,the utilization of introduced germplasms has been inefficient,therefore.The genetic diversity of hulless barley cultivated across the Qinghai-Tibet plateau is relatively lower.（2）Theβ-glucan content of 203 hulless barley accessions was ranged from3.22%-9.50%,with an average of 5.11%.Theβ-glucan content of 96.06%accessions was ranged from 4.00%to 7.00%.The starch content of the investigated accessions was ranged from 42.28%to 59.91%,with an average of 50.46%.The starch content of93.10%accessions was ranged from 45.00%to 55.00%.The averageβ-glucan content of the accessions derived from Tibet,Qinghai,Sichuan and Gansu was 5.23%、5.07%、5.05%and 5.21%,respectively.There was no significant difference among the averageβ-glucan contents of accessions from different areas.The average starch content of the accessions derived from Tibet,Qinghai,Sichuan and Gansu was 50.30%、50.86%、50.53%and 48.91%,respectively.There was significant difference between the average starch content of accessions from Qinghai and Gansu province.11 hulless barley accessions with significant difference inβ-glucan content were selected from the 203hulless barley accessions,including 3 varieties and 8 landraces,which were used to analyse the association between theβ-glucan content and the Hv Csl F6 gene.（3）16 polymorphic sites of Hv Csl F6 gene were detected within the selected accessions.Two SNPs were located at 5’UTR,8 SNPs at intron 1,5 SNPs at intron 2and 1 SNP at exon 3.A phylogenetic tree was constructed on the basis of the sequences of Hv Csl F6 gene.According to the phylogenetic tree,the investigated accessions were divided into three groups.there was no significant difference among the averageβ-glucan contents of different groups.Therefore,it was concluded that the relationship between the polymorphic sites of Hv Csl F6 gene and theβ-glucan content in matured grain was not obvious.（4）During the endosperm development,at the sampling time of 22^nd DAP（Day After Pollination）,there was significant positive correlation between the transcript abundance of Hv Csl F6 gene and theβ-glucan content in the matured grain.For waxy or non-waxy barley accessions,at the sampling time of 22^nd DAP,there was significant or highly significant positive correlation between the transcript abundance of Hv Csl F6gene and theβ-glucan content in the matured grain,which indicated that the high transcript abundance of Hv Csl F6 gene during the middle stage of endosperm development was one of the causes that led to the highβ-glucan content of some hulless barley accessions.（5）At the sampling time of 22^nd DAP,the Hv Csl F6 gene’s transcript abundance of waxy accessions was lower than that of the non-waxy accessions,whereas theβ-glucan content was higher than or similar with that of the non-waxy accessions.Therefore,the trait of waxy might be the another cause that led to the highβ-glucan content.Moreover,there might be a superimposed efffect created by the trait of waxy and the trait of high transcript abundance of Hv Csl F6 gene during the middle stage of endosperm development,which led theβ-glucan content of Z999 reach 9.50%.（6）To verify this speculation,waxy barley accession Z1191 and non-waxy barley varieties ZQ25 were used as parents to develop a population of 101 F₆-derived RILs.Based on the results of two-year field experiments of this RILs,4 quality traits of this RILs were analysed,includingβ-glucan and starch content,the ratio of amylose and the weight of thousand grains.The averageβ-glucan content of waxy parent and non-waxy parent was 5.78%and 6.83%,respectively and theβ-glucan content of this RILs was ranged from 4.11%to 9.08%,with an average of 6.51%.For 65 non-waxy lines,theβ-glucan content was ranged from 4.11%to 7.79%,with an average of 5.62%,and for36 waxy lines,theβ-glucan content was ranged from 6.69%to 9.08%,with an average of 8.11%.There was highly significant negative correlation between theβ-glucan content and the ratio of amylose（r=-0.83）.Therefore,it was concluded that the trait of waxy was one of the causes that led to the highβ-glucan content.The averageβ-glucan content of the waxy lines was 44.31%significantly higher than that of the non-waxy lines,which was 40.31%and 18.74%higher than that of their waxy and non-waxy parents,respectively;there were 3 waxy lines with extremely highβ-glucan content（>9.00%）;which was 55.71%and 31.77%higher than that of their waxy and non-waxy parents,respectively.This results indicated that for the waxy lines,there was the superimposed effect between the trait of waxy and the trait of high transcript abundance of Hv Csl F6 gene during the middle stage of endosperm development.Based on the results of the RILs,making a cross between a waxy barley accession and a non-waxy barley accession with higherβ-glucan content could be a feasible method of breeding hulless barley varieties with higherβ-glucan content.更多还原